RSV F vaccine formulation

Abstract

Infectious diseases remain a global challenge. Although progress has been made in developing vaccines against some pathogens, many remain a threat to human health. The development of vaccines that prevent or reduce the severity of life-threatening infectious diseases such as RSV is desirable. Disclosed herein are RSV F glycoproteins and nanoparticles comprising the same suitable for use in vaccines. The nanoparticles present pathogen-derived antigens surrounded by and associated with a surfactant core, which results in enhanced stability and good immunogenicity. Dosages, formulations, and methods for preparing vaccines and nanoparticles are also disclosed.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 483,119, filed February 3, 2023, which is incorporated herein in its entirety for all purposes.

[0002] Description of electronically submitted text files The contents of the electronic sequence listing (NOVV_104_01WO_SeqList_ST26.xml, size: 852,414 bytes, and creation date: February 3, 2024) are incorporated herein by reference in their entirety.

[0003] The present disclosure generally relates to RSV F glycoproteins and nanoparticles useful for stimulating an immune response. The nanoparticles provide an antigen, such as a glycoprotein antigen, associated with a surfactant core and are typically produced using recombinant techniques. The nanoparticles have improved stability and enhanced epitope presentation. The present disclosure also provides compositions containing nanoparticles, methods for producing them, and methods for stimulating an immune response. [Background technology]

[0004] Infectious diseases remain a challenge worldwide. While progress has been made in developing vaccines against some pathogens, many remain a threat to human health. Respiratory syncytial virus (RSV) causes 6,000–10,000 deaths annually in adults aged 65 years and older and 100–300 deaths in children under the age of five. The development of vaccines to prevent or reduce the severity of life-threatening infectious diseases such as RSV is desirable. Summary of the Invention

[0005] The present disclosure provides a non-naturally occurring respiratory syncytial virus (RSV) fusion (F) glycoprotein suitable for inducing an immune response against RSV. The present disclosure also provides nanoparticles containing the glycoprotein and methods for stimulating an immune response against RSV.

[0006] Provided herein is a RSV F glycoprotein comprising an F1 domain, an F2 domain, and p27, wherein the glycoprotein comprises one or more modifications selected from the group consisting of (a) one or more deletions of amino acids 137-146, (b) an inactivated first furin cleavage site, (c) an inactivated second furin cleavage site, (d) S155C, (e) S290C, (f) S190F, (g) V207L, and (h) N116Q, wherein the modifications are numbered based on the RSV F glycoprotein of SEQ ID NO: 330. In some embodiments, the RSV F glycoprotein comprises the S155C and S290C mutations. In some embodiments, the RSV F glycoprotein comprises the S190F and V207L ​​mutations. In some embodiments, the RSV F glycoprotein comprises the S155C, S290C, S190F, and V207L ​​mutations. In some embodiments, the RSV F glycoprotein comprises an N116Q mutation. In some embodiments, the RSV F glycoprotein comprises an inactivated primary furin cleavage site having the amino acid sequence of KKQKQQ (SEQ ID NO: 342), an S190F mutation, a V207 mutation, and a deletion of amino acids 137-146. In some embodiments, the RSV F glycoprotein comprises an inactivated primary furin cleavage site having the amino acid sequence of KKQKQQ (SEQ ID NO: 342), an S190F mutation, a V207 mutation, and a deletion of amino acids 137-146, wherein the RSV F glycoprotein has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the RSV F glycoprotein of SEQ ID NO: 337.In some embodiments, the RSV F glycoprotein comprises (i) an F1 domain having an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the F1 domain of SEQ ID NO: 352; and (ii) an F2 domain having an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the F2 domain of SEQ ID NO: 357. In several embodiments, the RSV F glycoprotein is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 333-340 and 364-366. In some embodiments, the RSV F glycoprotein is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the RSV F glycoprotein of SEQ ID NO: 337. [Brief explanation of the drawings]

[0007] [Figure 1] 1 shows the binding of the BV2279 glycoprotein to antibodies that recognize site II (palivizumab), site IV (RSV.42, designated "RSV.42.2"), site φ (D25), and site VIII (hRSV90) epitopes. [Figure 2] Shown is the anti-RSV F IgG titer in cotton rat serum 42 days after immunization with either the BV2279 (designated "prefusion RSV F Cav1") or BV1184 (designated "prefusogenic RSV F") vaccine compositions of Example 2. [Figure 3] Figure 1 shows RSV neutralization titers against RSV A (Long) and RSV B (18537) strains after immunization with either the BV2279 (designated "pre-fusion RSV F Cav1") or BV1184 (designated "pre-fusion RSV F") vaccine compositions of Example 2. [Figure 4] Shown are the titers of competitive antibodies induced by immunization with either the BV2279 (referred to as "pre-fusion RSV F Cav1") or BV1184 (referred to as "pre-fusion RSV F") vaccine compositions of Example 2. [Figure 5] Figure 1 shows RSV virus titers in cotton rat tissues 4 days after viral infection. The rats were immunized with either the BV2279 (referred to as "pre-fusion RSV F Cav1") or BV1184 (referred to as "pre-fusion RSV F") vaccine compositions of Example 2. [Figure 6] Transmission electron microscopy (TEM) images of influenza HA surfactant core nanoparticles alone (left), saponin adjuvant (i.e., first iscom particles containing Quillaja Saponaria Molina fraction A but not Quillaja Saponaria Molina fraction C, and second iscom particles containing Quillaja Saponaria Molina fraction C but not Quillaja Saponaria Molina fraction A, where fraction A accounts for 85% of the combined weight of Quillaja Saponaria Molina fraction A and Quillaja Saponaria Molina fraction C in the adjuvant, and Quillaja Saponaria Molina fraction C accounts for the remainder) (center), and hemagglutinin saponin matrix nanoparticles (HaSMaN) (right). [Figure 7] A shows the primary structure of the wild-type SARS-CoV-2 S polypeptide containing the signal peptide, numbered with respect to SEQ ID NO: 1. B shows the primary structure of the wild-type SARS-CoV-2 S polypeptide without the signal peptide, numbered with respect to SEQ ID NO: 2. DETAILED DESCRIPTION OF THE INVENTION

[0008] definition As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a protein" can refer to one protein or a mixture of such proteins, and reference to "the method" includes equivalent steps and / or methods known to those skilled in the art, and so forth.

[0009] As used herein, the term "adjuvant" refers to a compound that, when used in combination with an immunogen, amplifies or otherwise alters or modifies the immune response elicited against the immunogen. Modification of the immune response can include enhancing or broadening the specificity of either or both the antibody and cellular immune responses.

[0010] As used herein, the terms "about" or "approximately," when preceding a numerical value, indicate a range of that value plus or minus 10%. For example, "about 100" includes 90 and 110.

[0011] As used herein, the terms "immunogen," "antigen," and "epitope" refer to substances, such as proteins and peptides, including glycoproteins, that are capable of eliciting an immune response.

[0012] As used herein, an "immunogenic composition" is a composition that contains an antigen, and administration of the composition to a subject results in the subject generating a humoral and / or cellular immune response against the antigen.

[0013] As used herein, "substantially" refers to the isolation of a substance (e.g., a compound, polynucleotide, or polypeptide) such that the substance forms a majority proportion of the sample in which it is contained. For example, in a sample, a substantially purified component constitutes 85% of the sample, preferably 85% to 90%, more preferably at least 95% to 99.5%, and most preferably at least 99%. When a component is substantially replaced, the amount remaining in the sample is about 0.5% to about 10% or less, preferably about 0.5% to about 1.0% or less.

[0014] The terms "treat," "treatment," and "treating," as used herein, refer to an approach for obtaining a beneficial or desired result, e.g., a clinical result. For purposes of this disclosure, a beneficial or desired result may include inhibiting or suppressing the onset or progression of an infection or disease, ameliorating symptoms of or reducing the occurrence of an infection or disease, or a combination thereof.

[0015] "Prevention," as used herein, is used interchangeably with "prophylaxis" and can mean the complete prevention of an infection or disease, or the prevention of the onset of symptoms of that infection or disease; a delay in the onset of an infection or disease or its symptoms; or a reduction in the severity of a later-occurring infection or disease or its symptoms.

[0016] As used herein, "effective dose" or "effective amount" refers to an amount of an immunogen sufficient to induce an immune response that alleviates at least one symptom of a pathogen infection. An effective dose or amount can be determined, for example, by measuring the amount of neutralizing secretory and / or serum antibodies, e.g., by plaque neutralization, complement fixation, enzyme-linked immunosorbent (ELISA), or microneutralization assays.

[0017] As used herein, the term "vaccine" refers to an immunogenic composition, such as an immunogen derived from a pathogen, used to induce an immune response against a pathogen that results in protective immunity (e.g., immunity that protects a subject from infection with the pathogen and / or reduces the severity of a disease or condition caused by infection with the pathogen). A protective immune response can include the formation of antibodies and / or a cell-mediated response. Depending on the context, the term "vaccine" can also refer to a suspension or solution of immunogens that is administered to a subject to elicit protective immunity.

[0018] As used herein, the term "subject" includes humans and other animals. Typically, the subject is a human. For example, the subject can be an adult, a teenager, a child (ages 2-14), an infant (birth-2 years), or a newborn (up to 2 months). In certain aspects, the subject is up to 4 months of age, or up to 6 months of age. In some aspects, an adult is about 65 years of age or older, or an elderly person about 60 years of age or older. In some embodiments, the subject is 5 years of age or younger. In some embodiments, the subject is 65 years of age or older. In some aspects, the subject is a pregnant woman or a woman who is intending to become pregnant. In other aspects, the subject is not a human, e.g., a non-human primate, e.g., a baboon, chimpanzee, gorilla, or macaque. In certain aspects, the subject may be a companion animal, such as a dog or cat.

[0019] The term "percent identity" in the context of two or more nucleic acid or polypeptide sequences refers to two or more sequences or subsequences that have a specified percentage of nucleotides or amino acid residues that are the same when compared. Unless otherwise indicated, percent identity is calculated over the entire length of the sequences being compared using EMBOSS Needle, a tool available online at ebi.ac.uk / jdispatcher / psa / emboss_needle. When comparing the percent identity of two polypeptides, the following default parameters are used: Output Format=pair, Matrix=BLOSUM62, Gap Open=10, Gap Extend 0.5, End Gap=false, End Gap Open=10, End Gap Extend=0.5. When comparing the percent identity of two nucleic acids, the following default parameters are used: Output Format=pair, Matrix=DNAfull, Gap Open=10, Gap Extend 0.5, End Gap=false, End Gap Open=10, End Gap Extend=0.5.

[0020] As used herein, the term "vaccine" refers to an immunogenic composition, such as an immunogen derived from a pathogen, used to induce an immune response against a pathogen that results in protective immunity (e.g., immunity that protects a subject from infection with the pathogen and / or reduces the severity of a disease or condition caused by infection with the pathogen). A protective immune response can include the formation of antibodies and / or a cell-mediated response. Depending on the context, the term "vaccine" can also refer to a suspension or solution of immunogens that is administered to a subject to elicit protective immunity.

[0021] In some aspects, the subject is immunocompromised. In some embodiments, the immunocompromised subject is administered a medication that causes immunosuppression. Non-limiting examples of medications that cause immunosuppression include corticosteroids (e.g., prednisone), alkylating agents (e.g., cyclophosphamide), antimetabolites (e.g., azathioprine or 6-mercaptopurine), transplant-related immunosuppressants (e.g., cyclosporine, tacrolimus, sirolimus, or mycophenolate mofetil), mitoxantrone, chemotherapeutic agents, methotrexate, tumor necrosis factor (TNF) blockers (e.g., etanercept, adalimumab, infliximab). In some embodiments, the immunocompromised subject is infected with a virus (e.g., human immunodeficiency virus or Epstein-Barr virus). In some embodiments, the virus is a respiratory virus, such as respiratory syncytial virus, influenza, parainfluenza, adenovirus, or picornavirus. In some embodiments, the immunocompromised subject has acquired immune deficiency syndrome (AIDS). In some embodiments, the immunocompromised subject is infected with human immunodeficiency virus (HIV). In some embodiments, the immunocompromised subject is immunocompromised due to a treatment regimen designed to prevent inflammation or prevent transplant rejection. In some embodiments, the immunocompromised subject is a transplant recipient. In some embodiments, the immunocompromised subject has undergone radiation therapy or splenectomy.In embodiments, the immunocompromised subject is a patient with a condition including cancer, an autoimmune disease, tuberculosis, a substance use disorder (e.g., alcohol, opioid, or cocaine use disorder), stroke or cerebrovascular disease, solid organ or blood stem cell transplant, sickle cell disease, thalassemia, autoimmune lymphoproliferative syndrome (ALPS), autoimmune polyglandular syndrome type 1 (APS-1), B-cell proliferation with NF-κB and T-cell anergy (BENTA) disorder, caspase 8 deficiency state (CEDS), chronic granulomatous disease (CGD), common variable immunodeficiency (CVID), congenital neutropenic syndrome, cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) deficiency, DOCK8 deficiency, GATA2 deficiency, glycosylation disorders with immunodeficiency, hyperimmunoglobulin E syndrome (HGE-E), and / or steroid-resistant ... The subject has been diagnosed with: hyperimmunoglobulin M syndrome, diabetes, type 1 diabetes, type 2 diabetes, interferon gamma deficiency, interleukin-12 deficiency, interleukin-23 deficiency, leukocyte adhesion deficiency, lipopolysaccharide-responsive beige-like anchor (LRBA) deficiency, PI3 kinase disease, PLCG2-associated antibody deficiency / immunopathies (PLAID), severe combined immunodeficiency (SCID), STAT3 dominant-negative disease, STAT3 gain-of-function disease, warts, hypogammaglobulinemia, infection, and myeloid accumulation (WHIM) syndrome, Wiskott-Aldrich syndrome (WAS), X-linked agammaglobulinemia (XLA), X-linked lymphoproliferative disease (XLP), uremia, a nutritional disorder, or an XMEN disease. In some embodiments, the immunocompromised subject is a current or former cigarette smoker. In embodiments, the immunocompromised subject has a B cell deficiency, a T cell deficiency, a macrophage deficiency, a cytokine deficiency, a phagocyte deficiency, a phagocyte dysfunction, a complement deficiency, or a combination thereof.

[0022] In some embodiments, the subject is overweight or obese. In some embodiments, the overweight subject is overweight or obese below 25 kg / m 2 Over 30kg / m 2 In some embodiments, an obese subject has a body mass index (BMI) of less than 30 kg / m 2In some embodiments, the subject has a psychiatric disorder. In some embodiments, the psychiatric disorder is depression, schizophrenia, or anxiety.

[0023] As used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency of the U.S. federal or state government, or listed in the U.S. Pharmacopoeia, the European Pharmacopoeia, or other pharmacopoeias generally recognized for use in mammals, more particularly humans. These compositions may be useful as vaccines and / or antigenic compositions for inducing a protective immune response in vertebrates.

[0024] As used herein, the term "modification," when referring to the RSV F glycoprotein, refers to the mutation, deletion, or addition of one or more amino acids of the RSV F glycoprotein. The location of the modification within the RSV F glycoprotein can be determined by aligning the sequence of the RSV F glycoprotein to a native RSV F glycoprotein (e.g., SEQ ID NO: 330 (RSV F glycoprotein containing a signal peptide) or SEQ ID NO: 329 (mature RSV F glycoprotein lacking a signal peptide). The location of the modification within the SARS-CoV-2 spike (S) glycoprotein (alternatively referred to herein as "CoV S glycoprotein") can be determined by aligning the polypeptide sequence to SEQ ID NO: 1 (CoV S glycoprotein containing a signal peptide) or SEQ ID NO: 2 (mature CoV S glycoprotein lacking a signal peptide). The sequences of native RSV F glycoproteins having the amino acid sequences of SEQ ID NOs: 329 and 330, and native CoV S glycoproteins having the amino acid sequences of SEQ ID NOs: 1 and 2, are found in the table below. [Table 1-1] [Table 1-2]

[0025] The term "efficacy" of the immunogenic or vaccine compositions described herein refers to the percentage reduction in infection (e.g., of RSV virus) in a group administered the immunogenic composition compared to a group not administered the immunogenic composition. In embodiments, efficacy (E) is calculated using the following formula: E(%) = (1 - RR) x 100, where RR = the relative risk of infection between a group administered the immunogenic composition and a group not administered the immunogenic composition. In embodiments, the immunogenic compositions described herein have an efficacy against RSV virus of at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 100%, at least about 101%, at least about 102%, at least about 103%, at least about 104%, at least about 105%, at least about 106%, at least about 107%, at least about 108%, at least about 109%, at least about 110%, at least about 111%, at least about 112%, at least about 113%, at least about 114%, at least about 115%, at least about 116%, at least about 117%, at least about 118%, at least about 119%, at least about 120%, at least about 121%, at least about 122%, at least about 123%, at least about 124%, at least about 125%, at least about 126%, at least about 127%, at least about 128%, at least about 129%, at least about 130%, at least about 131%, at least The compound has an efficacy of about 97%, at least about 98%, at least about 99%, about 50% to about 99%, about 50% to about 98%, about 60% to about 99%, about 60% to about 98%, about 70% to about 98%, about 70% to about 95%, about 70% to about 99%, about 80% to about 99%, about 80% to about 98%, about 80% to about 95%, about 85% to about 99%, about 85% to about 98%, about 85% to about 95%, about 90% to about 95%, about 90% to 98%, or about 90% to about 99%.

[0026] The term SARS-CoV-2 "variant," used interchangeably herein with "heterologous SARS-CoV-2 strain," refers to a SARS-CoV-2 virus that includes a CoV S polypeptide that has one or more modifications compared to the SARS-CoV S polypeptide having the amino acid sequence of SEQ ID NO:2. For example, a SARS-CoV-2 variant can have at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, at least about 25, at least about 26, at least about 27, at least about 28, at least about 29, at least about 30, at least about 31, at least about 32, at least about 33, at least about 34, or at least about 35 modifications compared to a CoV S polypeptide having the amino acid sequence of SEQ ID NO:2. For example, a SARS-CoV-2 variant can have at least 1 and up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9, up to 10, up to 11, up to 12, up to 13, up to 14, up to 15, up to 16, up to 17, up to 18, up to 19, up to 20, up to 21, up to 22, up to 23, up to 24, up to 25, up to 26, up to 27, up to 28, up to 29, up to 30, up to 31, up to 32, up to 33, up to 34, up to 35 modifications, up to 40 modifications, up to 45 modifications, up to 50 modifications, up to 55 modifications, up to 60 modifications, up to 65 modifications, up to 70 modifications, up to 75 modifications, up to 80 modifications, up to 85 modifications, up to 90 modifications, up to 95 modifications, or up to 100 modifications compared to a CoV S polypeptide having the amino acid sequence of SEQ ID NO:2.In embodiments, a SARS-CoV-2 variant can have about 2 to about 35 modifications, about 5 to about 10 modifications, about 5 to about 20 modifications, about 10 to about 20 modifications, about 15 to about 25 modifications, about 20 to 30 modifications, about 20 to about 40 modifications, about 25 to about 45 modifications, about 25 to about 100 modifications, about 25 to about 45 modifications, or about 35 to about 100 modifications compared to a CoV S polypeptide having the amino acid sequence of SEQ ID NO:2.

[0027] As used herein, the terms "intranasal administration" and "nasal administration" refer to administration of the immunogenic compositions described herein to the nasal cavity of a subject. In embodiments, intranasal administration is achieved using a liquid preparation (e.g., an aqueous preparation), an aerosol preparation, or a dry powder preparation. In embodiments, the liquid preparation, the aerosol preparation, or the dry powder preparation is administered via an externally driven nasal delivery device. In embodiments, the liquid preparation, the aerosol preparation, or the dry powder preparation is administered via a self-driven (i.e., by inhalation) nasal delivery device. In embodiments, the liquid preparation, the aerosol preparation, or the dry powder preparation is administered via nasal insufflation (where the immunogenic composition is blown into the nose) or nasal drops (where the immunogenic composition is instilled into the nose). In some embodiments, the liquid, aerosol, or dry powder preparation is administered via a gel, cream, ointment, lotion, or paste applied to one or more sites of the nasal epithelium (e.g., the olfactory epithelium or the nasal respiratory epithelium). In some embodiments, the immunogenic composition is applied to the mucus in the nasal cavity of the subject.

[0028] As used herein, the term "non-invasive nasal delivery device" refers to an instrument that can deliver an immunogenic composition to the nasal cavity without piercing the epithelium of a subject. Non-limiting examples of non-invasive nasal delivery devices include propellant (e.g., pressurized inhalers) and non-propellant (e.g., pump-type inhalers) aerosol or spray devices, particle dispersion devices, nebulizers, and pressurized olfactory delivery devices for the delivery of liquid or powder formulations.

[0029] As used herein, the term "dry powder composition" refers to a lyophilized or spray-dried form of the immunogenic compositions described herein. In embodiments, the dry powder composition contains less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less residual moisture.

[0030] As used herein, the term "permeability enhancer" refers to a component of an immunogenic composition formulated for intranasal administration that facilitates passage of viral glycoproteins across the nasal epithelium.

[0031] As used herein, the terms "bedside mix," "bedside formulation," "bedside vaccine composition," "bedside vial," and "bedside vial formulation" refer to a vaccine formulation that is prepared immediately prior to administration. Such a vaccine formulation contains a viral antigen and an adjuvant, which are stored separately in different containers and administered to a subject (e.g., by administering two sequential injections, or by combining the antigen and adjuvant into a single injection and then administering it).

[0032] As used herein, the terms "combination mix," "combination," "combination immunogenic composition," "prefilled syringe," and "premix" refer to vaccine formulations prepared for short- to long-term storage prior to administration to a subject. Such vaccine formulations contain a combination of antigen and adjuvant in the same container and are prepared prior to administration. In embodiments, the formulation contains a hemagglutinin and an adjuvant (e.g., a saponin adjuvant) to form HaSMaN (hemagglutinin saponin matrix nanoparticles).

[0033] As used herein, the term "split virion" refers to a virus (e.g., influenza virus or SARS-CoV-2 virus) that has had its viral membrane disrupted with a surfactant. Examples of surfactants are described throughout this disclosure. Split virions have not undergone further purification and therefore typically contain multiple viral proteins.

[0034] As used herein, the term "recombinant" refers to a protein (e.g., hemagglutinin) produced in a cell by transcription and translation of a nucleic acid introduced into the cell. The nucleic acid may be introduced via a vector or virus encoding the nucleic acid.

[0035] As used herein, the term "whole influenza virus" refers to a virus including all of the envelope, viral membranes, nucleocapsid, and genetic material. In embodiments, the whole influenza virus is inactivated.

[0036] As used herein, the term "inactivated virus" refers to a virus that has undergone a process that substantially reduces or eliminates pathogenicity compared to the wild-type virus.

[0037] The term "bronchoalveolar lavage fluid," also referred to as "BAL," refers to a fluid sample collected from a patient's lungs. In some embodiments, BAL is collected during a bronchoscopy. During a bronchoscopy, a bronchoscope containing a solution (e.g., saline) is passed through the mouth or nose into the lungs. The solution is then withdrawn from the lungs.

[0038] The term "mucosal immunity" refers to cellular and humoral immune responses that occur in the mucosa. In embodiments, the methods of the disclosure provided herein result in mucosal immunity in the respiratory system.

[0039] The term "atomization" refers to the production of fine, respirable droplets of a liquid, with typical dimensions in the range of a few microns.

[0040] The term "aerosol" refers to a dispersion of solid or liquid particles in a gas phase. In embodiments, the gas phase is air.

[0041] Immunogenic and vaccine compositions containing RSV F glycoprotein The present disclosure provides non-naturally occurring RSV F glycoproteins, nanoparticles containing the RSV F glycoprotein, and immunogenic and vaccine compositions containing either the non-naturally occurring RSV F glycoprotein or nanoparticles containing the RSV F glycoprotein. In several embodiments, methods for stimulating an immune response to the RSV virus using the RSV F glycoprotein, nanoparticles comprising the same, immunogenic compositions comprising the RSV F glycoprotein and / or nanoparticles, and vaccine compositions are provided herein.

[0042] Also provided herein are methods for producing nanoparticles and vaccine compositions. Advantageously, the methods provide nanoparticles that are substantially free of contamination with other proteins, such as proteins associated with recombinant expression of proteins in insect cells. In some embodiments, expression is carried out in the baculovirus / Sf9 system.

[0043] Non-naturally occurring RSV F glycoprotein antigens In several embodiments, RSV F glycoproteins having one or more modifications compared to the native (also referred to as "wild-type") RSV F glycoprotein are provided herein. The wild-type RSV F glycoprotein is synthesized as a single-chain inactive precursor designated F0, which contains three subunits: F1, F2, and a 27-amino acid glycopeptide designated pep27 ("p27"). The F1 subunit is located at amino acids 137-574 of the RSV F glycoprotein of SEQ ID NO: 330. The F2 subunit is located at amino acids 26-110 of the RSV F glycoprotein of SEQ ID NO: 330. p27 is located at amino acids 111-136 of the RSV F glycoprotein of SEQ ID NO: 330. The C-terminal F1 subunit contains a transmembrane domain, two heptad repeats, and an N-terminal fusion peptide. The F0 precursor is cleaved by a furin-like protease to form a mature fusion-competent protein. The mature RSV F protein contains an F1 subunit and an F2 subunit connected by a disulfide bond. Further sources describing the RSV F protein structure are found in Swanson et al. A Monomeric Uncleaved Respiratory Syncytial Virus F Antigen Retains Prefusion-Specific Neutralizing Epitopes. Journal of Virology, 2014, 88, 11802-11810. Jason S. McLellan et al. Structure of RSV Fusion Glycoprotein Trimer Bound to a Prefusion-Specific Neutralizing Antibody. Science, 2013, 340, 1113-1117.

[0044] In some embodiments, the RSV F glycoprotein from the wild-type RSV F A2 strain is a native RSV F glycoprotein. The RSV F glycoprotein from the wild-type RSV F A2 strain comprises the amino acid sequence of SEQ ID NO: 330. The wild-type RSV F A2 strain has sequencing errors (A102P, V379I, V447M). These sequencing errors are corrected in the non-naturally occurring RSV F glycoprotein described herein, i.e., amino acid 102 is alanine, amino acid 379 is valine, and amino acid 447 is valine.

[0045] In some embodiments, the RSV F glycoprotein is (a) deletion of 1 to 10 amino acids from the fusion peptide; (b) inactivation of the primary furin cleavage site (also called site II) by mutation of one or more of amino acids 131 to 136; (c) inactivation of the secondary furin cleavage site (also called site I) by mutation of one or more of amino acids 106 to 109; (d) S155C, (e) S290C, (f) S190F, (g) V207L, and (h)N116Q These modifications have one or more modifications selected from the group consisting of:

[0046] In some embodiments, the RSV F glycoprotein is (a) deletion of 1 to 10 amino acids from the fusion peptide; (b) inactivation of the primary furin cleavage site (also called site II) by mutation of one or more of amino acids 131 to 136; (c) inactivation of the secondary furin cleavage site (also called site I) by mutation of one or more of amino acids 106 to 109; (d) S155C, (e) S290C, (f) S190F, and (g)V207L These modifications have one or more modifications selected from the group consisting of:

[0047] In some embodiments, the RSV F glycoproteins described herein exhibit one or more of antigenic sites Φ, II, IV, and V. In some embodiments, the RSV F proteins described herein exhibit mutations that result in the presence of one or more of the RSV F antigenic sites Φ, II, IV, and V. The RSV F antigenic site Φ, located at the membrane distal end of the pre-fusion RSV F trimer, is targeted by potent RSV F neutralizing antibodies, including 5C4, AM22, and D25. The RSV F antigenic site II, also referred to herein as the palivizumab site, is the target of the antibodies pavilizumab and motavizumab. Antigenic site II consists of a helix-turn-helix motif spanning residues 253-278 of the RSV F glycoprotein of SEQ ID NO: 330. The RSV F antigenic site IV is located on the Fl subunit of the RSV F glycoprotein and encompasses residues 422-471 of the RSV F glycoprotein of SEQ ID NO: 330. The RSV F antigen site V is composed of α2 to α3 and β3 to β4.

[0048] In some embodiments, a particular antigenic site contains an epitope associated with a pre-fusion or post-fusion structure. For example, RSV F antigenic sites Φ and V are expressed in the pre-fusion structure of the RSV F glycoprotein. In contrast, RSV F antigenic sites II and IV are expressed in both the pre-fusion and post-fusion structures of the RSV F glycoprotein. In some embodiments, the RSV F protein contains an antigenic site that induces the formation of neutralizing antibodies against the pre-fusion form of the RSV F glycoprotein. In some embodiments, the RSV F glycoprotein contains an antigenic site that induces the formation of neutralizing antibodies against the post-fusion form of the RSV F glycoprotein. In some embodiments, the RSV F glycoprotein described herein contains an antigenic site that induces the production of neutralizing antibodies against both the pre-fusion and post-fusion forms of the RSV F glycoprotein.

[0049] In some embodiments, the RSV F glycoprotein comprises a deletion of amino acids 137-146, an inactivated first furin cleavage site, an S155C mutation, and an S290C mutation, where these modifications are numbered based on the RSV F glycoprotein of SEQ ID NO: 330. In some embodiments, the RSV F glycoprotein comprises a deletion of amino acids 137-146, an inactivated first furin cleavage site, an S155C mutation, an S290C mutation, an S190F mutation, and a V207L ​​mutation, where these modifications are numbered based on the RSV F glycoprotein of SEQ ID NO: 330. In some embodiments, the RSV F glycoprotein comprises a deletion of amino acids 137-146, an inactivated first furin cleavage site, an S190F mutation, and a V207L ​​mutation, where these modifications are numbered based on the RSV F glycoprotein of SEQ ID NO: 330.

[0050] In some embodiments, the RSV F glycoprotein comprises a deletion of amino acids 137-146, an inactivated first furin cleavage site, and an N116Q mutation, where these modifications are numbered based on the RSV F glycoprotein of SEQ ID NO: 330. In some embodiments, the RSV F glycoprotein comprises a deletion of amino acids 137-146, an inactivated first furin cleavage site, an S155C mutation, an S290C mutation, and an N116Q mutation, where these modifications are numbered based on the RSV F glycoprotein of SEQ ID NO: 330. In some embodiments, the RSV F glycoprotein comprises a deletion of amino acids 137-146, an inactivated first furin cleavage site, an S190F mutation, a V207L ​​mutation, and an N116Q mutation, where these modifications are numbered based on the RSV F glycoprotein of SEQ ID NO: 330. In some embodiments, the RSV F glycoprotein contains a deletion of amino acids 137-146, an inactivated primary furin cleavage site, an S155C mutation, an S290C mutation, an S190F mutation, a V207L ​​mutation, and an N116Q mutation, wherein these modifications are numbered based on the RSV F glycoprotein of SEQ ID NO: 330.

[0051] Non-naturally occurring RSV F glycoprotein antigen-furin cleavage site mutations In some embodiments, certain mutations cause the mature RSV F protein of the present disclosure to retain p27 at the N-terminus of the F1 subunit. The RSV F protein retaining p27 contains a p27-F1 subunit and an F2 subunit connected by a disulfide bond. In some embodiments, a mutation in the primary furin cleavage site results in retention of p27.

[0052] The primary furin cleavage site, also referred to herein as site II, is located at residues 131-136 of SEQ ID NO: 330. The wild-type primary furin cleavage site has the amino acid sequence KKRKRR (SEQ ID NO: 341). Inactivation of the primary fusion cleavage site can be achieved by mutating residues at that site so that furin can no longer recognize the consensus site. For example, inactivation of the primary furin cleavage site can be achieved by introducing at least one, at least two, or three amino acid substitutions into positions corresponding to arginine 133, arginine 135, and arginine 136 of the wild-type RSV F protein (SEQ ID NO: 330). In certain embodiments, one, two, or all three arginines are mutated to glutamine. In other aspects, inactivation is achieved by mutating the wild-type primary furin cleavage site to one of the following sequences: KKQKQQ (SEQ ID NO: 342), QKQKQQ (SEQ ID NO: 343), KKQKRQ (SEQ ID NO: 344), and GRRQQR (SEQ ID NO: 345).

[0053] In some embodiments, the RSV F glycoprotein described herein comprises a wild-type secondary furin cleavage site. The secondary furin cleavage site, also referred to herein as site I, is located at residues 106-109 of SEQ ID NO: 330. The wild-type secondary furin cleavage site has the amino acid sequence RARR (SEQ ID NO: 6). In some embodiments, the RSV F glycoprotein described herein comprises an inactive secondary furin cleavage site. In some embodiments, inactivation of the secondary furin cleavage site is achieved by mutating the wild-type site to one of the following sequences: QQAQ (SEQ ID NO: 7), RAQQ (SEQ ID NO: 348), or RANN (SEQ ID NO: 349). In some embodiments, the inactivated secondary furin cleavage site comprises the amino acid sequence of any one of SEQ ID NOs: 7-34, 97, 111, 348, and 349. In some embodiments, the inactivated secondary furin cleavage site comprises the amino acid sequence of QQAQ (SEQ ID NO: 7).

[0054] Non-naturally occurring modifications to the RSV F glycoprotein antigen-F1 domain In various embodiments, the non-naturally occurring RSV F glycoprotein described herein may have a deletion, insertion, or mutation of up to about 1, up to about 2, up to about 3, up to about 4, up to about 5, up to about 10, up to about 15, up to about 20, up to about 25, or up to about 30 amino acids compared to the amino acid sequence of the F1 domain of SEQ ID NO: 330. The wild-type F1 domain has the amino acid sequence of SEQ ID NO: 350. In various embodiments, the RSV F glycoprotein described herein comprises an F1 domain having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to the F1 domain of SEQ ID NO: 350. The F1 domain of the RSV F glycoprotein herein may have a deletion, insertion, or mutation of 1 to about 30 amino acids, 1 to about 25 amino acids, 1 to about 20 amino acids, 1 to about 15 amino acids, 1 to about 10 amino acids, about 1 to about 5 amino acids, about 3 to about 10 amino acids, about 5 to 10 amino acids, about 8 to 12 amino acids, about 10 to 15 amino acids, about 12 to 17 amino acids, about 15 to 20 amino acids, about 18 to 23 amino acids, about 20 to 25 amino acids, about 22 to about 27 amino acids, or about 25 to 30 amino acids, compared to the F1 domain of SEQ ID NO: 350.

[0055] In some embodiments, the F1 domain of the RSV F glycoprotein comprises one or more mutations selected from the group consisting of S190F, V207L, S155C, S290C, I379A, and M447V, where these mutations are numbered based on the RSV F glycoprotein of SEQ ID NO: 330. In some embodiments, the F1 domain of the RSV F glycoprotein comprises S190F and V207L ​​mutations, where these mutations are numbered based on the RSV F glycoprotein of SEQ ID NO: 330. In some embodiments, the F1 domain of the RSV F glycoprotein comprises S155C and S290C mutations, where these mutations are numbered based on the RSV F glycoprotein of SEQ ID NO: 2. In some embodiments, the F1 domain of the RSV F glycoprotein comprises S190F, V207L, S155C, and S290C mutations, where these mutations are numbered based on the RSV F glycoprotein of SEQ ID NO: 330.

[0056] In some embodiments, the F1 domain of the RSV F glycoprotein comprises an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to any one of SEQ ID NOs: 350-355.

[0057] Deletions in non-naturally occurring RSV F glycoprotein antigen-F1 domain fusion peptides In several embodiments, the RSV F glycoprotein of the present disclosure lacks one or more amino acids of the RSV F fusion peptide (amino acids 137-146 of SEQ ID NO: 330). The fusion peptide of the RSV F glycoprotein of SEQ ID NO: 330 has the amino acid sequence FLGFLLGVGS (SEQ ID NO: 363). In several embodiments, RSV F glycoproteins lacking a fusion peptide are provided herein. In several embodiments, RSV F glycoproteins lacking 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, or 1-2 consecutive amino acids of the fusion peptide are provided herein. In several embodiments, RSV F glycoproteins lacking 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, or 1-2 non-consecutive amino acids of the fusion peptide are provided herein. In embodiments, up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9, or up to 10 amino acids may be deleted in the fusion peptide. In embodiments, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 amino acids, but not more than 10 amino acids, of the fusion peptide are deleted.

[0058] Non-naturally occurring modifications to the RSV F glycoprotein antigen-F2 domain In various embodiments, the non-naturally occurring RSV F glycoprotein described herein may have a deletion, insertion, or mutation of up to about 1, up to about 2, up to about 3, up to about 4, up to about 5, up to about 10, up to about 15, up to about 20, up to about 25, or up to about 30 amino acids compared to the amino acid sequence of the F2 domain of SEQ ID NO: 330. The wild-type F2 domain has the amino acid sequence of SEQ ID NO: 356. In various embodiments, the RSV F glycoprotein described herein comprises an F1 domain having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to the F2 domain of SEQ ID NO: 356. The F2 domain of the RSV F glycoprotein herein may have a deletion, insertion, or mutation of about 1 to about 5 amino acids, about 3 to about 10 amino acids, about 5 to 10 amino acids, about 8 to 12 amino acids, about 10 to 15 amino acids, about 12 to 17 amino acids, about 15 to 20 amino acids, about 18 to 23 amino acids, about 20 to 25 amino acids, about 22 to about 27 amino acids, or about 25 to 30 amino acids compared to the F2 domain of SEQ ID NO: 356.

[0059] In some embodiments, the F2 domain of the RSV F glycoprotein may include a P102A mutation, which is numbered based on the RSV F glycoprotein of SEQ ID NO: 330.

[0060] In various embodiments, the F2 domain of the RSV F glycoprotein comprises an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to any one of SEQ ID NOs: 356-357.

[0061] Non-naturally occurring modifications to the RSV F glycoprotein antigen-p27 In various embodiments, the non-naturally occurring RSV F glycoprotein described herein may have a deletion, insertion, or mutation of up to about 1, up to about 2, up to about 3, up to about 4, up to about 5, up to about 10, up to about 15, up to about 20, up to about 25, or up to about 30 amino acids compared to the amino acid sequence of p27 of SEQ ID NO: 330. Wild-type p27 has the amino acid sequence of SEQ ID NO: 359. In various embodiments, the RSV F glycoprotein described herein includes a p27 having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to p27 of SEQ ID NO: 330. The p27 of the RSV F glycoprotein herein may have a deletion, insertion, or mutation of about 1 to about 5 amino acids, about 3 to about 10 amino acids, about 5 to 10 amino acids, about 8 to 12 amino acids, about 10 to 15 amino acids, about 12 to 17 amino acids, about 15 to 20 amino acids, about 18 to 23 amino acids, about 20 to 25 amino acids, about 22 to about 27 amino acids, or about 25 and 30 amino acids compared to p27 of SEQ ID NO: 330.

[0062] In some embodiments, the p27 domain of the RSV F glycoprotein may include an N116Q mutation, which is numbered based on the RSV F glycoprotein of SEQ ID NO: 330.

[0063] In various embodiments, the RSV F glycoprotein p27 comprises an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to any one of SEQ ID NOs: 358-359.

[0064] Non-naturally occurring RSV F glycoprotein antigen-transmembrane domain In some embodiments, the RSV F glycoprotein described herein comprises a transmembrane domain. In some embodiments, the transmembrane domain has the sequence ITTIIIVIIVILLSLIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN (SEQ ID NO: 362). In some embodiments, the RSV F glycoprotein described herein may have a deletion, insertion, or mutation of up to about 1, up to about 2, up to about 3, up to about 4, up to about 5, up to about 10, up to about 15, up to about 20, up to about 25, or up to about 30 amino acids compared to the amino acid sequence of the transmembrane domain. In some embodiments, the RSV F glycoprotein lacks a transmembrane domain.

[0065] Non-naturally occurring RSV F glycoprotein antigen-signal peptide In some embodiments, the RSV F glycoprotein described herein is expressed with an N-terminal signal peptide. In some embodiments, the N-terminal signal peptide has the amino acid sequence of SEQ ID NO: 360 (MELLILKANAITTILTAVTFCFASG). In some embodiments, the signal peptide can be replaced with any signal peptide that enables expression of the RSV F glycoprotein. In some embodiments, one or more of the RSV F glycoprotein signal peptide amino acids can be deleted or mutated. An initial methionine residue is maintained to initiate expression. In some embodiments, the RSV F glycoprotein is encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NO: 361.

[0066] After expression of the RSV F glycoprotein in a host cell, the N-terminal signal peptide is cleaved to produce the mature RSV F glycoprotein sequence (SEQ ID NOs: 331 and 337-340). In some embodiments, the signal peptide is cleaved by a host cell protease. In some aspects, the full-length protein is isolated from the host cell, after which the signal peptide is cleaved.

[0067] After cleavage of the signal peptide from the RSV F glycoprotein having an amino acid sequence corresponding to SEQ ID NOs: 332-336 during expression and purification, a mature polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 331 and 337-340 is obtained, which is used to produce a RSV F nanoparticle vaccine or RSV F nanoparticles.

[0068] Advantageously, the disclosed RSV F glycoproteins can have enhanced protein expression, stability, and immunogenicity compared to the native RSV F glycoprotein.

[0069] In some embodiments, the RSV F glycoprotein described herein contains additional modifications from the native RSV F glycoprotein (SEQ ID NO: 330). In some embodiments, the RSV F glycoprotein described herein exhibits at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the native RSV F glycoprotein.

[0070] In some embodiments, the RSV F glycoproteins described herein are at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to a RSV F glycoprotein having an amino acid sequence selected from the group consisting of SEQ ID NOs: 329 to 340. In some embodiments, RSV F glycoproteins having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to the RSV glycoprotein of SEQ ID NO: 337 are provided herein. The RSV F glycoprotein may have a deletion, insertion, or mutation of up to about 1, up to about 2, up to about 3, up to about 4, up to about 5, up to about 10, up to about 15, up to about 20, up to about 25, up to about 30, up to about 35, up to about 40, up to about 45, or up to about 50 amino acids compared to the amino acid sequence of the RSV F glycoprotein having any one of the amino acid sequences of SEQ ID NOs: 329-340. The RSV F glycoprotein may have a deletion, insertion, or mutation of about 1 to about 5 amino acids, about 3 to about 10 amino acids, about 5 to 10 amino acids, about 8 to 12 amino acids, about 10 to 15 amino acids, about 12 to 17 amino acids, about 15 to 20 amino acids, about 18 to 23 amino acids, about 20 to 25 amino acids, about 22 to about 27 amino acids, about 25 to 30 amino acids, about 30 to 35 amino acids, about 35 to 40 amino acids, about 40 to 45 amino acids, or about 45 to 50 amino acids, compared to the RSV F glycoprotein having any one of the amino acid sequences of SEQ ID NOs: 329 to 340. In various embodiments, the RSV F glycoprotein described herein includes about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, or about 25 modifications compared to the RSV F glycoprotein having any one of the amino acid sequences of SEQ ID NOs: 329-340.

[0071] In some embodiments, the RSV F glycoprotein is extended at the N-terminus, C-terminus, or both the N-terminus and C-terminus. In some aspects, the extension is a tag useful for functions such as purification or detection. In some aspects, the tag contains an epitope. For example, the tag can be a polyglutamate tag, a FLAG tag, an HA tag, a polyHis tag (having approximately 5-10 histidines) (SEQ ID NO: 34), a hexahistidine tag (SEQ ID NO: 35), an 8xHis tag (having 8 histidines) (SEQ ID NO: 36), a Myc tag, a glutathione-S-transferase tag, a green fluorescent protein tag, a maltose-binding protein tag, a thioredoxin tag, or an Fc tag. In other aspects, the extension can be an N-terminal signal peptide fused to the protein to enhance expression. Such signal peptides are often cleaved during expression in cells, but some nanoparticles may contain antigens with an intact signal peptide. Thus, when a nanoparticle contains an antigen, the antigen may contain an extension portion and thus may be a fusion protein when incorporated into the nanoparticle. For purposes of calculating sequence identity, the extension portion is not included. In some embodiments, the tag is a protease cleavage site. Non-limiting examples of protease cleavage sites include HRV3C protease cleavage site, chymotrypsin, trypsin, elastase, endopeptidase, caspase-1, caspase-2, caspase-3, caspase-4, caspase-5, caspase-6, caspase-7, caspase-8, caspase-9, caspase-10, enterokinase, factor Xa, granzyme B, TEV protease, and thrombin. In some embodiments, the protease cleavage site is an HRV3C protease cleavage site.

[0072] In some embodiments, the RSV F glycoprotein comprises a fusion protein. In some embodiments, the RSV F glycoprotein comprises an N-terminal fusion protein. In some embodiments, the RSV F glycoprotein comprises a C-terminal fusion protein. In some embodiments, the fusion protein includes a tag useful for protein expression, purification, or detection. In some embodiments, the tag is a poly-His tag (having about 5-10 histidines) (SEQ ID NO: 34), a Myc tag, a glutathione-S-transferase tag, a green fluorescent protein tag, a maltose binding protein tag, a thioredoxin tag, a Strep tag, a Twin-Strep tag, or an Fc tag. In some embodiments, the tag is an Fc tag. In some embodiments, the Fc tag is a monomer, dimer, or trimer. In some embodiments, the tag is a hexahistidine tag, such as a poly-His tag containing six histidines (SEQ ID NO: 35).

[0073] Immunogenic and / or vaccine compositions containing RSV F glycoprotein and additional viral glycoproteins In some embodiments, provided herein are immunogenic compositions comprising the RSV F glycoprotein. In some embodiments, the immunogenic composition is a vaccine composition. In some embodiments, provided herein are immunogenic compositions and vaccine compositions comprising nanoparticles comprising the RSV F glycoprotein. In some embodiments, the immunogenic composition comprises about 1 to about 50, about 1 to about 45, about 1 to about 40, 1 to about 35, about 1 to about 30, 1 to about 25, 1 to about 20, 1 to about 15, 1 to about 10, or 1 to about 5 different RSV F glycoproteins.

[0074] In another embodiment, the present disclosure provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the vaccine composition.

[0075] The compositions disclosed herein may be used for either prophylactic or therapeutic purposes, but are typically prophylactic. Accordingly, the present disclosure includes methods for treating or preventing infectious diseases. The methods involve administering to a subject a therapeutic or prophylactic amount of an immunogenic composition of the present disclosure. Preferably, the pharmaceutical composition is a vaccine composition that provides a protective effect. In other aspects, the protective effect can include an improvement in symptoms associated with infectious diseases in a proportion of an exposed population. For example, the composition may prevent or reduce one or more symptoms of viral disease selected from fever, fatigue, muscle pain, headache, sore throat, vomiting, diarrhea, rash, symptoms of impaired kidney and liver function, and internal and external bleeding, compared to untreated subjects.

[0076] The nanoparticles can be formulated for administration as a vaccine in the presence of various excipients, buffers, etc. For example, the vaccine composition can contain sodium phosphate, sodium chloride, and / or histidine. Sodium phosphate can be present at about 10 mM to about 50 mM, about 15 mM to about 25 mM, or about 25 mM. In certain cases, about 22 mM sodium phosphate is present. Histidine can be present at about 0.1% (w / v), about 0.5% (w / v), about 0.7% (w / v), about 1% (w / v), about 1.5% (w / v), about 2% (w / v), or about 2.5% (w / v). Sodium chloride, if present, can be about 150 mM. In certain compositions, sodium chloride can be present at higher concentrations, e.g., about 200 mM to about 500 mM. In embodiments, sodium chloride is present at a high concentration, including but not limited to, about 200 mM, about 250 mM, about 300 mM, about 350 mM, about 400 mM, about 450 mM, or about 500 mM.

[0077] In embodiments, the nanoparticles described herein have improved stability at certain pH levels. In embodiments, the nanoparticles are stable at slightly acidic pH levels. For example, nanoparticles are stable at slightly acidic pHs, such as pH 5.8 to pH 7.0. In embodiments, the nanoparticles and compositions containing the nanoparticles may be stable at a pH range of about pH 5.8 to about pH 7.0, including about pH 5.9 to about pH 6.8, about pH 6.0 to about pH 6.5, about pH 6.1 to about pH 6.4, about pH 6.1 to about pH 6.3, or about pH 6.2. In embodiments, the nanoparticles and compositions described herein are stable at neutral pHs, including about pH 7.0 to about pH 7.4. In embodiments, the nanoparticles and compositions described herein are stable at slightly alkaline pHs, such as about pH 7.0 to about pH 8.5, about pH 7.0 to about pH 8.0, or about pH 7.0 to about pH 7.5, including all values ​​and ranges therebetween.

[0078] In some embodiments, in addition to the RSV F glycoprotein, the compositions described herein comprise one or more additional viral glycoproteins. In some embodiments, the additional viral glycoprotein is selected from the group consisting of a SARS-CoV-2 S glycoprotein, an influenza hemagglutinin glycoprotein, and an influenza neuraminidase. In some embodiments, the immunogenic composition comprises a RSV F glycoprotein, an influenza glycoprotein, and a SARS-CoV-2 S glycoprotein described herein. In some embodiments, the immunogenic composition comprises a RSV F glycoprotein and an influenza glycoprotein described herein. In some embodiments, the immunogenic composition comprises a RSV F glycoprotein and a SARS-CoV-2 S glycoprotein described herein.

[0079] In some embodiments, the immunogenic composition comprises about 1 to about 50, about 1 to about 45, about 1 to about 40, about 1 to about 35, about 1 to about 30, about 1 to about 25, about 1 to about 20, about 1 to about 15, about 1 to about 10, or about 1 to about 5 different viral glycoproteins. "Different" viral glycoproteins have different amino acid sequences. In some embodiments, the different viral glycoproteins may be derived from different viruses, different strains, or subtypes of the same virus, or may differ from each other by mutation.

[0080] In some embodiments, in addition to the RSV F glycoprotein, the immunogenic composition comprises from 1 to about 50 different influenza glycoproteins (e.g., hemagglutinin and / or neuraminidase), hi some embodiments, the influenza glycoproteins (e.g., hemagglutinin or neuraminidase) may be derived from any influenza virus strain.

[0081] In embodiments, the immunogenic compositions comprise 1, 2, 3, 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, or about 50 influenza glycoproteins (including all values ​​and ranges therebetween).

[0082] In some embodiments, the influenza glycoprotein is hemagglutinin. In some embodiments, the influenza HA glycoprotein is selected from any one of the following subtypes: H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, H17, and H18. Phylogenetically, influenza is divided into groups. In some embodiments, the HA glycoprotein is a Group 1 HA glycoprotein (i.e., a glycoprotein from an H1, H2, H5, H6, H8, H9, H11, H12, H13, H16, H17, or H18 subtype). In some embodiments, the HA glycoprotein is a Group 2 HA glycoprotein (i.e., a glycoprotein from an H3, H4, H7, H10, H14, or H15 subtype).

[0083] Several examples of influenza HA glycoproteins are described in the following patent documents: WO 2017 / 041100 and WO 2019 / 022930, each of which is incorporated herein by reference in its entirety for all purposes.

[0084] In embodiments, the immunogenic compositions of the present disclosure comprise at least three different HA glycoproteins or at least four different HA glycoproteins. In embodiments, the immunogenic compositions of the present disclosure comprise three or four different HA glycoproteins. In embodiments, each of the HA glycoproteins is derived from a different influenza strain. In embodiments, three HA glycoproteins are derived from influenza A strains and one HA glycoprotein is derived from an influenza B strain. In embodiments, two HA glycoproteins are derived from influenza A strains and two HA glycoproteins are derived from influenza B strains. In embodiments, two HA glycoproteins are derived from influenza A strains and one HA glycoprotein is derived from an influenza B strain.

[0085] In some embodiments, each of the at least three different HA glycoproteins is separately isolated using an egg-based production method, hi some embodiments, the egg-based production method comprises (a) growing influenza virus in eggs and (b) recovering the influenza virus.

[0086] In embodiments, the influenza virus is a live virus. In embodiments, the influenza virus is a weakened or "attenuated" virus. In embodiments, the influenza virus is optimized for growth in eggs. In embodiments, the optimized influenza virus lacks the polybasic cleavage site of hemagglutinin. The following paper describes in detail the development of optimized influenza viruses (referred to as "vaccine candidate viruses") and is incorporated herein by reference in its entirety: Belser et al. Virology. 2017 Nov;511:135-141.

[0087] In embodiments, the eggs are chicken eggs. In embodiments, the chicken eggs are embryonated chicken eggs. In embodiments, the eggs are pathogen-free eggs. In embodiments, the influenza virus is propagated by inoculating the virus into the allantoic cavity of an embryonated chicken egg. The following article describes exemplary inoculation methods and is incorporated herein by reference in its entirety: Brauer et al. J Vis Exp. 2015;(97):52421.

[0088] In embodiments, the egg-based manufacturing method includes purifying the influenza virus. In embodiments, the influenza virus is purified using any of the following techniques: centrifugation, chromatography, precipitation, or nanofiltration. In embodiments, the centrifugation technique is ultracentrifugation. In embodiments, the virus is purified using zonal centrifugation. In embodiments, the zonal centrifugation is continuous-flow zonal centrifugation.

[0089] In embodiments, the egg-based manufacturing method includes inactivating (also referred to herein as "killing") the influenza virus. In embodiments, the influenza virus is inactivated using low pH (e.g., a pH of about 3.5 to 5.5), heat, ethanol, ultraviolet light, exposure to a detergent (e.g., octylphenol), or exposure to chemicals (e.g., 2-propanol, ethanol, povidone-iodine). In embodiments, the purified virus is inactivated using ultraviolet light, beta-propiolactone, sodium deoxycholate, formaldehyde, or any combination thereof.

[0090] In some embodiments, the egg-based production method includes exposing the influenza virus to a surfactant. The surfactant can be sodium taurodeoxycholate, octylphenol ethoxylate (Triton®-X100), or cetyltrimethylammonium bromide. Exposing the influenza virus to the surfactant results in the formation of influenza split virions. In some embodiments, the at least three HA glycoproteins are in the form of influenza split virions.

[0091] In embodiments, the egg-based manufacturing method includes purifying influenza antigens (eg, hemagglutinin) from the virus.

[0092] The following U.S. Food and Drug Administration (FDA)-approved influenza vaccines are made using an egg-based manufacturing method: AFLURIA® tetravalent, FLUARIX® tetravalent, FLULAVAL tetravalent, FLUZONE® tetravalent, FLUZONE® High Dose tetravalent, FLUMIST® tetravalent, FLUAD®, and FLUAD® tetravalent.

[0093] In some embodiments, each of the at least three HA glycoproteins is isolated using a cell culture-based method. In some embodiments, the cell culture-based method comprises (i) growing influenza virus in cells and (ii) recovering the virus from the cells. In some embodiments, the cell culture-based method comprises (i) transfecting the cells with a vector comprising a hemagglutinin and (ii) recovering the hemagglutinin from the cells. In some embodiments, the cell culture-based method comprises (i) transducing the cells with a virus encoding the hemagglutinin and (ii) recovering the hemagglutinin from the cells. In some embodiments, the recovered hemagglutinin is a recombinant hemagglutinin.

[0094] In some embodiments, the cell is an animal cell, a bacterial cell, an insect cell, or a fungal cell. In some embodiments, the animal is a human, a bird (e.g., a chicken), a dog, a reptile, a goat, a pig, a mouse, a rabbit, or a rat.

[0095] In embodiments, the hemagglutinin-encoding virus is a baculovirus, a lentivirus, or an adeno-associated virus.

[0096] In embodiments, influenza viruses produced using cell culture-based methods are purified. Any of the purification techniques described for purifying influenza viruses produced using egg-based manufacturing methods can be used to purify influenza viruses produced using cell culture-based methods.

[0097] In embodiments, hemagglutinins produced using cell culture-based methods are purified. Purification techniques include chromatography, centrifugation, precipitation, and nanofiltration.

[0098] FLUCELVAX® tetravalent, a US Food and Drug Administration (FDA)-approved influenza vaccine, is produced using a cell culture-based method.

[0099] In embodiments, each of the at least three HA glycoproteins is a recombinant hemagglutinin. The U.S. Food and Drug Administration (FDA)-approved influenza vaccine, FLUBLOK® tetravalent, is a recombinant hemagglutinin.

[0100] In some embodiments, the at least three hemagglutinins are in the form of recombinant hemagglutinins. The recombinant hemagglutinins are isolated from cells that produce the hemagglutinins. In some embodiments, the cells that produce the hemagglutinins are transfected with a vector encoding the hemagglutinins. In some embodiments, the cells that produce the hemagglutinins are transduced with a virus that encodes the hemagglutinins.

[0101] In embodiments, the compositions disclosed herein comprise surfactant-core nanoparticles comprising hemagglutinin from influenza virus, which are described in detail in U.S. Patent No. 10,426,829, which is incorporated herein by reference in its entirety for all purposes.

[0102] The surfactant-core nanoparticles comprise influenza virus-derived hemagglutinin associated with a surfactant core. In some embodiments, the hemagglutinin is a trimer. Each nanoparticle may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 trimers. In some embodiments, the nanoparticles contain about 2 to about 9, about 2 to about 6, or about 5 hemagglutinin trimers. The hemagglutinin is associated with the nonionic surfactant-containing core of the nanoparticle. In some embodiments, the surfactant is selected from polysorbate-20 (PS20), polysorbate-40 (PS40), polysorbate-60 (PS60), polysorbate-65 (PS65), and polysorbate-80 (PS80). The presence of the surfactant facilitates nanoparticle formation by forming a core that organizes and presents antigens. In some embodiments, the nanoparticles may contain antigens assembled into multi-oligomeric glycoprotein-PS80 protein surfactant nanoparticles in which the head regions protrude outward and the hydrophobic regions and PS80 surfactant form a central core surrounded by the antigen.

[0103] In embodiments, surfactant-core nanoparticles or HaSMaNs are trypsin-resistant nanoparticles made using neutral pH purification. Trypsin resistance is achieved by a neutral pH range of greater than 6.9 to 8.5 during the purification and formulation of HA nanoparticles. Trypsin-resistant influenza glycoproteins and trypsin-resistant influenza nanoparticles, and methods for making them, are described in detail in U.S. Patent No. 10,426,829.

[0104] In some embodiments, the hemagglutinin of the surfactant core nanoparticles or HaSMaN described herein comprises the full-length wild-type hemagglutinin amino acid sequence. In some embodiments, the hemagglutinin is a hemagglutinin variant. In some embodiments, the hemagglutinin exhibits at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity to the wild-type hemagglutinin protein.

[0105] HA is a homotrimer, with each monomer consisting of approximately 550 amino acid residues. Each HA monomer is conceptually divided into three domains: an ectodomain of approximately 515 residues that constitutes the extraviral portion of the molecule, a single stretch of 27 residues that defines the transmembrane (TM) domain, and approximately 10 residues that constitute the cytoplasmic tail (CT). While some modifications may be made to hemagglutinin, the complete transmembrane (TM) domain is required for the formation of both surfactant-core nanoparticles and HaSMaNs. Thus, in certain instances, the modified HA protein sequence may have 100% identity to the wild-type TM and CT domains, with some flexibility in the remaining ectodomain portion, where identity may be at least 90% or at least 95%. The domains can be identified by homology to the amino acid sequences of the TM and CT domains of Japan / 305 / 57 HA shown in Figure 1 of Melikyan et al. (Mol Biol Cell. 1999 Jun;10(6):1821-1836), although it should be noted that the boundaries between the ectodomain, TM domain, and CT domain may differ by up to three amino acids from one HA protein to another.

[0106] In some embodiments, the immunogenic and vaccine compositions described herein comprise HaSMaN (hemagglutinin saponin matrix nanoparticles). Figure 6 (right panel) shows the HaSMaN structure as observed under an electron microscope. HA glycoprotein coats the matrix cage-like structure. HaSMaN structures are formed by preparing surfactant-core nanoparticles containing influenza virus-derived hemagglutinin and then incubating them with ISCOM matrix adjuvant particles for a period of time. ISCOM matrix particles are shown in the middle panel of Figure 6. Notably, HaSMaN readily forms with influenza A HA protein but not with influenza B HA protein.

[0107] The HaSMaNs disclosed herein are prepared by incubating surfactant core nanoparticles with an ISCOM matrix adjuvant comprising a saponin fraction, cholesterol, and phospholipids. In some embodiments, the HaSMaNs are formed by incubating surfactant core nanoparticles with the ISCOM matrix adjuvant for about 24 to about 48 hours. For example, surfactant core nanoparticles may be incubated with the ISCOM matrix adjuvant for about 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, or longer. In some embodiments, HaSMaN are formed by incubating surfactant core nanoparticles with ISCOM matrix adjuvant for at least about 24 hours, at least about 25 hours, at least about 26 hours, at least about 27 hours, at least about 28 hours, at least about 29 hours, at least about 30 hours, at least about 31 hours, at least about 32 hours, at least about 33 hours, at least about 34 hours, at least about 35 hours, at least about 36 hours, at least about 37 hours, at least about 38 hours, at least about 39 hours, at least about 40 hours, at least about 41 hours, at least about 42 hours, at least about 43 hours, at least about 44 hours, at least about 45 hours, at least about 46 hours, at least about 47 hours, or at least about 48 hours. In several embodiments, HaSMaN are formed by incubating surfactant core nanoparticles with ISCOM matrix adjuvant at a temperature of about 4°C to about 25°C. For example, HaSMaNs can be formed by incubating surfactant core nanoparticles with ISCOM matrix adjuvant at temperatures of about 4°C, about 5°C, about 6°C, about 7°C, about 8°C, about 9°C, about 10°C, about 11°C, about 12°C, about 13°C, about 14°C, about 15°C, about 16°C, about 17°C, about 18°C, about 19°C, about 20°C, about 21°C, about 22°C, about 23°C, about 24°C, about 25°C, or higher.In some embodiments, HaSMaN is formed by incubating surfactant-core nanoparticles with an ISCOM matrix adjuvant at a temperature of at least about 4°C, at least about 5°C, at least about 6°C, at least about 7°C, at least about 8°C, at least about 9°C, at least about 10°C, at least about 11°C, at least about 12°C, at least about 13°C, at least about 14°C, at least about 15°C, at least about 16°C, at least about 17°C, at least about 18°C, at least about 19°C, at least about 20°C, at least about 21°C, at least about 22°C, at least about 23°C, at least about 24°C, or at least about 25°C. Typically, about 24-48 hours of incubation at 4°C or 25°C is required for formation. Formation of HaSMaN is facilitated by higher temperatures. In some embodiments, HaSMaN is formed by incubating surfactant-core nanoparticles with an ISCOM matrix adjuvant at about 25°C for at least 24 hours. Mixing surfactant-core nanoparticles with ISCOM matrix adjuvant immediately before administration to a subject, i.e., bedside mix, does not produce HaSMaN. Longer incubation periods do not adversely affect HaSMaN formation.

[0108] In some embodiments, in addition to the RSV F glycoprotein, the immunogenic composition comprises from 1 to about 50 different SARS-CoV-2 S glycoproteins ("CoV S glycoproteins"). In some embodiments, in addition to the RSV F glycoprotein, the immunogenic composition comprises about 5 different SARS-CoV-2 CoV S glycoproteins. The CoV S glycoprotein is synthesized as an inactive precursor (SO), which is proteolytically cleaved at a furin cleavage site into S1 and S2 subunits, which remain non-covalently associated to form a prefusion trimer. The S2 domain of the CoV S glycoprotein contains a fusion peptide (FP), two heptad repeats (HR1 and HR2), a transmembrane (TM) domain, and a cytoplasmic tail (CT). SARS-CoV-2 The S1 domain of the S protein folds into four separate domains: an N-terminal domain (NTD) and a C-terminal domain containing a receptor-binding domain (RBD) and two subdomains, SD1 and SD2. The pre-fusion SARS-CoV-2 S protein trimer undergoes structural rearrangement from a pre-fusion conformation to a post-fusion conformation upon S protein receptor binding and cleavage. In some embodiments, the CoV S polypeptide is a glycoprotein due to post-translational glycosylation. The glycoprotein comprises one or more domains, including a signal peptide, an S1 subunit, an S2 subunit, an NTD, an RBD, two subdomains (SD1 and SD2, shown as SD1 / 2 in Figures 1A-B and referred to herein as "SD1 / 2"), a complete or modified fusion peptide, an HR1 domain, an HR2 domain, a TM, and a CD. In some embodiments, the amino acids of each domain are shown in Figure 7A (shown based on SEQ ID NO: 1) and Figure 7B (shown based on SEQ ID NO: 2). The SARS-CoV-2 S glycoprotein also contains a furin cleavage site.

[0109] In embodiments, each of the CoV S glycoproteins described herein contains prolines at amino acid positions 973 and 974 and includes an inactive furin cleavage site, where the CoV S glycoproteins are numbered based on the CoV S glycoprotein of SEQ ID NO:2.

[0110] In embodiments, the CoV S glycoprotein described herein is expressed with an N-terminal signal peptide. In embodiments, the N-terminal signal peptide has the amino acid sequence of SEQ ID NO: 5 (MFVFLVLLPLVSS). In embodiments, the N-terminal signal peptide has the amino acid sequence of SEQ ID NO: 117 (MFVFLVLLPLVSI). In embodiments, the N-terminal signal peptide has the amino acid sequence of SEQ ID NO: 154 (MFVFFVLLPLVSS). In embodiments, the N-terminal signal peptide has the amino acid sequence of SEQ ID NO: 193 (MFGFLVLLPLVSS). In embodiments, the signal peptide can be replaced with any signal peptide that allows for expression of the CoV S glycoprotein. In embodiments, one or more of the CoV S glycoprotein signal peptide amino acids can be deleted or mutated. An initial methionine residue is maintained to initiate expression. In embodiments, the CoV S glycoprotein is encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:95, SEQ ID NO:43, SEQ ID NO:47, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:96, SEQ ID NO:60, SEQ ID NO:131, SEQ ID NO:135, SEQ ID NO:142, SEQ ID NO:145, SEQ ID NO:148, SEQ ID NO:150, SEQ ID NO:196, SEQ ID NO:197, SEQ ID NO:198, SEQ ID NO:199, SEQ ID NO:201, SEQ ID NO:202, SEQ ID NO:204, SEQ ID NO:206, SEQ ID NO:208, SEQ ID NO:210, SEQ ID NO:212, SEQ ID NO:214, and SEQ ID NO:216. In embodiments, the N-terminal signal peptide of the CoV S glycoprotein contains a mutation at Ser-13 compared to the native CoV spike (S) signal polypeptide (SEQ ID NO:5). In embodiments, Ser-13 is mutated to any naturally occurring amino acid. In embodiments, Ser-13 is mutated to alanine, methionine, isoleucine, leucine, threonine, or valine. In embodiments, Ser-13 is mutated to isoleucine.

[0111] After expression of the CoV S glycoprotein in host cells, the N-terminal signal peptide is cleaved to yield the mature CoV glycoprotein sequence (SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:106, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:132, SEQ ID NO:144, SEQ ID NO:151, SEQ ID NO:153, SEQ ID NO:156, SEQ ID NO:158, SEQ ID NO:174, SEQ ID NO:175, SEQ ID NO:176, SEQ ID NO:181, SEQ ID NO:182, SEQ ID NO:183, SEQ ID NO:184, SEQ ID NO:186, SEQ ID NO:188, SEQ ID NO:190, SEQ ID NO:192, SEQ ID NO:195, SEQ ID NO:217, SEQ ID NO:218, SEQ ID NO:219, SEQ ID NO:220, SEQ ID NO:221, SEQ ID NO:222, SEQ ID NO:223, SEQ ID NO:224, SEQ ID NO:225, SEQ ID NO:226, SEQ ID NO:227, SEQ ID NO:228, SEQ ID NO:229, SEQ ID NO:230, SEQ ID NO:231, SEQ ID NO:232, SEQ ID NO:233, SEQ ID NO:234, SEQ ID NO:235, SEQ ID NO:236, SEQ ID NO:237, SEQ ID NO:238, SEQ ID NO:239, SEQ ID NO:240, SEQ ID NO:241, SEQ ID NO:242, SEQ ID NO:243, SEQ ID NO:244, SEQ 20, SEQ ID NO:221, SEQ ID NO:222, SEQ ID NO:223, SEQ ID NO:224, SEQ ID NO:225, SEQ ID NO:226, SEQ ID NO:227, SEQ ID NO:228, SEQ ID NO:233, SEQ ID NO:234, SEQ ID NO:235, SEQ ID NO:236, SEQ ID NO:260, SEQ ID NO:261, SEQ ID NO:262, SEQ ID NO:263, SEQ ID NO:264, SEQ ID NO:274, SEQ ID NO:276, SEQ ID NO:278, SEQ ID NO:280, SEQ ID NO:284, SEQ ID NO:288, SEQ ID NO:292, SEQ ID NO:296, SEQ ID NO:300, SEQ ID NO:304, SEQ ID NO:308, SEQ ID NO:312, SEQ ID NO:316, SEQ ID NO:320, SEQ ID NO:324, SEQ ID NO:328). In embodiments, the signal peptide is cleaved by a host cell protease. In aspects, the full-length protein may be isolated from the host cell, after which the signal peptide is cleaved.

[0112] SEQ ID NO:88, SEQ ID NO:105, SEQ ID NO:130, SEQ ID NO:136, SEQ ID NO:143, SEQ ID NO:149, SEQ ID NO:152, SEQ ID NO:155, SEQ ID NO:157, SEQ ID NO:159, SEQ ID NO:160, SEQ ID NO:167, SEQ ID NO:170, SEQ ID NO:173, SEQ ID NO:177, SEQ ID NO:178, SEQ ID NO:179, SEQ ID NO:180, SEQ ID NO:185, SEQ ID NO:187, SEQ ID NO:189, SEQ ID NO:191, SEQ ID NO:194, SEQ ID NO:200, SEQ ID NO:203, SEQ ID NO:205, SEQ ID NO:207, SEQ ID NO:209, SEQ ID NO:211, SEQ ID NO:213 during expression and purification , SEQ ID NO:215, SEQ ID NO:229, SEQ ID NO:230, SEQ ID NO:231, SEQ ID NO:232, SEQ ID NO:242, SEQ ID NO:243, SEQ ID NO:244, SEQ ID NO:250, SEQ ID NO:251, SEQ ID NO:252, SEQ ID NO:253, SEQ ID NO:254, SEQ ID NO:266, SEQ ID NO:268, SEQ ID NO:270, SEQ ID NO:272, SEQ ID NO:282, SEQ ID NO:286, SEQ ID NO:290, SEQ ID NO:295, SEQ ID NO:299, SEQ ID NO:303, SEQ ID NO:307, SEQ ID NO:311, SEQ ID NO:315, SEQ ID NO:319, SEQ ID NO:323, and SEQ ID NO:327 After cleavage of the signal peptide from the CoV spike (S) polypeptide having the sequences SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:106, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:132, SEQ ID NO:144, SEQ ID NO:151, SEQ ID NO:153, SEQ ID NO:156, SEQ ID NO:158, SEQ ID NO:174, SEQ ID NO:175, SEQ ID NO:176, SEQ ID NO:181, SEQ ID NO:182, SEQ ID NO:183, SEQ ID NO:184, SEQ ID NO:186, SEQ ID NO:188, SEQ ID NO:190, SEQ ID NO:192, SEQ ID NO:195, SEQ ID NO:217, SEQ ID NO:218 8, SEQ ID NO:219, SEQ ID NO:220, SEQ ID NO:221, SEQ ID NO:222, SEQ ID NO:223, SEQ ID NO:224, SEQ ID NO:225, SEQ ID NO:226, SEQ ID NO:227, SEQ ID NO:228, SEQ ID NO:233, SEQ ID NO:234, SEQ ID NO:235, SEQ ID NO:236, SEQ ID NO:260, SEQ ID NO:261, SEQ ID NO:262, SEQ ID NO:263, SEQ ID NO:264, SEQ ID NO:274, SEQ ID NO:276, SEQ ID NO:278, SEQ ID NO:280, SEQ ID NO:284, SEQ ID NO:288, SEQ ID NO:292, SEQ ID NO:296, SEQ ID NO:300, SEQ ID NO:304, SEQ ID NO:308, SEQ ID NO:312,A mature polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 316, SEQ ID NO: 320, SEQ ID NO: 324, and SEQ ID NO: 328 is obtained and used to produce a CoV S nanoparticle vaccine or CoV S nanoparticles.

[0113] Advantageously, the disclosed CoV S polypeptides can have enhanced protein expression and stability compared to native CoV spike (S) proteins.

[0114] In embodiments, the CoV S polypeptides described herein contain additional modifications from a native coronavirus S protein (e.g., SEQ ID NO: 2). In embodiments, the coronavirus S proteins described herein exhibit at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a native CoV S glycoprotein. One of skill in the art would use known techniques to calculate the percent identity of a recombinant coronavirus S protein to either a native protein or a CoV S glycoprotein described herein.

[0115] In embodiments, the CoV S polypeptides described herein are at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to a CoV S polypeptide having the amino acid sequence of any one of SEQ ID NO:87, SEQ ID NO:174, SEQ ID NO:175, SEQ ID NO:176, SEQ ID NOs:181-184, SEQ ID NO:186, SEQ ID NO:188, SEQ ID NO:190, SEQ ID NO:195, SEQ ID NOs:217-228, SEQ ID NOs:233-236, and SEQ ID NOs:243, SEQ ID NOs:255-328. The CoV S polypeptide may have a deletion, insertion, or mutation of up to about 1, up to about 2, up to about 3, up to about 4, up to about 5, up to about 10, up to about 15, up to about 20, up to about 25, up to about 30, up to about 35, up to about 40, up to about 45, or up to about 50 amino acids compared to the amino acid sequence of a CoV S polypeptide having any one of the amino acid sequences of SEQ ID NO:87, SEQ ID NO:174, SEQ ID NO:175, SEQ ID NO:176, SEQ ID NOs:181 to 184, SEQ ID NO:186, SEQ ID NO:188, SEQ ID NO:190, SEQ ID NO:195, SEQ ID NOs:217 to 228, SEQ ID NOs:233 to 236, SEQ ID NO:243, and SEQ ID NOs:255 to 328. The CoV S polypeptide may have a deletion, insertion, or mutation of 1 to about 70 amino acids, 1 to about 5 amino acids, about 3 to about 10 amino acids, about 5 to about 10 amino acids, about 8 to about 12 amino acids, about 10 to about 15 amino acids, about 12 to about 17 amino acids, about 15 to about 20 amino acids, about 18 to about 23 amino acids, about 20 to about 25 amino acids, about 22 to about 27 amino acids, about 25 to about 30 amino acids, about 30 to about 35 amino acids, about 35 to about 40 amino acids, about 40 to about 45 amino acids, or about 45 to about 50 amino acids compared to a CoV S polypeptide having the amino acid sequence of any one of SEQ ID NO: 87, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NOs: 181 to 184, SEQ ID NO: 186, SEQ ID NO: 188, SEQ ID NO: 190, SEQ ID NO: 195, SEQ ID NOs: 217 to 228, SEQ ID NOs: 233 to 236, SEQ ID NO: 243, and SEQ ID NOs: 255 to 328.In embodiments, the CoV S polypeptides described herein have a length of about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 51, about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, about 60, about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71, about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 79, about 80, about 81, about 82, about 83, about 84, about 85, about 86, about 87, about 88, about 89, about 90, about 91, about 92, about 93, about 94, about 95, about 96, about 97, about 98, about 99, about 100, about 10 about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 51, about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, about 60, about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71, about 72, about 73, about 74, or about 75 deletions, insertions, or mutations.

[0116] In embodiments, the coronavirus S polypeptide is extended at the N-terminus, C-terminus, or both the N-terminus and C-terminus. In some aspects, the extension is a tag useful for functions such as purification or detection. In some aspects, the tag contains an epitope. For example, the tag can be a polyglutamate tag, a FLAG tag, an HA tag, a polyHis tag (having approximately 5-10 histidines) (SEQ ID NO: 101), a hexahistidine tag (SEQ ID NO: 100), an 8xHis tag (having 8 histidines) (SEQ ID NO: 102), a Myc tag, a glutathione-S-transferase tag, a green fluorescent protein tag, a maltose-binding protein tag, a thioredoxin tag, or an Fc tag. In other aspects, the extension can be an N-terminal signal peptide fused to the protein to enhance expression. Such signal peptides are often cleaved during expression in cells, but some nanoparticles may contain antigens with an intact signal peptide. Thus, when a nanoparticle comprises an antigen, the antigen may contain an extension portion and thus may be a fusion protein when incorporated into the nanoparticle. For purposes of calculating sequence identity, the extension portion is not included. In some embodiments, the tag is a protease cleavage site. Non-limiting examples of protease cleavage sites include HRV3C protease cleavage site, chymotrypsin, trypsin, elastase, endopeptidase, caspase-1, caspase-2, caspase-3, caspase-4, caspase-5, caspase-6, caspase-7, caspase-8, caspase-9, caspase-10, enterokinase, factor Xa, granzyme B, TEV protease, and thrombin. In some embodiments, the protease cleavage site is an HRV3C protease cleavage site. In some embodiments, the protease cleavage site comprises the amino acid sequence of SEQ ID NO:98.

[0117] In some embodiments, the CoV S glycoprotein comprises a fusion protein. In some embodiments, the CoV S glycoprotein comprises an N-terminal fusion protein. In some embodiments, the CoV S glycoprotein comprises a C-terminal fusion protein. In some embodiments, the fusion protein includes a tag useful for protein expression, purification, or detection. In some embodiments, the tag is a poly-His tag (having about 5-10 histidines), a Myc tag, a glutathione-S-transferase tag, a green fluorescent protein tag, a maltose-binding protein tag, a thioredoxin tag, a Strep tag, a Twin-Strep tag, or an Fc tag. In some embodiments, the tag is an Fc tag. In some embodiments, the Fc tag is a monomer, a dimer, or a trimer. In some embodiments, the tag is a hexahistidine tag, e.g., a poly-His tag containing six histidines (SEQ ID NO: 100). In some embodiments, the tag is a Twin-Strep tag having the amino acid sequence of SEQ ID NO: 99.

[0118] In some embodiments, the CoV S polypeptide is a fusion protein that includes another coronavirus protein. In some embodiments, the other coronavirus protein is from the same coronavirus. In some embodiments, the other coronavirus protein is from a different coronavirus.

[0119] In several embodiments, the CoV S glycoprotein can be truncated. For example, the N-terminus can be truncated by about 10, 30, 50, 75, 100, or 200 amino acids. The C-terminus can be truncated instead of or in addition to the N-terminus. For example, the C-terminus can be truncated by about 10, 30, 50, 75, 100, or 200 amino acids. For purposes of calculating identity to a protein having a truncation, identity is measured across the remainder of the protein.

[0120] In embodiments, the CoV S glycoprotein contains one or more modifications to the S1 subunit having the amino acid sequence of SEQ ID NO:121.

[0121] The amino acid sequence of the S1 subunit (SEQ ID NO: 121) is shown below.

[0122] QCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEG KQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFAS VYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFEL LHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPRRAR

[0123] In embodiments, the CoV S polypeptides described herein comprise an S1 subunit having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to the S1 subunit of SEQ ID NO: 1 or SEQ ID NO: 2. The S1 subunit can have deletions, insertions, or mutations of up to about 1, up to about 2, up to about 3, up to about 4, up to about 5, up to about 10, up to about 15, up to about 20, up to about 25, up to about 30, up to about 35, up to about 40, up to about 45, or up to about 50 amino acids compared to the amino acid sequence of the S1 subunit of SEQ ID NO: 1 or SEQ ID NO: 2. The S1 subunit may have a deletion, insertion, or mutation of about 1 to about 5 amino acids, about 3 to about 10 amino acids, about 5 to 10 amino acids, about 8 to 12 amino acids, about 10 to 15 amino acids, about 12 to 17 amino acids, about 15 to 20 amino acids, about 18 to 23 amino acids, about 20 to 25 amino acids, about 22 to about 27 amino acids, or about 25 to 30 amino acids compared to the S1 subunit of SEQ ID NO: 1 or SEQ ID NO: 2.

[0124] In embodiments, the S1 subunit can contain any combination of the modifications shown in Table 1A. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6] [Table 2-7] [Table 2-8] [Table 2-9] [Table 2-10]

[0125] In embodiments, the CoV S polypeptide contains one or more modifications to the NTD, hi embodiments, the NTD has the amino acid sequence of SEQ ID NO: 118, which corresponds to amino acids 14-305 of SEQ ID NO: 1 or amino acids 1-292 of SEQ ID NO: 2.

[0126] The amino acid sequence of the NTD (SEQ ID NO: 118) is shown below.

[0127] QCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRV YSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKS

[0128] In embodiments, the NTD has the amino acid sequence of SEQ ID NO: 45, which corresponds to amino acids 14 to 331 of SEQ ID NO: 1 or amino acids 1 to 318 of SEQ ID NO: 2. The amino acid sequence of the NTD (SEQ ID NO: 45) is shown below.

[0129] QCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVS QPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPN

[0130] In embodiments, the NTD and RBD overlap by at most about 1 amino acid, at most about 5 amino acids, at most about 10 amino acids, or at most about 20 amino acids.

[0131] In embodiments, the NTDs provided herein may be extended at the C-terminus by up to 5, up to 10, up to 15, up to 20, up to 25, or up to 30 amino acids.

[0132] In embodiments, the CoV S polypeptides described herein comprise an NTD having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to the NTD of SEQ ID NO: 1 or SEQ ID NO: 2. The NTD can have up to about 1, up to about 2, up to about 3, up to about 4, up to about 5, up to about 10, up to about 15, up to about 20, up to about 25, or up to about 30 amino acid deletions, insertions, or mutations compared to the amino acid sequence of the NTD of SEQ ID NO: 1 or SEQ ID NO: 2. The NTD may have a deletion, insertion, or mutation of about 1 to about 5 amino acids, about 3 to about 10 amino acids, about 5 to 10 amino acids, about 8 to 12 amino acids, about 10 to 15 amino acids, about 12 to 17 amino acids, about 15 to 20 amino acids, about 18 to 23 amino acids, about 20 to 25 amino acids, about 22 to about 27 amino acids, or about 25 to 30 amino acids compared to the NTD of SEQ ID NO: 1 or SEQ ID NO: 2.

[0133] In embodiments, the CoV S polypeptide contains a deletion of one or more amino acids from the N-terminal domain (NTD) (corresponding to amino acids 1-292 of SEQ ID NO: 2). In embodiments, the CoV S polypeptide contains a deletion of up to about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 292 amino acids of the NTD.

[0134] In embodiments, the CoV S polypeptide contains a deletion of one or more amino acids from the NTD (corresponding to amino acids 1-318 of SEQ ID NO:2). In embodiments, the CoV S polypeptide contains a deletion of amino acids 1-318 of the NTD of SEQ ID NO:2. In embodiments, the deletion of the NTD enhances protein expression of the CoV spike (S) polypeptide. In embodiments, the CoV S polypeptide having an NTD deletion has an amino acid sequence represented by SEQ ID NOs:46, 48, 49, 51, 52, and 54. In embodiments, the CoV S polypeptide having an NTD deletion is encoded by an isolated nucleic acid sequence selected from the group consisting of SEQ ID NO:47, SEQ ID NO:50, and SEQ ID NO:53.

[0135] In embodiments, the NTD may contain any combination of the modifications shown in Table 1B. The modifications are shown with reference to the mature S polypeptide sequence, SEQ ID NO:2. [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5] [Table 3-6]

[0136] In embodiments, the CoV S polypeptide contains one or more modifications to the RBD.

[0137] In some embodiments, the RBD has the amino acid sequence of SEQ ID NO: 126, which corresponds to amino acids 331-527 of SEQ ID NO: 1 or amino acids 318-514 of SEQ ID NO:2.

[0138] The amino acid sequence of RBD (SEQ ID NO: 126) is shown below:

[0139] NITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGP

[0140] In some embodiments, the RBD has the amino acid sequence of SEQ ID NO: 116, which corresponds to amino acids 335-530 of SEQ ID NO: 1 or amino acids 322-517 of SEQ ID NO:2.

[0141] The amino acid sequence of the RBD (SEQ ID NO: 116) is shown below.

[0142] LCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKS

[0143] In embodiments, the RBDs provided herein may be extended at the N- or C-terminus by up to 1 amino acid, up to 5 amino acids, up to 10 amino acids, up to 15 amino acids, up to 20 amino acids, up to 25 amino acids, or up to 30 amino acids.

[0144] In embodiments, the CoV S polypeptides described herein comprise an RBD that has at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to the RBD of SEQ ID NO: 1 or SEQ ID NO: 2. The RBD may have deletions, insertions, or mutations of up to about 1, up to about 2, up to about 3, up to about 4, up to about 5, up to about 10, up to about 15, up to about 20, up to about 25, or up to about 30 amino acids compared to the amino acid sequence of the RBD of SEQ ID NO: 1 or SEQ ID NO: 2. The RBD may have deletions, insertions, or mutations of about 1 to about 50 amino acids, about 1 to about 5 amino acids, about 3 to about 10 amino acids, about 5 to about 10 amino acids, about 8 to about 12 amino acids, about 10 to about 15 amino acids, about 12 to about 17 amino acids, about 15 to about 20 amino acids, about 18 to about 23 amino acids, about 20 to about 25 amino acids, about 22 to about 27 amino acids, or about 25 to about 30 amino acids compared to the RBD of SEQ ID NO: 1 or SEQ ID NO: 2.

[0145] In embodiments, the CoV S polypeptide has at least 1, at least 2, at least 3, at least 4, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 modifications in the RBD. In embodiments, there are up to about 20, up to about 25, up to about 30, up to about 35, up to about 40, up to about 45, or up to about 50 modifications in the RBD. In embodiments, the RBD can contain any combination of modifications shown in Table 1C. [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4]

[0146] In embodiments, the CoV S polypeptide contains one or more modifications to SD1 / 2 having the amino acid sequence of SEQ ID NO: 122, which corresponds to amino acids 542-681 of SEQ ID NO: 1 or amino acids 529-668 of SEQ ID NO: 2.

[0147] The amino acid sequence of SD1 / 2 (SEQ ID NO: 122) is shown below.

[0148] NFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSP

[0149] In embodiments, the CoV S polypeptides described herein comprise an SD1 / 2 that has at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to the SD1 / 2 of SEQ ID NO: 1 or SEQ ID NO: 2. The SD1 / 2 can have up to about 1, up to about 2, up to about 3, up to about 4, up to about 5, up to about 10, up to about 15, up to about 20, up to about 25, or up to about 30 amino acid deletions, insertions, or mutations compared to the amino acid sequence of SD1 / 2 of SEQ ID NO: 1 or SEQ ID NO: 2. SD1 / 2 may have a deletion, insertion, or mutation of about 1 to about 5 amino acids, about 3 to about 10 amino acids, about 5 to 10 amino acids, about 8 to 12 amino acids, about 10 to 15 amino acids, about 12 to 17 amino acids, about 15 to 20 amino acids, about 18 to 23 amino acids, about 20 to 25 amino acids, about 22 to about 27 amino acids, or about 25 to 30 amino acids compared to SD1 / 2 of SEQ ID NO: 1 or SEQ ID NO: 2.

[0150] In embodiments, the CoV S polypeptide has at least 1, at least 2, at least 3, at least 4, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 modifications in SD1 / 2. In embodiments, there are up to about 20, up to about 25, up to about 30, up to about 35, up to about 40, up to about 45, or up to about 50 modifications in SD1 / 2. In embodiments, SD1 / 2 can contain any combination of modifications shown in Table ID. [Table 5-1] [Table 5-2]

[0151] In embodiments, the CoV S polypeptide contains a furin site (RRAR) corresponding to amino acids 682-685 of SEQ ID NO: 1 or amino acids 669-672 of SEQ ID NO: 2 that is inactivated by one or more mutations. Inactivation of the furin cleavage site prevents furin from cleaving the CoV S polypeptide. In embodiments, the CoV S polypeptide described herein containing an inactivated furin cleavage site is expressed as a single chain.

[0152] In some embodiments, one or more of the amino acids contained in the native furin cleavage site are mutated to any naturally occurring amino acid. In some embodiments, the amino acids are L-amino acids. Non-limiting examples of amino acids include alanine, arginine, glycine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, serine, threonine, histidine, lysine, methionine, proline, valine, isoleucine, leucine, tyrosine, tryptophan, and phenylalanine.

[0153] In some embodiments, one or more of the amino acids contained in the native furin cleavage site are mutated to glutamine. In some embodiments, one, two, three, or four amino acids may be mutated to glutamine. In some embodiments, one of the arginines contained in the native furin cleavage site is mutated to glutamine. In some embodiments, two of the arginines contained in the native furin cleavage site are mutated to glutamine. In some embodiments, three of the arginines contained in the native furin cleavage site are mutated to glutamine.

[0154] In some embodiments, one or more of the amino acids contained in the native furin cleavage site are mutated to alanine. In some embodiments, one, two, three, or four amino acids may be mutated to alanine. In some embodiments, one of the arginines contained in the native furin cleavage site is mutated to alanine. In some embodiments, two of the arginines contained in the native furin cleavage site are mutated to alanine. In some embodiments, three of the arginines contained in the native furin cleavage site are mutated to alanine.

[0155] In some embodiments, one or more of the amino acids contained in the native furin cleavage site are mutated to glycine. In some embodiments, one, two, three, or four amino acids may be mutated to glycine. In some embodiments, one of the arginines contained in the native furin cleavage site is mutated to glycine. In some embodiments, two of the arginines contained in the native furin cleavage site are mutated to glycine. In some embodiments, three of the arginines contained in the native furin cleavage site are mutated to glycine.

[0156] In some embodiments, one or more of the amino acids contained in the natural furin cleavage site are mutated to asparagine. For example, one, two, three, or four amino acids may be mutated to asparagine. In some embodiments, one of the arginines contained in the natural furin cleavage site is mutated to asparagine. In some embodiments, two of the arginines contained in the natural furin cleavage site are mutated to asparagine. In some embodiments, three of the arginines contained in the natural furin cleavage site are mutated to asparagine.

[0157] In embodiments, instead of an active furin cleavage site (SEQ ID NO: 6), the CoV S polypeptides described herein contain an inactivated furin cleavage site. In embodiments, the amino acid sequence of the inactivated furin cleavage site is represented by any one of SEQ ID NOs: 7-34 or SEQ ID NO: 97. In embodiments, the amino acid sequence of the inactivated furin cleavage site is QQAQ (SEQ ID NO: 7). In embodiments, the amino acid sequence of the inactivated furin cleavage site is GSAS (SEQ ID NO: 97). In embodiments, the amino acid sequence of the inactivated furin cleavage site is GSGA (SEQ ID NO: 111). In embodiments, the amino acid sequence of the inactivated furin cleavage site is GG, GGG (SEQ ID NO: 127), GGGG (SEQ ID NO: 128), or GGGGG (SEQ ID NO: 129).

[0158] Non-limiting examples of amino acid sequences of inactivated furin sites contained in CoV S polypeptides are found in Table 1E. [Table 6]

[0159] In embodiments, the CoV S polypeptide contains one or more modifications to the S2 subunit having the amino acid sequence of SEQ ID NO: 120, which corresponds to amino acids 686 to 1273 of SEQ ID NO: 1 or amino acids 673 to 1260 of SEQ ID NO: 2.

[0160] The amino acid sequence of the S2 subunit (SEQ ID NO: 120) is shown below.

[0161] SVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT

[0162] In embodiments, the CoV S polypeptides described herein comprise an S2 subunit having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.7%, at least 99.9%, or 100% identity to the S2 subunit of SEQ ID NO: 1 or SEQ ID NO: 2. The S2 subunit can have up to about 1, up to about 2, up to about 3, up to about 4, up to about 5, up to about 10, up to about 15, up to about 20, up to about 25, or up to about 30 amino acid deletions, insertions, or mutations compared to the amino acid sequence of the S2 subunit of SEQ ID NO: 1 or SEQ ID NO: 2. The S2 subunit may have deletions, insertions, or mutations of about 1 to about 50 amino acids, about 1 to about 5 amino acids, about 3 to about 10 amino acids, about 5 to 10 amino acids, about 8 to 12 amino acids, about 10 to 15 amino acids, about 12 to 17 amino acids, about 15 to 20 amino acids, about 18 to 23 amino acids, about 20 to 25 amino acids, about 22 to about 27 amino acids, or about 25 to 30 amino acids compared to the S2 subunit of SEQ ID NO: 1 or SEQ ID NO: 2.

[0163] In embodiments, the CoV S polypeptide contains a mutation at Lys-973 of the native CoV spike (S) polypeptide (SEQ ID NO: 2). In embodiments, Lys-973 is mutated to any native amino acid. In embodiments, Lys-973 is mutated to proline. In embodiments, Lys-973 is mutated to glycine. In embodiments, the CoV S polypeptide containing a mutation at amino acid 973 is selected from the group consisting of SEQ ID NOs: 84-89, 105-106, and 109-110.

[0164] In embodiments, the CoV S polypeptide contains a mutation at Val-974 of a native CoV spike (S) polypeptide (SEQ ID NO: 2). In embodiments, Val-974 is mutated to any native amino acid. In embodiments, Val-974 is mutated to proline. In embodiments, Val-974 is mutated to glycine. In embodiments, the CoV S polypeptide containing a mutation at amino acid 974 is selected from the group consisting of SEQ ID NOs: 84-89, 105-106, and 109-110.

[0165] In some embodiments, the CoV S polypeptide contains mutations at Lys-973 and Val-974 of a native CoV spike (S) polypeptide (SEQ ID NO: 2). In some embodiments, Lys-973 and Val-974 are mutated to any native amino acid. In some embodiments, Lys-973 and Val-974 are mutated to proline. In some embodiments, the CoV S polypeptide containing mutations at amino acids 973 and 974 is selected from SEQ ID NOs: 84-89, 105-106, 109-110, 175, 220, and 217-228.

[0166] In embodiments, the S2 subunit can contain any combination of the modifications shown in Table 1F. [Table 7-1] [Table 7-2] [Table 7-3]

[0167] In embodiments, the immunogenic composition comprises 1, 2, 3, 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, or about 50 SARS-CoV-2 S glycoproteins (including all values ​​and ranges therebetween).

[0168] Several examples of SARS-CoV-2 S glycoproteins are described in the following patent documents, each of which is incorporated by reference in its entirety for all purposes: WO 2021 / 015220, WO 2022 / 020974, WO 2022 / 235662, and WO 2023 / 102448.

[0169] Nanoparticles containing viral glycoproteins In some embodiments, the immunogenic compositions and vaccine compositions described herein comprise nanoparticles. The nanoparticles of the present disclosure are non-naturally occurring products, and their components do not occur together in nature. In some embodiments, the nanoparticles are surfactant-core nanoparticles comprising a viral glycoprotein and a non-ionic surfactant. In some embodiments, provided herein are compositions comprising surfactant-core nanoparticles comprising an RSV F glycoprotein and a non-ionic surfactant. In some embodiments, provided herein are compositions comprising surfactant-core nanoparticles comprising an HA glycoprotein and a non-ionic surfactant. In some embodiments, provided herein are compositions comprising surfactant-core nanoparticles comprising a CoV S glycoprotein and a non-ionic surfactant.

[0170] In some embodiments, a single nanoparticle comprises one or more different viral glycoproteins. In some embodiments, provided herein are surfactant core nanoparticles comprising the RSV F glycoprotein and the HA glycoprotein. In some embodiments, provided herein are surfactant core nanoparticles comprising the RSV F glycoprotein and the CoV S glycoprotein. In some embodiments, provided herein are surfactant core nanoparticles comprising the RSV F glycoprotein, the HA glycoprotein, and the CoV S glycoprotein.

[0171] In embodiments, the surfactant-core nanoparticles comprise a viral glycoprotein having a transmembrane domain, wherein the viral glycoprotein is anchored to the surfactant core via the transmembrane domain of the viral glycoprotein, hi embodiments, the viral glycoprotein forms a trimer.

[0172] In several embodiments, surfactant-core nanoparticles are made using a surfactant exchange approach in which a first surfactant is used to isolate viral glycoproteins, and then the first surfactant is exchanged with a second surfactant to form nanoparticles.

[0173] The viral glycoproteins contained in the nanoparticles are typically produced by recombinant expression in a host cell. Standard recombinant techniques can be used. In embodiments, the viral glycoproteins are expressed in insect host cells using a baculovirus system. In embodiments, 1 to 50 viral glycoproteins are co-expressed in the host cell. In embodiments, the baculovirus is a cathepsin-L knockout baculovirus or a chitinase knockout baculovirus. Optionally, the baculovirus is a double knockout of both cathepsin-L and chitinase. High levels of expression can be obtained in insect cell expression systems. Non-limiting examples of insect cells are Spodoptera frugiperda (Sf) cells, e.g., Sf9, Sf21, Trichoplusia ni cells, e.g., High Five cells, and Drosophila S2 cells. In embodiments, the viral glycoproteins described herein are produced in any suitable host cell. In embodiments, the host cell is an insect cell. In embodiments, the insect cell is an Sf9 cell.

[0174] Typical transfection and cell growth methods can be used to culture the cells. A vector, such as a vector containing a polynucleotide encoding a fusion protein, can be transfected into a host cell according to methods well known in the art. For example, introducing a nucleic acid into a eukaryotic cell can be achieved by calcium phosphate co-precipitation, electroporation, microinjection, lipofection, and transfection using polyamine transfection reagents. In one embodiment, the vector is a recombinant baculovirus.

[0175] Methods for growing host cells include, but are not limited to, batch, batch-fed, continuous, and perfusion cell culture techniques. Cell culture refers to the growth and propagation of cells in a bioreactor (fermentation chamber), where cells grow and express proteins (e.g., recombinant proteins) for purification and isolation. Typically, cell culture is carried out in a bioreactor under sterile conditions and controlled temperature and atmospheric conditions. A bioreactor is a chamber used to culture cells, where environmental conditions such as temperature, atmosphere, agitation, and / or pH can be monitored. In one embodiment, the bioreactor is a stainless steel chamber. In another embodiment, the bioreactor is a sterilized plastic bag (e.g., Cellbag®, Wave Biotech, Bridgewater, NJ). In another embodiment, the sterilized plastic bag is approximately 50 L to 3500 L.

[0176] After host cell growth, proteins can be recovered from the host cells using detergents and purification protocols. In some embodiments, multiple viral glycoproteins are purified simultaneously. In some embodiments, host cells expressing multiple viral glycoproteins are pooled together. After growing the host cells for 48-96 hours, the cells are isolated from the medium and a detergent-containing solution is added to solubilize the cell membrane and release the proteins into a detergent extract. Triton® X-100 and TERGITOL® nonylphenol ethoxylate, also known as NP-9, are preferred detergents for extraction. The detergent can be added to a final concentration of about 0.1% to about 1.0%. For example, concentrations can be about 0.1%, about 0.2%, about 0.3%, about 0.5%, about 0.7%, about 0.8%, or about 1.0%. The range can be about 0.1% to about 0.3%. In some embodiments, the concentration is about 0.5%.

[0177] In other embodiments, various first detergents can be used to isolate proteins from host cells, such as bis(polyethylene glycol bis[imidazoylcarbonyl]), nonoxynol-9, bis(polyethylene glycol bis[imidazoylcarbonyl]), BRIJ® polyethylene glycol dodecyl ether 35, BRIJ® polyethylene glycol (3) cetyl ether 56, BRIJ® alcohol ethoxylate 72, BRIJ® polyoxyl 2 stearyl ether 76, BRIJ® polyethylene glycol monooleyl ether 92V, BRIJ® polyoxyethylene (10) oleyl ether 97, BRIJ® polyethylene glycol hexadecyl ether 58P, and CREMOPHOR® EL macrogol glycerol ricinoleate. , Decaethylene Glycol Monododecyl Ether, N-Decanoyl-N-Methylglucamine, n-Decyl Alpha-D Glucopyranoside, Decyl Beta-D-Maltopyranoside, n-Dodecanoyl-N-Methylglucamide, n-Dodecyl Alpha-D-Maltoside, n-Dodecyl Beta-D-Maltoside, n-Dodecyl Beta-D-Maltoside, Heptaethylene Glycol Monodecyl Ether, Heptaethylene Glycol Monododecyl Ether, Heptaethylene Glycol Monotetradecyl Ether, n-Hexadecyl Beta-D-Maltoside, Hexaethylene Glycol Monododecyl Ether, Hexaethylene Glycol Monohexadecyl Ether, Hexaethylene Glycol Monooctadecyl Ether, Hexaethylene Glycol Monotetradecyl Ether, Igepal CA-630, Igepal CA-630, methyl-6-0-(N-heptylcarbamoyl)-alpha-D-glucopyranoside, nonaethylene glycol monododecyl ether, N-nonanoyl-N-methylglucamine, N-nonanoyl N-methylglucamine, octaethylene glycol monodecyl ether, octaethylene glycol monododecyl ether, octaethylene glycol monohexadecyl ether, octaethylene glycol monooctadecyl ether, octaethylene glycol monotetradecyl ether, octyl-beta-D-glucopyranoside,Pentaethylene glycol monodecyl ether, pentaethylene glycol monododecyl ether, pentaethylene glycol monohexadecyl ether, pentaethylene glycol monohexyl ether, pentaethylene glycol monooctadecyl ether, pentaethylene glycol monooctyl ether, polyethylene glycol diglycidyl ether, polyethylene glycol ether W-1, polyoxyethylene 10 tridecyl ether, polyoxyethylene 100 stearate, polyoxyethylene 20 isohexadecyl ether, polyoxyethylene 20 oleyl ether, polyoxyethylene 40 stearate, polyoxyethylene 50 stearate, polyoxyethylene 8 stearate, polyoxyethylene bis(imidazolylcarbonyl), polyoxyethylene 25 propylene glycol stearate, Quillaja bark saponin, SPAN® 20 sorbitan laurate, SPAN® 40 sorbitan monopalmitate, SPAN® 60 sorbitan stearate, SPAN® 65 sorbitan tristearate, SPAN (R) 80 Sorbitan Monooleate, SPAN® 85 Sorbitan Trioleate, TERGITOL® Secondary Alcohol Ethoxylate (Type 15-S-12), TERGITOL® Secondary Alcohol Ethoxylate (Type 15-S-30), TERGITOL® Secondary Alcohol Ethoxylate (Type 15-S-5), TERGITOL® Secondary Alcohol Ethoxylate (Type 15-S-7), TERGITOL® Secondary Alcohol Ethoxylate (Type 1 5-S-9), TERGITOL® Nonylphenol Ethoxylate (Type NP-10), TERGITOL® Nonylphenol Ethoxylate (Type NP-4), TERGITOL® Nonylphenol Ethoxylate (Type NP-40), TERGITOL® Nonylphenol Ethoxylate (Type NP-7), TERGITOL® Nonylphenol Ethoxylate (Type NP-9), TERGITOL® Branched Secondary Alcohol Ethoxylate (Type TMN-10),The copolymer may be TERGITOL® branched secondary alcohol ethoxylate (type TMN-6), TRITON® X-100 polyethylene glycol tert-octylphenyl ether, or a combination thereof.

[0178] The nanoparticles can then be isolated from the cellular debris using centrifugation. In some embodiments, gradient centrifugation, such as using cesium chloride, sucrose, and iodixanol, can be used. Other techniques, such as standard purification techniques including ion exchange chromatography, affinity chromatography, and gel filtration chromatography, can alternatively or additionally be used.

[0179] For example, the first column can be an ion exchange chromatography resin such as FRACTOGEL® EMD methacrylate-based polymer beads TMAE (EMD Millipore), the second column can be a lentil (Lens culinaris) lectin affinity resin, and the third column can be a cation exchange column such as FRACTOGEL® EMD methacrylate-based polymer beads SO3 (EMD Millipore) resin. In other embodiments, the cation exchange column can be an MMC column or a Nuvia C Prime column (Bio-Rad Laboratories, Inc.). Preferably, the methods disclosed herein do not use detergent extraction columns, e.g., hydrophobic interaction columns. Such columns are often used to remove detergents during purification, which can adversely affect the methods disclosed herein.

[0180] To form surfactant-core nanoparticles, the first surfactant used to extract proteins from host cells is substantially replaced with a second surfactant to obtain the nanoparticle structure. In some embodiments, the first surfactant is NP-9. Typically, the nanoparticles contain no detectable NP-9 as measured by HPLC. The second surfactant is typically selected from the group consisting of PS20, PS40, PS60, PS65, and PS80. In some embodiments, the second surfactant is PS80.

[0181] In certain embodiments, detergent exchange is performed using affinity chromatography, which binds glycoproteins via their carbohydrate moieties. For example, affinity chromatography can use a legume lectin column. Legume lectins are proteins originally identified in plants and found to interact specifically and reversibly with carbohydrate residues. See, for example, Sharon and Lis, "Legume lectins—a large family of homologous proteins," FASEB J. 1990 Nov;4(14):3198-208; Liener, "The Lectins: Properties, Functions, and Applications in Biology and Medicine," Elsevier, 2012. Suitable lectins include concanavalin A (con A), pea lectin, sainfoin lectin, and lentil lectin. Lentil lectin is a preferred column for detergent exchange due to its binding properties. Lectin columns are commercially available, e.g., Capto lentil lectin is available from GE Healthcare. In certain embodiments, the lentil lectin column may use recombinant lectins. At the molecular level, it is believed that carbohydrate moieties bind to the lentil lectin, freeing amino acids from the protein to conjugate to the surfactant, resulting in the formation of a surfactant core that results in nanoparticles with multiple copies of the antigen, e.g., glycoprotein oligomers that may be dimers, trimers, or tetramers, immobilized on the surfactant. In some embodiments, the viral glycoprotein forms trimers. In some embodiments, the viral glycoprotein trimers are immobilized on the surfactant. In some embodiments, each viral glycoprotein nanoparticle contains at least one trimer associated with a non-ionic core.

[0182] Detergents can be present at up to about 0.1% (w / v) during early purification steps when incubated with proteins to form nanoparticles during detergent exchange; this amount can be reduced to achieve final nanoparticles with optimal stability. For example, nonionic detergents (e.g., PS80) can be present at about 0.005% (v / v) to about 0.1% (v / v), e.g., about 0.005% (v / v), about 0.006% (v / v), about 0.007% (v / v), about 0.008% (v / v), about 0.009% (v / v), about 0.01% (v / v), about 0.015% (v / v), about 0.02% (v / v), about 0.025% (v / v), about 0.03% (v / v), or about 0.04% (v / v). The nanoparticles may contain about 0.035% (v / v), about 0.04% (v / v), about 0.045% (v / v), about 0.05% (v / v), about 0.055% (v / v), about 0.06% (v / v), about 0.065% (v / v), about 0.07% (v / v), about 0.075% (v / v), about 0.08% (v / v), about 0.085% (v / v), about 0.09% (v / v), about 0.095% (v / v), or about 0.1% (v / v) PS80. In some embodiments, the nanoparticles contain about 0.03% to about 0.05% PS80. In some embodiments, the nanoparticles contain about 0.01% (v / v) PS80.

[0183] In some embodiments, the purified viral glycoprotein is dialyzed. In some embodiments, dialysis is performed after purification. In some embodiments, the viral glycoprotein is dialyzed in a solution comprising sodium phosphate, NaCl, and PS80. In some embodiments, the sodium phosphate-containing dialysis solution contains about 5 mM to about 100 mM sodium phosphate, e.g., about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, about 80 mM, about 85 mM, about 90 mM, about 95 mM, or about 100 mM sodium phosphate. In some embodiments, the pH of the sodium phosphate solution is about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, or about 7.5. In some embodiments, the sodium chloride dialysis solution is about 50 mM NaCl to about 750 mM NaCl, e.g., about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, about 100 mM, about 110 mM, about 120 mM, about 130 mM, about 140 mM, about 150 mM, about 160 mM, about 170 mM, about 180 mM, about 190 mM, about 200 mM, about 210 mM, about 220 mM, about 230 mM, about 240 mM, about 250 mM, about 260 mM, about 270 mM, about 280 mM, about 290 mM, about 300 mM, about 310 mM, about 320 mM, about 330 mM, about 340 mM, about 350 mM, about 360 mM, about 370 mM, about 380 mM, about 390 mM, about 400 mM, about 410 mM, about 420 mM, about 430 mM, about 440 mM, about 450 mM, about 460 mM, about 470 mM, about 480 mM, about 490 mM, about 500 mM, about 510 mM, about 520 mM, about 530 mM, about 540 mM, about 550 mM, about 560 mM, about 570 mM, about 580 mM, about 590 mM, about 600 mM, about 610 mM, about 6 20mM, about 230mM, about 240mM, about 250mM, about 260mM, about 270mM, about 280mM, about 290mM, about 300mM, about 310mM, about 320mM, about 330mM, about 340mM, about 350mM, about 360mM, about 370mM, about 380mM, about 390mM, about 400 mM, about 410 mM, about 420 mM, about 430 mM, about 440 mM, about 450 mM, about 460 mM, about 470 mM, about 480 mM, about 490 mM, about 500 mM, about 510 mM, about 520 mM, about 530 mM, about 540 mM, about 550 mM, about 560 mM, about 570 mM, About 580 mM, about 590 mM, about 600 mM, about 610 mM, about 620 mM, about 630 mM, about 640 mM, about 650 mM, about 660 mM, about 670 mM, about 680 mM, about 690, about 700 mM, about 710 mM, about 720 mM, about 730 mM, about 740 mM, or about 750 mM NaCl.In some embodiments, the dialysis solution containing PS80 is at about 0.005% (v / v), about 0.006% (v / v), about 0.007% (v / v), about 0.008% (v / v), about 0.009% (v / v), about 0.01% (v / v), about 0.015% (v / v), about 0.02% (v / v), about 0.025% (v / v), about 0.03% (v / v), about 0.035% (v / v), about 0.0 4% (v / v), about 0.045% (v / v), about 0.05% (v / v), about 0.055% (v / v), about 0.06% (v / v), about 0.065% (v / v), about 0.07% (v / v), about 0.075% (v / v), about 0.08% (v / v), about 0.085% (v / v), about 0.09% (v / v), about 0.095% (v / v), or about 0.1% (v / v) PS 80. In some embodiments, the dialysis solution comprises about 25 mM sodium phosphate (pH 7.2), about 300 mM NaCl, and about 0.01% (v / v) PS 80.

[0184] In embodiments, the pharmaceutically acceptable buffer comprises 10 mM sodium phosphate, 150 mM NaCl, 100 mM arginine, 5% trehalose, and 0.03% PS80 at a pH of 7.5. In embodiments, the pharmaceutically acceptable buffer comprises 25 mM sodium phosphate, 300 mM NaCl, and 0.03% PS80 at a pH of 7.2. In embodiments, the pharmaceutically acceptable buffer comprises 25 mM sodium phosphate, 600 mM NaCl, and 0.01% PS80 at a pH of 6.8.

[0185] Detergent exchange can be performed with proteins purified as discussed above, frozen for storage, and then thawed for detergent exchange.

[0186] The stability of the compositions disclosed herein can be measured by various means. In one approach, peptide maps can be prepared to determine the integrity of antigenic proteins after various treatments designed to stress nanoparticles by mimicking harsh storage conditions. Thus, a measure of stability is the relative abundance of antigenic peptides in stressed samples compared to control samples. For example, the stability of nanoparticles containing viral glycoproteins can be assessed by exposing them to various pHs, proteases, salts, oxidizing agents including, but not limited to, hydrogen peroxide, various temperatures, freeze / thaw cycles, and agitation. The location of the glycoprotein anchored to the surfactant core is believed to enhance stability by reducing undesirable interactions. For example, improved protection against protease-based degradation may be achieved by immobilizing the glycoprotein to the core at the molar ratios disclosed herein, resulting in a shielding effect that prevents protease access. Stability may also be measured by monitoring protein integrity.

[0187] In several embodiments, provided herein are immunogenic and vaccine compositions containing nanoparticles comprising the RSV F glycoprotein, the HA glycoprotein, the SARS-CoV-2 S glycoprotein, or a combination thereof.

[0188] In some embodiments, the mature RSV F glycoprotein is used to produce vaccines comprising RSV F glycoprotein nanoparticles. In some embodiments, the nanoparticles of the present disclosure comprise the RSV F glycoprotein described herein. In some embodiments, the immunogenic and vaccine compositions provided herein comprise nanoparticles comprising a CoV S glycoprotein, an influenza HA glycoprotein, a RSV F glycoprotein, or any combination thereof.

[0189] In some embodiments, the nanoparticles of the present disclosure comprise a viral glycoprotein (e.g., RSV F, CoV S, influenza HA glycoprotein, or a combination thereof) associated with a surfactant core. The presence of the surfactant promotes nanoparticle formation by forming a core that organizes and presents antigens. In some embodiments, the nanoparticles may contain viral glycoproteins assembled into multi-oligomeric glycoprotein-surfactant (e.g., PS80) nanoparticles, in which the head region protrudes outward and the hydrophobic region and surfactant form a central core surrounded by the glycoprotein. In some embodiments, the viral glycoprotein essentially contains or is configured to contain a transmembrane domain that promotes association of the protein with the surfactant core. In some embodiments, the viral glycoprotein contains a head domain. Primarily, the transmembrane domain of the viral glycoprotein trimer associates with the surfactant, although other portions of the polypeptide may also interact. Advantageously, the nanoparticles have improved resistance to environmental stress, resulting in enhanced stability and / or improved presentation to the immune system due to the organization of multiple copies of the protein around the surfactant.

[0190] In some embodiments, the surfactant core is a non-ionic surfactant core. In some embodiments, the RSV F glycoprotein is associated with the non-ionic surfactant core. In some embodiments, the surfactant is selected from the group consisting of polysorbate-20 (PS20), polysorbate-40 (PS40), polysorbate-60 (PS60), polysorbate-65 (PS65), and polysorbate-80 (PS80).

[0191] In embodiments, the surfactant is PS80.

[0192] In some embodiments, the RSV F glycoprotein forms trimers. In some embodiments, the RSV F glycoprotein nanoparticles are composed of multiple polypeptide trimers surrounding a non-ionic surfactant core. In some embodiments, the nanoparticles contain at least about one trimer or more. In some embodiments, the nanoparticles contain at least about 5 trimers to about 30 trimers of spike protein. In some embodiments, each nanoparticle may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 15, 20, 25, or 30 trimers (including all values ​​and ranges therebetween). The compositions disclosed herein may contain nanoparticles with different numbers of trimers. For example, the compositions may contain nanoparticles with a number of trimers ranging from 2 to 9, and in some embodiments, the nanoparticles in the composition may contain 2 to 6 trimers. In some embodiments, the composition contains a heterogeneous population of nanoparticles having 2-6 trimers per nanoparticle or 2-9 trimers per nanoparticle. In some embodiments, the composition may contain a substantially homogeneous population of nanoparticles. For example, the population may contain about 95% of the nanoparticles having 5 trimers.

[0193] The nanoparticles disclosed herein vary in particle size. In embodiments, the nanoparticles disclosed herein have a particle size, based on the Z-average size, ranging from about 20 nm to about 60 nm, about 20 nm to about 50 nm, about 20 nm to about 45 nm, about 20 nm to about 35 nm, about 20 nm to about 30 nm, about 25 nm to about 35 nm, about 25 nm to about 45 nm, about 30 nm to about 120 nm, about 30 nm to about 80 nm, about 30 nm to about 60 nm, about 30 nm to about 65 nm, or about 30 nm to about 50 nm. Particle size (Z-average) is measured by dynamic light scattering (DLS) using a Zetasizer NanoZS (Malvern, UK) unless otherwise specified.

[0194] Several nanoparticle species can be included in the vaccine compositions disclosed herein. In some embodiments, the nanoparticle species are in the form of anisotropic rods, which can be dimeric or monomeric. In other embodiments, the nanoparticle species are spherical oligomers. In still other embodiments, the nanoparticles can be described as intermediate nanoparticles with sedimentation properties intermediate between the first two species. The formation of nanoparticle species can be regulated by controlling the surfactant and protein concentrations during the fabrication process. The nanoparticle species can be determined by measuring the sedimentation coefficient.

[0195] Amounts of viral glycoproteins in immunogenic and vaccine compositions In embodiments, the compositions described herein comprise from about 1 μg to about 400 μg, 1 μg to about 350 μg, 1 μg to about 300 μg, 1 μg to about 250 μg, 1 μg to about 200 μg, 1 μg to about 150 μg, 1 μg to about 100 μg, 1 μg to about 50 μg, or 1 μg to about 25 μg of each viral glycoprotein. In embodiments, the compositions comprise about 1 μg, about 2 μg, about 3 μg, about 4 μg, about 5 μg, about 6 μg, about 7 μg, about 8 μg, about 9 μg, about 10 μg, about 11 μg, about 12 μg, about 13 μg, about 14 μg, about 15 μg, about 16 μg, about 17 μg, about 18 μg, about 19 μg, about 20 μg, about 21 μg, about 22 μg, about 23 μg, about 24 μg, about 25 μg, about 26 μg, about 27 μg, about 28 μg, about 29 μg, about 30 μg, about 31 μg, about 32 μg, about 33 μg, about 34 μg, about 35 μg, about 36 μg, about 37 μg, about 38 μg, about 39 μg, about 40 μg, about 41 μg, about 42 μg, about 43 μg, about 44 μg, about 45 μg, about 46 μg, about 47 μg, about 48 μg, about 49 μg, about 50 μg, about 51 μg, about 52 μg, about 53 μg, about 54 μg, about 55 μg, about 56 μg, about 57 μg, about 58 μg, about 59 μg, about 60 μg, about 61 μg, about 62 μg, about 63 μg, about 64 μg, about 65 μg, about 66 μg, about 67 μg, about 68 μg, about 69 μg, about 70 μg, about 71 μg, about 72 μg, about 73 μg, about 7 g, about 36μg, about 37μg, about 38μg, about 39μg, about 40μg, about 41μg, about 42μg, about 43μg, about 44μg, about 45μg, about 46μg, about 47μg, about 48μg, about 49μg, about 50μg, about 51μg, about 52μg, about 53 54μg, 55μg, 56μg, 57μg, 58μg, 59μg, 60μg, 61μg, 62μg, 63μg, 64μg, 65μg, 66μg, 67μg, 68μg, 69μg, 70μg, 71 μg, approximately 72 μg, approximately 73 μg, approximately 74 μg, approximately 75 μg, approximately 76 μg, approximately 77 μg, approximately 78 μg, approximately 79 μg, approximately 80 μg, approximately 81 μg, approximately 82 μg, approximately 83 μg, approximately 84 μg, approximately 85 μg, approximately 86 μg, approximately 87 μg, approximately 88 μg, approximately 8 9μg, about 90μg, about 91μg, about 92μg, about 93μg, about 94μg, about 95μg, about 96μg, about 97μg, about 98μg, about 99μg, about 100μg, about 101μg, about 102μg, about 103μg, about 104μg, about 105μg, about 1 06μg, about 107μg, about 108μg, about 109μg, about 110μg, about 111μg, about 112μg, about 113μg, about 114μg, about 115μg, about 116μg, about 117μg, about 118μg, about 119μg, about 120μg, about 121 μg, approximately 122 μg, approximately 123 μg, approximately 124 μg, approximately 125 μg, approximately 126 μg, approximately 127 μg, approximately 128 μg, approximately 129 μg, approximately 130 μg, approximately 131 μg, approximately 132 μg, approximately 133 μg, approximately 134 μg, approximately 135 μg, approximately 136 μg,Approximately 137 μg, approximately 138 μg, approximately 139 μg, approximately 140 μg, approximately 141 μg, approximately 142 μg, approximately 143 μg, approximately 144 μg, approximately 145 μg, approximately 146 μg, approximately 147 μg, approximately 148 μg, approximately 149 μg, approximately 150 μg, approximately 151 μg, approximately 152 μg, approximately 153 μg, approximately 154 μg Approximately 155 μg, approximately 156 μg, approximately 157 μg, approximately 158 μg, approximately 159 μg, approximately 160 μg, approximately 161 μg, approximately 162 μg, approximately 163 μg, approximately 164 μg, approximately 165 μg, approximately 166 μg, approximately 167 μg, approximately 168 μg, approximately 169 μg, approximately 170 μg, approximately 171 μg, approximately 172 μg g, approximately 173μg, approximately 174μg, approximately 175μg, approximately 176μg, approximately 177μg, approximately 178μg, approximately 179μg, approximately 180μg, approximately 181μg, approximately 182μg, approximately 183μg, approximately 184μg, approximately 185μg, approximately 186μg, approximately 187μg, approximately 188μg, approximately 189μg, approximately 190 μg, approximately 191μg, approximately 192μg, approximately 193μg, approximately 194μg, approximately 195μg, approximately 196μg, approximately 197μg, approximately 198μg, approximately 199μg, approximately 200μg, approximately 201μg, approximately 202μg, approximately 203μg, approximately 204μg, approximately 205μg, approximately 206μg, approximately 207μg, approximately 20 8μg, approximately 209μg, approximately 210μg, approximately 211μg, approximately 212μg, approximately 213μg, approximately 214μg, approximately 215μg, approximately 216μg, approximately 217μg, approximately 218μg, approximately 219μg, approximately 220μg, approximately 221μg, approximately 222μg, approximately 223μg, approximately 224μg, approximately 225μg, approximately 2 26μg, approximately 227μg, approximately 228μg, approximately 229μg, approximately 230μg, approximately 231μg, approximately 232μg, approximately 233μg, approximately 234μg, approximately 235μg, approximately 236μg, approximately 237μg, approximately 238μg, approximately 239μg, approximately 240μg, approximately 241μg, approximately 242μg, approximately 243μg, approximately 244μg, approximately 245μg, approximately 246μg, approximately 247μg, approximately 248μg, approximately 249μg, approximately 250μg, approximately 251μg, approximately 252μg, approximately 253μg, approximately 254μg, approximately 255μg, approximately 256μg, approximately 257μg, approximately 258μg, approximately 259μg, approximately 260μg, approximately 261μg Approximately 262 μg, approximately 263 μg, approximately 264 μg, approximately 265 μg, approximately 266 μg, approximately 267 μg, approximately 268 μg, approximately 269 μg, approximately 270 μg, approximately 271 μg, approximately 272 μg, approximately 273 μg, approximately 274 μg, approximately 275 μg, approximately 276 μg, approximately 277 μg, approximately 278 μg, approximately 279 μgAbout 280μg, about 281μg, about 282μg, about 283μg, about 284μg, about 285μg, about 286μg, about 287μg, about 288μg, about 289μg, about 290μg, about 291μg, about 292μg, about 293μg, about 294μg, about 29 5μg, about 296μg, about 297μg, about 298μg, about 299μg, about 300μg, about 301μg, about 302μg, about 303μg, about 304μg, about 305μg, about 306μg, about 307μg, about 308μg, about 309μg, about 310μg , about 311μg, about 312μg, about 313μg, about 314μg, about 315μg, about 316μg, about 317μg, about 318μg, about 319μg, about 320μg, about 321μg, about 322μg, about 323μg, about 324μg, about 325μg, about 3 26μg, about 327μg, about 328μg, about 329μg, about 330μg, about 331μg, about 332μg, about 333μg, about 334μg, about 335μg, about 336μg, about 337μg, about 338μg, about 339μg, about 340μg, about 341μg g, approx. 342 μg, approx. 343 μg, approx. 344 μg, approx. 345 μg, approx. 346 μg, approx. 347 μg, approx. 348 μg, approx. 349 μg, approx. 357μg, about 358μg, about 359μg, about 360μg, about 361μg, about 362μg, about 363μg, about 364μg, about 365μg, about 366μg, about 367μg, about 368μg, about 369μg, about 370μg, about 371μg, about 372

[0033] The viral glycoproteins may be about 373 μg, about 374 μg, about 375 μg, about 376 μg, about 377 μg, about 378 μg, about 379 μg, about 380 μg, about 381 μg, about 382 μg, about 383 μg, about 384 μg, about 385 μg, about 386 μg, about 387 μg, about 388 μg, about 389 μg, about 390 μg, about 391 μg, about 392 μg, about 393 μg, about 394 μg, about 395 μg, about 396 μg, about 397 μg, about 398 μg, about 399 μg, or about 400 μg.

[0196] In some embodiments, the immunogenic composition comprises about 1 μg of each viral glycoprotein. In some embodiments, the immunogenic composition comprises about 5 μg of each viral glycoprotein. In some embodiments, the immunogenic composition comprises about 25 μg of each viral glycoprotein. In some embodiments, the immunogenic composition comprises about 60 μg of each viral glycoprotein. In some embodiments, the immunogenic composition comprises about 240 μg of each viral glycoprotein.

[0197] In some embodiments, the immunogenic compositions described herein comprise between about 1 μg and about 300 μg of each RSV F glycoprotein. In some embodiments, the immunogenic compositions described herein comprise between about 5 μg and about 60 μg of each RSV F glycoprotein. In some embodiments, the immunogenic compositions described herein comprise between about 24 μg and about 40 μg of each RSV F glycoprotein. In some embodiments, the immunogenic compositions described herein comprise between about 30 μg and about 40 μg of each RSV F glycoprotein. In some embodiments, the immunogenic compositions described herein comprise between about 30 μg and about 60 μg of each RSV F glycoprotein. In some embodiments, the immunogenic compositions described herein comprise between about 33 μg and about 39 μg of each RSV F glycoprotein. In some embodiments, the immunogenic compositions described herein comprise between about 20 μg and about 240 μg of each RSV F glycoprotein. In some embodiments, the immunogenic compositions described herein comprise between about 30 μg and about 60 μg of each RSV F glycoprotein. In some embodiments, the immunogenic compositions described herein contain between about 24 μg and about 40 μg of each RSV F glycoprotein. In embodiments, the immunogenic compositions comprise a concentration of about 1 μg, about 2 μg, about 3 μg, about 4 μg, about 5 μg, about 6 μg, about 7 μg, about 8 μg, about 9 μg, about 10 μg, about 11 μg, about 12 μg, about 13 μg, about 14 μg, about 15 μg, about 16 μg, about 17 μg, about 18 μg, about 19 μg, about 20 μg, about 21 μg, about 22 μg, about 23 μg, about 24 μg, about 25 μg, about 26 μg, about 27 μg, about 28 μg, about 29 μg, about 30 μg, about 31 μg, about 32 μg, about 33 μg, about 34 μg, about 35 μg, about 36 μg, about 37 μg, about 38 μg, about 39 μg, about 40 μg, about 41 μg, about 42 μg, about 43 μg, about 44 μg, about 45 μg, about 46 μg, about 47 μg, about 48 μg, about 49 μg, about 50 μg, about 51 μg, about 52 μg, about 53 μg, about 54 μg, about 55 μg, about 56 μg, about 57 μg, about 58 μg, about 59 μg, about 60 μg, about 61 μg, about 62 μg, about 63 μg, about 64 μg, about 65 μg, about 66 μg, about 67 μg, about 68 μg, about 69 μg, about 70 μg, about 71 μg, about 72 μg, about 73 μ g, about 41μg, about 42μg, about 43μg, about 44μg, about 45μg, about 46μg, about 47μg, about 48μg, about 49μg, about 50μg, Approximately 51μg, approximately 52μg, approximately 53μg, approximately 54μg, approximately 55μg, approximately 56μg, approximately 57μg, approximately 58μg, approximately 59μg, approximately 60μg, approximately 6 1μg, about 62μg, about 63μg, about 64μg, about 65μg, about 66μg, about 67μg, about 68μg, about 69μg, about 70μg, about 71μ g, about 72μg, about 73μg, about 74μg, about 75μg, about 76μg, about 77μg, about 78μg, about 79μg, about 80μg, about 81μg,Approximately 82 μg, approximately 83 μg, approximately 84 μg, approximately 85 μg, approximately 86 μg, approximately 87 μg, approximately 88 μg, approximately 89 μg, approximately 90 μg, approximately 91 μg, approximately 92 μg, approximately 93 μg, approximately 94 μg, approximately 95 μg, approximately 96 μg, approximately 97 μg, approximately 98 μg, approximately 99 μg, approximately 100 μg, approximately 101 μg, approximately 1 0.2 μg, approximately 103 μg, approximately 104 μg, approximately 105 μg, approximately 106 μg, approximately 107 μg, approximately 108 μg, approximately 109 μg, approximately 110 μg, approximately 111 μg, approximately 112 μg, approximately 113 μg, approximately 114 μg, approximately 115 μg, approximately 116 μg, approximately 117 μg, approximately 118 μg, approximately 119 μg, approximately 120μg, approximately 121μg, approximately 122μg, approximately 123μg, approximately 124μg, approximately 125μg, approximately 126μg, approximately 127μg, approximately 128μg, approximately 129μg, approximately 130μg, approximately 131μg, approximately 132μg, approximately 133μg, approximately 134μg, approximately 135μg, approximately 136μg, approximately 137μg Approximately 138 μg, approximately 139 μg, approximately 140 μg, approximately 141 μg, approximately 142 μg, approximately 143 μg, approximately 144 μg, approximately 145 μg, approximately 146 μg, approximately 147 μg, approximately 148 μg, approximately 149 μg, approximately 150 μg, approximately 151 μg, approximately 152 μg, approximately 153 μg, approximately 154 μg, approximately 155 μg g, approximately 156 μg, approximately 157 μg, approximately 158 μg, approximately 159 μg, approximately 160 μg, approximately 161 μg, approximately 162 μg, approximately 163 μg, approximately 164 μg, approximately 165 μg, approximately 166 μg, approximately 167 μg, approximately 168 μg, approximately 169 μg, approximately 170 μg, approximately 171 μg, approximately 172 μg, approximately 17 3μg, approximately 174μg, approximately 175μg, approximately 176μg, approximately 177μg, approximately 178μg, approximately 179μg, approximately 180μg, approximately 181μg, approximately 182μg, approximately 183μg, approximately 184μg, approximately 185μg, approximately 186μg, approximately 187μg, approximately 188μg, approximately 189μg, approximately 190μg, approximately 1 91μg, approximately 192μg, approximately 193μg, approximately 194μg, approximately 195μg, approximately 196μg, approximately 197μg, approximately 198μg, approximately 199μg, approximately 200μg, approximately 201μg, approximately 202μg, approximately 203μg, approximately 204μg, approximately 205μg, approximately 206μg, approximately 207μg, approximately 208μg, approximately 209μg, approximately 210μg, approximately 211μg, approximately 212μg, approximately 213μg, approximately 214μg, approximately 215μg, approximately 216μg, approximately 217μg, approximately 218μg, approximately 219μg, approximately 220μg, approximately 221μg, approximately 222μg, approximately 223μg, approximately 224μg, approximately 225μg, approximately 226μgApproximately 227μg, approximately 228μg, approximately 229μg, approximately 230μg, approximately 231μg, approximately 232μg, approximately 233μg, approximately 234μg, approximately 235μg, approximately 2 36μg, about 237μg, about 238μg, about 239μg, about 240μg, about 241μg, about 242μg, about 243μg, about 244μg, about 245μg g, about 246μg, about 247μg, about 248μg, about 249μg, about 250μg, about 251μg, about 252μg, about 253μg, about 254μg, Approximately 255μg, approximately 256μg, approximately 257μg, approximately 258μg, approximately 259μg, approximately 260μg, approximately 261μg, approximately 262μg, approximately 263μg, approximately 26 4μg, about 265μg, about 266μg, about 267μg, about 268μg, about 269μg, about 270μg, about 271μg, about 272μg, about 273μ g, about 274μg, about 275μg, about 276μg, about 277μg, about 278μg, about 279μg, about 280μg, about 281μg, about 282μg, about In some embodiments, the immunogenic compositions described herein comprise about 283 μg, about 284 μg, about 285 μg, about 286 μg, about 287 μg, about 288 μg, about 289 μg, about 290 μg, about 291 μg, about 292 μg, about 293 μg, about 294 μg, about 295 μg, about 296 μg, about 297 μg, about 298 μg, about 299 μg, or about 300 μg of each RSV F glycoprotein (including all values ​​and ranges therebetween). In some embodiments, the immunogenic compositions described herein comprise about 60 μg of each RSV F glycoprotein. In some embodiments, the immunogenic compositions described herein comprise about 240 μg of each RSV F glycoprotein.

[0198] In some embodiments, the immunogenic compositions described herein contain between about 1 μg and about 1000 μg of total RSV F glycoprotein. In embodiments, the immunogenic compositions comprise a concentration of about 1 μg, about 2 μg, about 3 μg, about 4 μg, about 5 μg, about 6 μg, about 7 μg, about 8 μg, about 9 μg, about 10 μg, about 11 μg, about 12 μg, about 13 μg, about 14 μg, about 15 μg, about 16 μg, about 17 μg, about 18 μg, about 19 μg, about 20 μg, about 21 μg, about 22 μg, about 23 μg, about 24 μg, about 25 μg, about 26 μg, about 27 μg, about 28 μg, about 29 μg, about 30 μg, about 31 μg, about 32 μg, about 33 μg, about 34 μg, about 35 μg, about 36 μg, about 37 μg, about 38 μg, about 39 μg, about 40 μg, about 41 μg, about 42 μg, about 43 μg, about 44 μg, about 45 μg, about 46 μg, about 47 μg, about 48 μg, about 49 μg, about 50 μg, about 51 μg, about 52 μg, about 53 μg, about 54 μg, about 55 μg, about 56 μg, about 57 μg, about 58 μg, about 59 μg, about 60 μg, about 61 μg, about 62 μg, about 63 μg, about 64 μg, about 65 μg, about 66 μg, about 67 μg, about 68 μg, about 69 μg, about 70 μg, about 71 μg, about 72 μg, about 73 μ μg, approx. 39 μg, approx. 40 μg, approx. 41 μg, approx. 42 μg, approx. 43 μg, approx. 44 μg, approx. 45 μg, approx. 46 μg, approx. 47 μg, approx. 48μg, about 49μg, about 50μg, about 51μg, about 52μg, about 53μg, about 54μg, about 55μg, about 56μg, about 57μg, Approximately 58μg, approximately 59μg, approximately 60μg, approximately 61μg, approximately 62μg, approximately 63μg, approximately 64μg, approximately 65μg, approximately 66μg, approximately 67μg , about 68μg, about 69μg, about 70μg, about 71μg, about 72μg, about 73μg, about 74μg, about 75μg, about 76μg, about 77μ g, about 78μg, about 79μg, about 80μg, about 81μg, about 82μg, about 83μg, about 84μg, about 85μg, about 86μg, about 8 7μg, about 88μg, about 89μg, about 90μg, about 91μg, about 92μg, about 93μg, about 94μg, about 95μg, about 96μg, about 97μg, about 98μg, about 99μg, about 100μg, about 101μg, about 102μg, about 103μg, about 104μg, about 105μg, Approximately 106μg, approximately 107μg, approximately 108μg, approximately 109μg, approximately 110μg, approximately 111μg, approximately 112μg, approximately 113μg, approximately 11 4 μg, approximately 115 μg, approximately 116 μg, approximately 117 μg, approximately 118 μg, approximately 119 μg, approximately 120 μg, approximately 121 μg, approximately 122 μg g, approx. 123 μg, approx. 124 μg, approx. 125 μg, approx. 126 μg, approx. 127 μg, approx. 128 μg, approx. 129 μg, approx. 130 μg, approx. 131μg, about 132μg, about 133μg, about 134μg, about 135μg, about 136μg, about 137μg, about 138μg, about 139 μg, approximately 140 μg, approximately 141 μg, approximately 142 μg, approximately 143 μg, approximately 144 μg, approximately 145 μg, approximately 146 μg, approximately 147 μg,Approximately 148 μg, approximately 149 μg, approximately 150 μg, approximately 151 μg, approximately 152 μg, approximately 153 μg, approximately 154 μg, approximately 155 μg, approximately 156 μg, approximately 157 μg, approximately 158 μg, approximately 159 μg, approximately 160 μg, approximately 161 μg, approximately 162 μg, approximately 163 μg, approximately 164 μg, approximately 165 μg Approximately 166 μg, approximately 167 μg, approximately 168 μg, approximately 169 μg, approximately 170 μg, approximately 171 μg, approximately 172 μg, approximately 173 μg, approximately 174 μg, approximately 175 μg, approximately 176 μg, approximately 177 μg, approximately 178 μg, approximately 179 μg, approximately 180 μg, approximately 181 μg, approximately 182 μg, approximately 183 μg g, approximately 184μg, approximately 185μg, approximately 186μg, approximately 187μg, approximately 188μg, approximately 189μg, approximately 190μg, approximately 191μg, approximately 192μg, approximately 193μg, approximately 194μg, approximately 195μg, approximately 196μg, approximately 197μg, approximately 198μg, approximately 199μg, approximately 200μg, approximately 201 μg, approximately 202μg, approximately 203μg, approximately 204μg, approximately 205μg, approximately 206μg, approximately 207μg, approximately 208μg, approximately 209μg, approximately 210μg, approximately 211μg, approximately 212μg, approximately 213μg, approximately 214μg, approximately 215μg, approximately 216μg, approximately 217μg, approximately 218μg, approximately 21 9μg, approximately 220μg, approximately 221μg, approximately 222μg, approximately 223μg, approximately 224μg, approximately 225μg, approximately 226μg, approximately 227μg, approximately 228μg, approximately 229μg, approximately 230μg, approximately 231μg, approximately 232μg, approximately 233μg, approximately 234μg, approximately 235μg, approximately 236μg, approximately 2 37μg, approximately 238μg, approximately 239μg, approximately 240μg, approximately 241μg, approximately 242μg, approximately 243μg, approximately 244μg, approximately 245μg, approximately 246μg, approximately 247μg, approximately 248μg, approximately 249μg, approximately 250μg, approximately 251μg, approximately 252μg, approximately 253μg, approximately 254μg, approximately 255μg, approximately 256μg, approximately 257μg, approximately 258μg, approximately 259μg, approximately 260μg, approximately 261μg, approximately 262μg, approximately 263μg, approximately 264μg, approximately 265μg, approximately 266μg, approximately 267μg, approximately 268μg, approximately 269μg, approximately 270μg, approximately 271μg, approximately 272μg Approximately 273 μg, approximately 274 μg, approximately 275 μg, approximately 276 μg, approximately 277 μg, approximately 278 μg, approximately 279 μg, approximately 280 μg, approximately 281 μg, approximately 282 μg, approximately 283 μg, approximately 284 μg, approximately 285 μg, approximately 286 μg, approximately 287 μg, approximately 288 μg, approximately 289 μg, approximately 290 μgApproximately 291 μg, approximately 292 μg, approximately 293 μg, approximately 294 μg, approximately 295 μg, approximately 296 μg, approximately 297 μg, approximately 298 μg, approximately 299 μg, approximately 300 μg, approximately 301 μg, approximately 302 μg, approximately 303 μg, approximately 304 μg, approximately 305 μg, approximately 306 μg, approximately 307 μg, approximately 308 μg Approximately 309 μg, approximately 310 μg, approximately 311 μg, approximately 312 μg, approximately 313 μg, approximately 314 μg, approximately 315 μg, approximately 316 μg, approximately 317 μg, approximately 318 μg, approximately 319 μg, approximately 320 μg, approximately 321 μg, approximately 322 μg, approximately 323 μg, approximately 324 μg, approximately 325 μg, approximately 326 μg g, approximately 327 μg, approximately 328 μg, approximately 329 μg, approximately 330 μg, approximately 331 μg, approximately 332 μg, approximately 333 μg, approximately 334 μg, approximately 335 μg, approximately 336 μg, approximately 337 μg, approximately 338 μg, approximately 339 μg, approximately 340 μg, approximately 341 μg, approximately 342 μg, approximately 343 μg, approximately 344 μg μg, approximately 345μg, approximately 346μg, approximately 347μg, approximately 348μg, approximately 349μg, approximately 350μg, approximately 351μg, approximately 352μg, approximately 353μg, approximately 354μg, approximately 355μg, approximately 356μg, approximately 357μg, approximately 358μg, approximately 359μg, approximately 360μg, approximately 361μg, approximately 36 2μg, approximately 363μg, approximately 364μg, approximately 365μg, approximately 366μg, approximately 367μg, approximately 368μg, approximately 369μg, approximately 370μg, approximately 371μg, approximately 372μg, approximately 373μg, approximately 374μg, approximately 375μg, approximately 376μg, approximately 377μg, approximately 378μg, approximately 379μg, approximately 3 80μg, approximately 381μg, approximately 382μg, approximately 383μg, approximately 384μg, approximately 385μg, approximately 386μg, approximately 387μg, approximately 388μg, approximately 389μg, approximately 390μg, approximately 391μg, approximately 392μg, approximately 393μg, approximately 394μg, approximately 395μg, approximately 396μg, approximately 397μg, approximately 398μg, approximately 399μg, approximately 400μg, approximately 401μg, approximately 402μg, approximately 403μg, approximately 404μg, approximately 405μg, approximately 406μg, approximately 407μg, approximately 408μg, approximately 409μg, approximately 410μg, approximately 411μg, approximately 412μg, approximately 413μg, approximately 414μg, approximately 415μg Approximately 416 μg, approximately 417 μg, approximately 418 μg, approximately 419 μg, approximately 420 μg, approximately 421 μg, approximately 422 μg, approximately 423 μg, approximately 424 μg, approximately 425 μg, approximately 426 μg, approximately 427 μg, approximately 428 μg, approximately 429 μg, approximately 430 μg, approximately 431 μg, approximately 432 μg, approximately 433 μgApproximately 434 μg, approximately 435 μg, approximately 436 μg, approximately 437 μg, approximately 438 μg, approximately 439 μg, approximately 440 μg, approximately 441 μg, approximately 442 μg, approximately 443 μg, approximately 444 μg, approximately 445 μg, approximately 446 μg, approximately 447 μg, approximately 448 μg, approximately 449 μg, approximately 450 μg, approximately 451 μg Approximately 452 μg, approximately 453 μg, approximately 454 μg, approximately 455 μg, approximately 456 μg, approximately 457 μg, approximately 458 μg, approximately 459 μg, approximately 460 μg, approximately 461 μg, approximately 462 μg, approximately 463 μg, approximately 464 μg, approximately 465 μg, approximately 466 μg, approximately 467 μg, approximately 468 μg, approximately 469 μg g, approximately 470μg, approximately 471μg, approximately 472μg, approximately 473μg, approximately 474μg, approximately 475μg, approximately 476μg, approximately 477μg, approximately 478μg, approximately 479μg, approximately 480μg, approximately 481μg, approximately 482μg, approximately 483μg, approximately 484μg, approximately 485μg, approximately 486μg, approximately 487 μg, approximately 488μg, approximately 489μg, approximately 490μg, approximately 491μg, approximately 492μg, approximately 493μg, approximately 494μg, approximately 495μg, approximately 496μg, approximately 497μg, approximately 498μg, approximately 499μg, approximately 500μg, approximately 501μg, approximately 502μg, approximately 503μg, approximately 504μg, approximately 50 5μg, approximately 506μg, approximately 507μg, approximately 508μg, approximately 509μg, approximately 510μg, approximately 511μg, approximately 512μg, approximately 513μg, approximately 514μg, approximately 515μg, approximately 516μg, approximately 517μg, approximately 518μg, approximately 519μg, approximately 520μg, approximately 521μg, approximately 522μg, approximately 5 23μg, approximately 524μg, approximately 525μg, approximately 526μg, approximately 527μg, approximately 528μg, approximately 529μg, approximately 530μg, approximately 531μg, approximately 532μg, approximately 533μg, approximately 534μg, approximately 535μg, approximately 536μg, approximately 537μg, approximately 538μg, approximately 539μg, approximately 540μg, approximately 541μg, approximately 542μg, approximately 543μg, approximately 544μg, approximately 545μg, approximately 546μg, approximately 547μg, approximately 548μg, approximately 549μg, approximately 550μg, approximately 551μg, approximately 552μg, approximately 553μg, approximately 554μg, approximately 555μg, approximately 556μg, approximately 557μg, approximately 558μg Approximately 559 μg, approximately 560 μg, approximately 561 μg, approximately 562 μg, approximately 563 μg, approximately 564 μg, approximately 565 μg, approximately 566 μg, approximately 567 μg, approximately 568 μg, approximately 569 μg, approximately 570 μg, approximately 571 μg, approximately 572 μg, approximately 573 μg, approximately 574 μg, approximately 575 μg, approximately 576 μgApproximately 577 μg, approximately 578 μg, approximately 579 μg, approximately 580 μg, approximately 581 μg, approximately 582 μg, approximately 583 μg, approximately 584 μg, approximately 585 μg, approximately 586 μg, approximately 587 μg, approximately 588 μg, approximately 589 μg, approximately 590 μg, approximately 591 μg, approximately 592 μg, approximately 593 μg, approximately 594 μg Approximately 595 μg, approximately 596 μg, approximately 597 μg, approximately 598 μg, approximately 599 μg, approximately 600 μg, approximately 601 μg, approximately 602 μg, approximately 603 μg, approximately 604 μg, approximately 605 μg, approximately 606 μg, approximately 607 μg, approximately 608 μg, approximately 609 μg, approximately 610 μg, approximately 611 μg, approximately 612 μg g, approximately 613 μg, approximately 614 μg, approximately 615 μg, approximately 616 μg, approximately 617 μg, approximately 618 μg, approximately 619 μg, approximately 620 μg, approximately 621 μg, approximately 622 μg, approximately 623 μg, approximately 624 μg, approximately 625 μg, approximately 626 μg, approximately 627 μg, approximately 628 μg, approximately 629 μg, approximately 630 μg μg, approximately 631μg, approximately 632μg, approximately 633μg, approximately 634μg, approximately 635μg, approximately 636μg, approximately 637μg, approximately 638μg, approximately 639μg, approximately 640μg, approximately 641μg, approximately 642μg, approximately 643μg, approximately 644μg, approximately 645μg, approximately 646μg, approximately 647μg, approximately 64 8μg, approximately 649μg, approximately 650μg, approximately 651μg, approximately 652μg, approximately 653μg, approximately 654μg, approximately 655μg, approximately 656μg, approximately 657μg, approximately 658μg, approximately 659μg, approximately 660μg, approximately 661μg, approximately 662μg, approximately 663μg, approximately 664μg, approximately 665μg, approximately 6 66μg, approximately 667μg, approximately 668μg, approximately 669μg, approximately 670μg, approximately 671μg, approximately 672μg, approximately 673μg, approximately 674μg, approximately 675μg, approximately 676μg, approximately 677μg, approximately 678μg, approximately 679μg, approximately 680μg, approximately 681μg, approximately 682μg, approximately 683μg, approximately 684μg, approximately 685μg, approximately 686μg, approximately 687μg, approximately 688μg, approximately 689μg, approximately 690μg, approximately 691μg, approximately 692μg, approximately 693μg, approximately 694μg, approximately 695μg, approximately 696μg, approximately 697μg, approximately 698μg, approximately 699μg, approximately 700μg, approximately 701μg Approximately 702 μg, approximately 703 μg, approximately 704 μg, approximately 705 μg, approximately 706 μg, approximately 707 μg, approximately 708 μg, approximately 709 μg, approximately 710 μg, approximately 711 μg, approximately 712 μg, approximately 713 μg, approximately 714 μg, approximately 715 μg, approximately 716 μg, approximately 717 μg, approximately 718 μg, approximately 719 μgApproximately 720 μg, approximately 721 μg, approximately 722 μg, approximately 723 μg, approximately 724 μg, approximately 725 μg, approximately 726 μg, approximately 727 μg, approximately 728 μg, approximately 729 μg, approximately 730 μg, approximately 731 μg, approximately 732 μg, approximately 733 μg, approximately 734 μg, approximately 735 μg, approximately 736 μg Approximately 737 μg, approximately 738 μg, approximately 739 μg, approximately 740 μg, approximately 741 μg, approximately 742 μg, approximately 743 μg, approximately 744 μg, approximately 745 μg, approximately 746 μg, approximately 747 μg, approximately 748 μg, approximately 749 μg, approximately 750 μg, approximately 751 μg, approximately 752 μg, approximately 753 μg, approximately 754 μg Approximately 755 μg, approximately 756 μg, approximately 757 μg, approximately 758 μg, approximately 759 μg, approximately 760 μg, approximately 761 μg, approximately 762 μg, approximately 763 μg, approximately 764 μg, approximately 765 μg, approximately 766 μg, approximately 767 μg, approximately 768 μg, approximately 769 μg, approximately 770 μg, approximately 771 μg, approximately 772 μg g, approximately 773 μg, approximately 774 μg, approximately 775 μg, approximately 776 μg, approximately 777 μg, approximately 778 μg, approximately 779 μg, approximately 780 μg, approximately 781 μg, approximately 782 μg, approximately 783 μg, approximately 784 μg, approximately 785 μg, approximately 786 μg, approximately 787 μg, approximately 788 μg, approximately 789 μg, approximately 790 μg μg, approximately 791μg, approximately 792μg, approximately 793μg, approximately 794μg, approximately 795μg, approximately 796μg, approximately 797μg, approximately 798μg, approximately 799μg, approximately 800μg, approximately 801μg, approximately 802μg, approximately 803μg, approximately 804μg, approximately 805μg, approximately 806μg, approximately 807μg, approximately 80 8μg, approximately 809μg, approximately 810μg, approximately 811μg, approximately 812μg, approximately 813μg, approximately 814μg, approximately 815μg, approximately 816μg, approximately 817μg, approximately 818μg, approximately 819μg, approximately 820μg, approximately 821μg, approximately 822μg, approximately 823μg, approximately 824μg, approximately 825μg, approximately 8 26μg, approximately 827μg, approximately 828μg, approximately 829μg, approximately 830μg, approximately 831μg, approximately 832μg, approximately 833μg, approximately 834μg, approximately 835μg, approximately 836μg, approximately 837μg, approximately 838μg, approximately 839μg, approximately 840μg, approximately 841μg, approximately 842μg, approximately 843μg, approximately 844μg, approximately 845μg, approximately 846μg, approximately 847μg, approximately 848μg, approximately 849μg, approximately 850μg, approximately 851μg, approximately 852μg, approximately 853μg, approximately 854μg, approximately 855μg, approximately 856μg, approximately 857μg, approximately 858μg, approximately 859μg, approximately 860μg, approximately 861μg Approximately 862 μg, approximately 863 μg, approximately 864 μg, approximately 865 μg, approximately 866 μg, approximately 867 μg, approximately 868 μg, approximately 869 μg, approximately 870 μg, approximately 871 μg, approximately 872 μg, approximately 873 μg, approximately 874 μg, approximately 875 μg, approximately 876 μg, approximately 877 μg, approximately 878 μg, approximately 879 μgAbout 880 μg, about 881 μg, about 882 μg, about 883 μg, about 884 μg, about 885 μg, about 886 μg, about 887 μg, about 888 μg, about 889 μg, about 890 μg, about 891 μg, about 892 μg, about 893 μg, about 894 μg, about 895 μg, about 896 μg, about 897 μg, about 898 μg, about 899 μg, about 900 μg, about 901 μg, about 902 μg, about 903 μg, about 904 μg, about 905 μg, about 906 μg, about 907 μg, about 908 μg, about 909 μg, 0μg, about 911μg, about 912μg, about 913μg, about 914μg, about 915μg, about 916μg, about 917μg, about 918μg, about 919μg, about 920μg, about 921μg, about 922μg, about 923μg, about 924μg, about 925 926μg, 927μg, 928μg, 929μg, 930μg, 931μg, 932μg, 933μg, 934μg, 935μg, 936μg, 937μg, 938μg, 939μg, 940μg , about 941μg, about 942μg, about 943μg, about 944μg, about 945μg, about 946μg, about 947μg, about 948μg, about 949μg, about 950μg, about 951μg, about 952μg, about 953μg, about 954μg, about 955μg, Approximately 956μg, approximately 957μg, approximately 958μg, approximately 959μg, approximately 960μg, approximately 961μg, approximately 962μg, approximately 963μg, approximately 964μg, approximately 965μg, approximately 966μg, approximately 967μg, approximately 968μg, approximately 969μg, approximately 970μg, approximately 9 71 μg, about 972 μg, about 973 μg, about 974 μg, about 975 μg, about 976 μg, about 977 μg, about 978 μg, about 979 μg, about 980 μg, about 981 μg, about 982 μg, about 983 μg, about 984 μg, about 985 μg, about 986 μg, about 987 μg, about 988 μg, about 989 μg, about 990 μg, about 991 μg, about 992 μg, about 993 μg, about 994 μg, about 995 μg, about 996 μg, about 997 μg, about 998 μg, about 999 μg, about 1000 μg of total RSV F glycoprotein (including all values ​​and ranges therebetween).

[0199] In embodiments, the immunogenic compositions described herein comprise between about 1 μg and about 300 μg of each HA glycoprotein. In embodiments, the immunogenic compositions described herein comprise between about 5 μg and about 60 μg of each HA glycoprotein. In embodiments, the immunogenic compositions described herein comprise between about 24 μg and about 40 μg of each HA glycoprotein. In embodiments, the immunogenic compositions described herein comprise between about 30 μg and about 40 μg of each HA glycoprotein. In embodiments, the immunogenic compositions described herein comprise between about 30 μg and about 60 μg of each HA glycoprotein. In embodiments, the immunogenic compositions described herein comprise between about 33 μg and about 39 μg of each HA glycoprotein. In embodiments, the immunogenic compositions described herein comprise between about 20 μg and about 240 μg of each HA glycoprotein. In embodiments, the immunogenic compositions described herein comprise between about 30 μg and about 60 μg of each HA glycoprotein. In some embodiments, the immunogenic compositions described herein comprise about 24 μg to about 40 μg of each HA glycoprotein. In some embodiments, the immunogenic compositions comprise about 1 μg, about 2 μg, about 3 μg, about 4 μg, about 5 μg, about 6 μg, about 7 μg, about 8 μg, about 9 μg, about 10 μg, about 11 μg, about 12 μg, about 13 μg, about 14 μg, about 15 μg, about 16 μg, about 17 μg, about 18 μg, about 19 μg, about 20 μg, or about 22 μg of each HA glycoprotein. Approximately 21μg, approximately 22μg, approximately 23μg, approximately 24μg, approximately 25μg, approximately 26μg, approximately 27μg, approximately 28μg, approximately 29μg, approximately 30μg, approximately 31μg , about 32μg, about 33μg, about 34μg, about 35μg, about 36μg, about 37μg, about 38μg, about 39μg, about 40μg, about 41μg, about 42μ g, about 43μg, about 44μg, about 45μg, about 46μg, about 47μg, about 48μg, about 49μg, about 50μg, about 51μg, about 52μg, about 53 μg, approximately 54 μg, approximately 55 μg, approximately 56 μg, approximately 57 μg, approximately 58 μg, approximately 59 μg, approximately 60 μg, approximately 61 μg, approximately 62 μg, approximately 63 μg, approximately 6 4μg, about 65μg, about 66μg, about 67μg, about 68μg, about 69μg, about 70μg, about 71μg, about 72μg, about 73μg, about 74μg, about 75μg, about 76μg, about 77μg, about 78μg, about 79μg, about 80μg, about 81μg, about 82μg, about 83μg, about 84μg, about 85μg,Approximately 86 μg, approximately 87 μg, approximately 88 μg, approximately 89 μg, approximately 90 μg, approximately 91 μg, approximately 92 μg, approximately 93 μg, approximately 94 μg, approximately 95 μg, approximately 96 μg, approximately 97 μg, approximately 98 μg, approximately 99 μg, approximately 100 μg, approximately 101 μg, approximately 102 μg, approximately 103 μg, approximately 104 μg, approximately 105 μg Approximately 106 μg, approximately 107 μg, approximately 108 μg, approximately 109 μg, approximately 110 μg, approximately 111 μg, approximately 112 μg, approximately 113 μg, approximately 114 μg, approximately 115 μg, approximately 116 μg, approximately 117 μg, approximately 118 μg, approximately 119 μg, approximately 120 μg, approximately 121 μg, approximately 122 μg, approximately 123 μg g, approximately 124 μg, approximately 125 μg, approximately 126 μg, approximately 127 μg, approximately 128 μg, approximately 129 μg, approximately 130 μg, approximately 131 μg, approximately 132 μg, approximately 133 μg, approximately 134 μg, approximately 135 μg, approximately 136 μg, approximately 137 μg, approximately 138 μg, approximately 139 μg, approximately 140 μg, approximately 141 μg μg, approximately 142μg, approximately 143μg, approximately 144μg, approximately 145μg, approximately 146μg, approximately 147μg, approximately 148μg, approximately 149μg, approximately 150μg, approximately 151μg, approximately 152μg, approximately 153μg, approximately 154μg, approximately 155μg, approximately 156μg, approximately 157μg, approximately 158μg, approximately 15 9μg, approximately 160μg, approximately 161μg, approximately 162μg, approximately 163μg, approximately 164μg, approximately 165μg, approximately 166μg, approximately 167μg, approximately 168μg, approximately 169μg, approximately 170μg, approximately 171μg, approximately 172μg, approximately 173μg, approximately 174μg, approximately 175μg, approximately 176μg, approximately 1 77μg, approximately 178μg, approximately 179μg, approximately 180μg, approximately 181μg, approximately 182μg, approximately 183μg, approximately 184μg, approximately 185μg, approximately 186μg, approximately 187μg, approximately 188μg, approximately 189μg, approximately 190μg, approximately 191μg, approximately 192μg, approximately 193μg, approximately 194μg, approximately 195μg, approximately 196μg, approximately 197μg, approximately 198μg, approximately 199μg, approximately 200μg, approximately 201μg, approximately 202μg, approximately 203μg, approximately 204μg, approximately 205μg, approximately 206μg, approximately 207μg, approximately 208μg, approximately 209μg, approximately 210μg, approximately 211μg, approximately 212μg Approximately 213 μg, approximately 214 μg, approximately 215 μg, approximately 216 μg, approximately 217 μg, approximately 218 μg, approximately 219 μg, approximately 220 μg, approximately 221 μg, approximately 222 μg, approximately 223 μg, approximately 224 μg, approximately 225 μg, approximately 226 μg, approximately 227 μg, approximately 228 μg, approximately 229 μg, approximately 230 μgApproximately 231μg, approximately 232μg, approximately 233μg, approximately 234μg, approximately 235μg, approximately 236μg, approximately 237μg, approximately 238μg, approximately 239μg, approximately 2 40μg, about 241μg, about 242μg, about 243μg, about 244μg, about 245μg, about 246μg, about 247μg, about 248μg, about 249μ g, about 250μg, about 251μg, about 252μg, about 253μg, about 254μg, about 255μg, about 256μg, about 257μg, about 258μg, Approximately 259μg, approximately 260μg, approximately 261μg, approximately 262μg, approximately 263μg, approximately 264μg, approximately 265μg, approximately 266μg, approximately 267μg, approximately 26 8 μg, about 269 μg, about 270 μg, about 271 μg, about 272 μg, about 273 μg, about 274 μg, about 275 μg, about 276 μg, about 277 μg, about 278 μg, about 279 μg, about 280 μg, about 281 μg, about 282 μg, about 283 μg, about 284 μg, about 285 μg, about 286 μg, about 287 μg, about 288 μg, about 289 μg, about 290 μg, about 291 μg, about 292 μg, about 293 μg, about 294 μg, about 295 μg, about 296 μg, about 297 μg, about 298 μg, about 299 μg, or about 300 μg of each HA glycoprotein (including all values ​​and ranges therebetween). In embodiments, the immunogenic compositions described herein comprise about 30 μg of each HA glycoprotein. In embodiments, the immunogenic compositions described herein comprise about 33 μg of each HA glycoprotein. In embodiments, the immunogenic compositions described herein comprise about 39 μg of each HA glycoprotein. In embodiments, the immunogenic compositions described herein comprise about 54 μg of each HA glycoprotein. In embodiments, the immunogenic compositions described herein comprise about 60 μg of each HA glycoprotein. In embodiments, the immunogenic compositions described herein comprise about 240 μg of each HA glycoprotein.

[0200] In embodiments, the immunogenic compositions described herein comprise from about 1 μg to about 1000 μg of total HA glycoprotein. In embodiments, the immunogenic compositions comprise a concentration of about 1 μg, about 2 μg, about 3 μg, about 4 μg, about 5 μg, about 6 μg, about 7 μg, about 8 μg, about 9 μg, about 10 μg, about 11 μg, about 12 μg, about 13 μg, about 14 μg, about 15 μg, about 16 μg, about 17 μg, about 18 μg, about 19 μg, about 20 μg, about 21 μg, about 22 μg, about 23 μg, about 24 μg, about 25 μg, about 26 μg, about 27 μg, about 28 μg, about 29 μg, about 30 μg, about 31 μg, about 32 μg, about 33 μg, about 34 μg, about 35 μg, about 36 μg, about 37 μg, about 38 μg, about 40 μg, about 41 μg, about 42 μg, about 43 μg, about 44 μg, about 45 μg, about 46 μg, about 47 μg, about 48 μg, about 49 μg, about 50 μg, about 51 μg, about 52 μg, about 53 μg, about 54 μg, about 55 μg, about 56 μg, about 57 μg, about 58 μg, about 59 μg, about 60 μg, about 61 μg, about 62 μg, about 63 μg, about 64 μg, about 65 μg, about 66 μg, about 67 μg, about 68 μg, about 69 μg, about 70 μg, about 71 μg, about 72 μg, about 73 μg, about 74 μ g, about 39μg, about 40μg, about 41μg, about 42μg, about 43μg, about 44μg, about 45μg, about 46μg, about 47μg, about 48 μg, approx. 49 μg, approx. 50 μg, approx. 51 μg, approx. 52 μg, approx. 53 μg, approx. 54 μg, approx. 55 μg, approx. 56 μg, approx. 57 μg, approx. 5 8μg, about 59μg, about 60μg, about 61μg, about 62μg, about 63μg, about 64μg, about 65μg, about 66μg, about 67μg, about 68μg, about 69μg, about 70μg, about 71μg, about 72μg, about 73μg, about 74μg, about 75μg, about 76μg, about 77μg, about 78μg, about 79μg, about 80μg, about 81μg, about 82μg, about 83μg, about 84μg, about 85μg, about 86μg, about 87μg, Approximately 88μg, approximately 89μg, approximately 90μg, approximately 91μg, approximately 92μg, approximately 93μg, approximately 94μg, approximately 95μg, approximately 96μg, approximately 97μg , about 98μg, about 99μg, about 100μg, about 101μg, about 102μg, about 103μg, about 104μg, about 105μg, about 106 μg, approx. 107 μg, approx. 108 μg, approx. 109 μg, approx. 110 μg, approx. 111 μg, approx. 112 μg, approx. 113 μg, approx. 114 μg, approx. 115μg, about 116μg, about 117μg, about 118μg, about 119μg, about 120μg, about 121μg, about 122μg, about 123 μg, approximately 124 μg, approximately 125 μg, approximately 126 μg, approximately 127 μg, approximately 128 μg, approximately 129 μg, approximately 130 μg, approximately 131 μg, Approximately 132μg, approximately 133μg, approximately 134μg, approximately 135μg, approximately 136μg, approximately 137μg, approximately 138μg, approximately 139μg, approximately 14 0μg, about 141μg, about 142μg, about 143μg, about 144μg, about 145μg, about 146μg, about 147μg, about 148μg,Approximately 149 μg, approximately 150 μg, approximately 151 μg, approximately 152 μg, approximately 153 μg, approximately 154 μg, approximately 155 μg, approximately 156 μg, approximately 157 μg, approximately 158 μg, approximately 159 μg, approximately 160 μg, approximately 161 μg, approximately 162 μg, approximately 163 μg, approximately 164 μg, approximately 165 μg, approximately 166 μg Approximately 167 μg, approximately 168 μg, approximately 169 μg, approximately 170 μg, approximately 171 μg, approximately 172 μg, approximately 173 μg, approximately 174 μg, approximately 175 μg, approximately 176 μg, approximately 177 μg, approximately 178 μg, approximately 179 μg, approximately 180 μg, approximately 181 μg, approximately 182 μg, approximately 183 μg, approximately 184 μg g, approximately 185μg, approximately 186μg, approximately 187μg, approximately 188μg, approximately 189μg, approximately 190μg, approximately 191μg, approximately 192μg, approximately 193μg, approximately 194μg, approximately 195μg, approximately 196μg, approximately 197μg, approximately 198μg, approximately 199μg, approximately 200μg, approximately 201μg, approximately 202 μg, approximately 203μg, approximately 204μg, approximately 205μg, approximately 206μg, approximately 207μg, approximately 208μg, approximately 209μg, approximately 210μg, approximately 211μg, approximately 212μg, approximately 213μg, approximately 214μg, approximately 215μg, approximately 216μg, approximately 217μg, approximately 218μg, approximately 219μg, approximately 22μg 0 μg, approximately 221 μg, approximately 222 μg, approximately 223 μg, approximately 224 μg, approximately 225 μg, approximately 226 μg, approximately 227 μg, approximately 228 μg, approximately 229 μg, approximately 230 μg, approximately 231 μg, approximately 232 μg, approximately 233 μg, approximately 234 μg, approximately 235 μg, approximately 236 μg, approximately 237 μg, approximately 2 38μg, approximately 239μg, approximately 240μg, approximately 241μg, approximately 242μg, approximately 243μg, approximately 244μg, approximately 245μg, approximately 246μg, approximately 247μg, approximately 248μg, approximately 249μg, approximately 250μg, approximately 251μg, approximately 252μg, approximately 253μg, approximately 254μg, approximately 255μg, approximately 256μg, approximately 257μg, approximately 258μg, approximately 259μg, approximately 260μg, approximately 261μg, approximately 262μg, approximately 263μg, approximately 264μg, approximately 265μg, approximately 266μg, approximately 267μg, approximately 268μg, approximately 269μg, approximately 270μg, approximately 271μg, approximately 272μg, approximately 273μg Approximately 274 μg, approximately 275 μg, approximately 276 μg, approximately 277 μg, approximately 278 μg, approximately 279 μg, approximately 280 μg, approximately 281 μg, approximately 282 μg, approximately 283 μg, approximately 284 μg, approximately 285 μg, approximately 286 μg, approximately 287 μg, approximately 288 μg, approximately 289 μg, approximately 290 μg, approximately 291 μgApproximately 292 μg, approximately 293 μg, approximately 294 μg, approximately 295 μg, approximately 296 μg, approximately 297 μg, approximately 298 μg, approximately 299 μg, approximately 300 μg, approximately 301 μg, approximately 302 μg, approximately 303 μg, approximately 304 μg, approximately 305 μg, approximately 306 μg, approximately 307 μg, approximately 308 μg, approximately 309 μg Approximately 310 μg, approximately 311 μg, approximately 312 μg, approximately 313 μg, approximately 314 μg, approximately 315 μg, approximately 316 μg, approximately 317 μg, approximately 318 μg, approximately 319 μg, approximately 320 μg, approximately 321 μg, approximately 322 μg, approximately 323 μg, approximately 324 μg, approximately 325 μg, approximately 326 μg, approximately 327 μg g, approximately 328 μg, approximately 329 μg, approximately 330 μg, approximately 331 μg, approximately 332 μg, approximately 333 μg, approximately 334 μg, approximately 335 μg, approximately 336 μg, approximately 337 μg, approximately 338 μg, approximately 339 μg, approximately 340 μg, approximately 341 μg, approximately 342 μg, approximately 343 μg, approximately 344 μg, approximately 345 μg μg, approximately 346μg, approximately 347μg, approximately 348μg, approximately 349μg, approximately 350μg, approximately 351μg, approximately 352μg, approximately 353μg, approximately 354μg, approximately 355μg, approximately 356μg, approximately 357μg, approximately 358μg, approximately 359μg, approximately 360μg, approximately 361μg, approximately 362μg, approximately 36 3μg, approximately 364μg, approximately 365μg, approximately 366μg, approximately 367μg, approximately 368μg, approximately 369μg, approximately 370μg, approximately 371μg, approximately 372μg, approximately 373μg, approximately 374μg, approximately 375μg, approximately 376μg, approximately 377μg, approximately 378μg, approximately 379μg, approximately 380μg, approximately 3 81μg, approximately 382μg, approximately 383μg, approximately 384μg, approximately 385μg, approximately 386μg, approximately 387μg, approximately 388μg, approximately 389μg, approximately 390μg, approximately 391μg, approximately 392μg, approximately 393μg, approximately 394μg, approximately 395μg, approximately 396μg, approximately 397μg, approximately 398μg, approximately 399μg, approximately 400μg, approximately 401μg, approximately 402μg, approximately 403μg, approximately 404μg, approximately 405μg, approximately 406μg, approximately 407μg, approximately 408μg, approximately 409μg, approximately 410μg, approximately 411μg, approximately 412μg, approximately 413μg, approximately 414μg, approximately 415μg, approximately 416μg Approximately 417 μg, approximately 418 μg, approximately 419 μg, approximately 420 μg, approximately 421 μg, approximately 422 μg, approximately 423 μg, approximately 424 μg, approximately 425 μg, approximately 426 μg, approximately 427 μg, approximately 428 μg, approximately 429 μg, approximately 430 μg, approximately 431 μg, approximately 432 μg, approximately 433 μg, approximately 434 μgApproximately 435 μg, approximately 436 μg, approximately 437 μg, approximately 438 μg, approximately 439 μg, approximately 440 μg, approximately 441 μg, approximately 442 μg, approximately 443 μg, approximately 444 μg, approximately 445 μg, approximately 446 μg, approximately 447 μg, approximately 448 μg, approximately 449 μg, approximately 450 μg, approximately 451 μg, approximately 452 μg Approximately 453 μg, approximately 454 μg, approximately 455 μg, approximately 456 μg, approximately 457 μg, approximately 458 μg, approximately 459 μg, approximately 460 μg, approximately 461 μg, approximately 462 μg, approximately 463 μg, approximately 464 μg, approximately 465 μg, approximately 466 μg, approximately 467 μg, approximately 468 μg, approximately 469 μg, approximately 470 μg g, approximately 471μg, approximately 472μg, approximately 473μg, approximately 474μg, approximately 475μg, approximately 476μg, approximately 477μg, approximately 478μg, approximately 479μg, approximately 480μg, approximately 481μg, approximately 482μg, approximately 483μg, approximately 484μg, approximately 485μg, approximately 486μg, approximately 487μg, approximately 488 μg, approximately 489μg, approximately 490μg, approximately 491μg, approximately 492μg, approximately 493μg, approximately 494μg, approximately 495μg, approximately 496μg, approximately 497μg, approximately 498μg, approximately 499μg, approximately 500μg, approximately 501μg, approximately 502μg, approximately 503μg, approximately 504μg, approximately 505μg, approximately 50 6μg, approximately 507μg, approximately 508μg, approximately 509μg, approximately 510μg, approximately 511μg, approximately 512μg, approximately 513μg, approximately 514μg, approximately 515μg, approximately 516μg, approximately 517μg, approximately 518μg, approximately 519μg, approximately 520μg, approximately 521μg, approximately 522μg, approximately 523μg, approximately 5 24μg, approximately 525μg, approximately 526μg, approximately 527μg, approximately 528μg, approximately 529μg, approximately 530μg, approximately 531μg, approximately 532μg, approximately 533μg, approximately 534μg, approximately 535μg, approximately 536μg, approximately 537μg, approximately 538μg, approximately 539μg, approximately 540μg, approximately 541μg, approximately 542μg, approximately 543μg, approximately 544μg, approximately 545μg, approximately 546μg, approximately 547μg, approximately 548μg, approximately 549μg, approximately 550μg, approximately 551μg, approximately 552μg, approximately 553μg, approximately 554μg, approximately 555μg, approximately 556μg, approximately 557μg, approximately 558μg, approximately 559μg Approximately 560 μg, approximately 561 μg, approximately 562 μg, approximately 563 μg, approximately 564 μg, approximately 565 μg, approximately 566 μg, approximately 567 μg, approximately 568 μg, approximately 569 μg, approximately 570 μg, approximately 571 μg, approximately 572 μg, approximately 573 μg, approximately 574 μg, approximately 575 μg, approximately 576 μg, approximately 577 μgApproximately 578 μg, approximately 579 μg, approximately 580 μg, approximately 581 μg, approximately 582 μg, approximately 583 μg, approximately 584 μg, approximately 585 μg, approximately 586 μg, approximately 587 μg, approximately 588 μg, approximately 589 μg, approximately 590 μg, approximately 591 μg, approximately 592 μg, approximately 593 μg, approximately 594 μg, approximately 595 μg Approximately 596 μg, approximately 597 μg, approximately 598 μg, approximately 599 μg, approximately 600 μg, approximately 601 μg, approximately 602 μg, approximately 603 μg, approximately 604 μg, approximately 605 μg, approximately 606 μg, approximately 607 μg, approximately 608 μg, approximately 609 μg, approximately 610 μg, approximately 611 μg, approximately 612 μg, approximately 613 μg g, approximately 614 μg, approximately 615 μg, approximately 616 μg, approximately 617 μg, approximately 618 μg, approximately 619 μg, approximately 620 μg, approximately 621 μg, approximately 622 μg, approximately 623 μg, approximately 624 μg, approximately 625 μg, approximately 626 μg, approximately 627 μg, approximately 628 μg, approximately 629 μg, approximately 630 μg, approximately 631 μg μg, approximately 632μg, approximately 633μg, approximately 634μg, approximately 635μg, approximately 636μg, approximately 637μg, approximately 638μg, approximately 639μg, approximately 640μg, approximately 641μg, approximately 642μg, approximately 643μg, approximately 644μg, approximately 645μg, approximately 646μg, approximately 647μg, approximately 648μg, approximately 64 9μg, approximately 650μg, approximately 651μg, approximately 652μg, approximately 653μg, approximately 654μg, approximately 655μg, approximately 656μg, approximately 657μg, approximately 658μg, approximately 659μg, approximately 660μg, approximately 661μg, approximately 662μg, approximately 663μg, approximately 664μg, approximately 665μg, approximately 666μg, approximately 6 67μg, approximately 668μg, approximately 669μg, approximately 670μg, approximately 671μg, approximately 672μg, approximately 673μg, approximately 674μg, approximately 675μg, approximately 676μg, approximately 677μg, approximately 678μg, approximately 679μg, approximately 680μg, approximately 681μg, approximately 682μg, approximately 683μg, approximately 684μg, approximately 685μg, approximately 686μg, approximately 687μg, approximately 688μg, approximately 689μg, approximately 690μg, approximately 691μg, approximately 692μg, approximately 693μg, approximately 694μg, approximately 695μg, approximately 696μg, approximately 697μg, approximately 698μg, approximately 699μg, approximately 700μg, approximately 701μg, approximately 702μg Approximately 703 μg, approximately 704 μg, approximately 705 μg, approximately 706 μg, approximately 707 μg, approximately 708 μg, approximately 709 μg, approximately 710 μg, approximately 711 μg, approximately 712 μg, approximately 713 μg, approximately 714 μg, approximately 715 μg, approximately 716 μg, approximately 717 μg, approximately 718 μg, approximately 719 μg, approximately 720 μgApproximately 721 μg, approximately 722 μg, approximately 723 μg, approximately 724 μg, approximately 725 μg, approximately 726 μg, approximately 727 μg, approximately 728 μg, approximately 729 μg, approximately 730 μg, approximately 731 μg, approximately 732 μg, approximately 733 μg, approximately 734 μg, approximately 735 μg, approximately 736 μg, approximately 73... 7μg, approximately 738μg, approximately 739μg, approximately 740μg, approximately 741μg, approximately 742μg, approximately 743μg, approximately 744μg, approximately 745μg, approximately 746μg, approximately 747μg, approximately 748μg, approximately 749μg, approximately 750μg, approximately 751μg, approximately 752μg, approximately 753μg, approximately 754μg, approximately 755μg, approximately 756μg, approximately 757μg, approximately 758μg, approximately 759μg, approximately 760μg, approximately 761μg, approximately 762μg, approximately 763μg, approximately 764μg, approximately 765μg, approximately 766μg, approximately 767μg, approximately 768μg, approximately 769μg, approximately 770μg, approximately 771μg, approximately 772μg Approximately 773 μg, approximately 774 μg, approximately 775 μg, approximately 776 μg, approximately 777 μg, approximately 778 μg, approximately 779 μg, approximately 780 μg, approximately 781 μg, approximately 782 μg, approximately 783 μg, approximately 784 μg, approximately 785 μg, approximately 786 μg, approximately 787 μg, approximately 788 μg, approximately 789 μg, approximately 790 μg Approximately 791 μg, approximately 792 μg, approximately 793 μg, approximately 794 μg, approximately 795 μg, approximately 796 μg, approximately 797 μg, approximately 798 μg, approximately 799 μg, approximately 800 μg, approximately 801 μg, approximately 802 μg, approximately 803 μg, approximately 804 μg, approximately 805 μg, approximately 806 μg, approximately 807 μg, approximately 808 μg g, approximately 809 μg, approximately 810 μg, approximately 811 μg, approximately 812 μg, approximately 813 μg, approximately 814 μg, approximately 815 μg, approximately 816 μg, approximately 817 μg, approximately 818 μg, approximately 819 μg, approximately 820 μg, approximately 821 μg, approximately 822 μg, approximately 823 μg, approximately 824 μg, approximately 825 μg, approximately 82 6μg, approximately 827μg, approximately 828μg, approximately 829μg, approximately 830μg, approximately 831μg, approximately 832μg, approximately 833μg, approximately 834μg, approximately 835μg, approximately 836μg, approximately 837μg, approximately 838μg, approximately 839μg, approximately 840μg, approximately 841μg, approximately 842μg, approximately 843μg, approximately 8 44μg, approximately 845μg, approximately 846μg, approximately 847μg, approximately 848μg, approximately 849μg, approximately 850μg, approximately 851μg, approximately 852μg, approximately 853μg, approximately 854μg, approximately 855μg, approximately 856μg, approximately 857μg, approximately 858μg, approximately 859μg, approximately 860μg, approximately 861μg, approximately 862μg, approximately 863μg, approximately 864μg, approximately 865μg, approximately 866μg, approximately 867μg, approximately 868μg, approximately 869μg, approximately 870μg, approximately 871μg, approximately 872μg, approximately 873μg, approximately 874μg, approximately 875μg, approximately 876μg, approximately 877μg, approximately 878μg, approximately 879μgAbout 880 μg, about 881 μg, about 882 μg, about 883 μg, about 884 μg, about 885 μg, about 886 μg, about 887 μg, about 888 μg, about 889 μg, about 890 μg, about 891 μg, about 892 μg, about 893 μg, about 894 μg, about 895 μg, about 896 μg, about 897 μg, about 898 μg, about 899 μg, about 900 μg, about 901 μg, about 902 μg, about 903 μg, about 904 μg, about 905 μg, about 906 μg, about 907 μg, about 908 μg, about 909 μg, about 910 μg, about 91 1μg, about 912μg, about 913μg, about 914μg, about 915μg, about 916μg, about 917μg, about 918μg, about 919μg, about 920μg, about 921μg, about 922μg, about 923μg, about 924μg, about 925μg, about 926μg, about 927μg, about 928μg, about 929μg, about 930μg, about 931μg, about 932μg, about 933μg, about 934μg, about 935μg, about 936μg, about 937μg, about 938μg, about 939μg, about 940μg, about 941μg, about 942μg , about 943μg, about 944μg, about 945μg, about 946μg, about 947μg, about 948μg, about 949μg, about 950μg, about 951μg, about 952μg, about 953μg, about 954μg, about 955μg, about 956μg, about 957μg, about 958 μg, approximately 959 μg, approximately 960 μg, approximately 961 μg, approximately 962 μg, approximately 963 μg, approximately 964 μg, approximately 965 μg, approximately 966 μg, approximately 967 μg, approximately 968 μg, approximately 969 μg, approximately 970 μg, approximately 971 μg, approximately 972 μg, approximately 973 μg, approximately 9 74 μg, about 975 μg, about 976 μg, about 977 μg, about 978 μg, about 979 μg, about 980 μg, about 981 μg, about 982 μg, about 983 μg, about 984 μg, about 985 μg, about 986 μg, about 987 μg, about 988 μg, about 989 μg, about 990 μg, about 991 μg, about 992 μg, about 993 μg, about 994 μg, about 995 μg, about 996 μg, about 997 μg, about 998 μg, about 999 μg, about 1000 μg of total HA glycoprotein (including all values ​​and ranges therebetween).

[0201] In embodiments, the immunogenic compositions described herein comprise between about 1 μg and about 100 μg of each CoV S glycoprotein. In embodiments, the immunogenic compositions described herein comprise between about 2.5 μg and about 35 μg of each CoV S glycoprotein. In embodiments, the immunogenic compositions described herein comprise between about 5 μg and about 25 μg of each CoV S glycoprotein. In embodiments, the immunogenic compositions described herein comprise between about 14 μg and about 15 μg of each CoV S glycoprotein. In embodiments, the immunogenic compositions described herein comprise between about 15 μg and about 35 μg of each CoV S glycoprotein. In embodiments, the immunogenic compositions described herein comprise between about 1 μg and about 100 μg of total CoV S glycoprotein. In embodiments, the immunogenic composition comprises about 1 μg, about 2 μg, about 3 μg, about 4 μg, about 5 μg, about 6 μg, about 7 μg, about 8 μg, about 9 μg, about 10 μg, about 11 μg, about 12 μg, about 13 μg, about 14 μg, about 15 μg, about 16 μg, about 17 μg, about 18 μg, about 19 μg, about 20 μg, about 21 μg, about 22 μg, about 23 μg, about 24 μg, about 25 μg g, about 26 μg, about 27 μg, about 28 μg, about 29 μg, about 30 μg, about 31 μg, about 32 μg, about 33 μg, about 34 μg, about 35 μg, about 36 μg, about 37 μg, about 38 μg , about 39μg, about 40μg, about 41μg, about 42μg, about 43μg, about 44μg, about 45μg, about 46μg, about 47μg, about 48μg, about 49μg, about 50μg, about 51μg, about 52μg, about 53μg, about 54μg, about 55μg, about 56μg, about 57μg, about 58μg, about 59μg, about 60μg, about 61μg, about 62μg, about 63μg, about 64μg, about 6 5μg, about 66μg, about 67μg, about 68μg, about 69μg, about 70μg, about 71μg, about 72μg, about 73μg, about 74μg, about 75μg, about 76μg, about 77μg, about 78 The compositions of the present invention comprise about 79 μg, about 80 μg, about 81 μg, about 82 μg, about 83 μg, about 84 μg, about 85 μg, about 86 μg, about 87 μg, about 88 μg, about 89 μg, about 90 μg, about 91 μg, about 92 μg, about 93 μg, about 94 μg, about 95 μg, about 96 μg, about 97 μg, about 98 μg, about 99 μg, about 100 μg (including all values ​​and ranges therebetween) of each CoV S glycoprotein.In embodiments, the immunogenic composition comprises about 1 μg, about 2 μg, about 3 μg, about 4 μg, about 5 μg, about 6 μg, about 7 μg, about 8 μg, about 9 μg, about 10 μg, about 11 μg, about 12 μg, about 13 μg, about 14 μg, about 15 μg, about 16 μg, about 17 μg, about 18 μg, about 19 μg, about 20 μg, about 21 μg, about 22 μg, about 23 μg, about 24 μg , about 25μg, about 26μg, about 27μg, about 28μg, about 29μg, about 30μg, about 31μg, about 32μg, about 33μg, about 34μg, about 35μg, about 36μg, about 37μ g, about 38μg, about 39μg, about 40μg, about 41μg, about 42μg, about 43μg, about 44μg, about 45μg, about 46μg, about 47μg, about 48μg, about 49μg, about 50 μg, approximately 51 μg, approximately 52 μg, approximately 53 μg, approximately 54 μg, approximately 55 μg, approximately 56 μg, approximately 57 μg, approximately 58 μg, approximately 59 μg, approximately 60 μg, approximately 61 μg, approximately 62 μg, approximately 6 3μg, about 64μg, about 65μg, about 66μg, about 67μg, about 68μg, about 69μg, about 70μg, about 71μg, about 72μg, about 73μg, about 74μg, about 75μg, about In some embodiments, the immunogenic composition comprises about 76 μg, about 77 μg, about 78 μg, about 79 μg, about 80 μg, about 81 μg, about 82 μg, about 83 μg, about 84 μg, about 85 μg, about 86 μg, about 87 μg, about 88 μg, about 89 μg, about 90 μg, about 91 μg, about 92 μg, about 93 μg, about 94 μg, about 95 μg, about 96 μg, about 97 μg, about 98 μg, about 99 μg, or about 100 μg of total CoV S glycoprotein (including all values ​​and ranges therebetween). In some embodiments, the immunogenic composition comprises about 1 μg of CoV S glycoprotein. In some embodiments, the immunogenic composition comprises about 13 μg of CoV S glycoprotein. In some embodiments, the immunogenic composition comprises about 5 μg of CoV S glycoprotein. In embodiments, the immunogenic composition comprises about 25 μg of CoV S glycoprotein.

[0202] Adjuvants in immunogenic and vaccine compositions In certain embodiments, the compositions disclosed herein comprise one or more adjuvants.In several embodiments, the one or more adjuvants enhance immune response.In other embodiments, the compositions are prepared without adjuvants, and thus can be used for administration as adjuvant-free compositions.Advantageously, the adjuvant-free compositions disclosed herein can produce protective immune responses when administered as a single dose.

[0203] Aluminum-based adjuvants In some embodiments, the adjuvant can be alum (e.g., AlPO4 or Al(OH)3). Typically, the nanoparticles are substantially bound to alum. For example, the nanoparticles can be at least 80% bound, at least 85% bound, at least 90% bound, or at least 95% bound to alum. Often, the nanoparticles are 92% to 97% bound to alum in the composition. The amount of alum present per dose typically ranges from about 400 μg to about 1250 μg. For example, alum can be present in an amount of about 300 μg to about 900 μg, about 400 μg to about 800 μg, about 500 μg to about 700 μg, about 400 μg to about 600 μg, or about 400 μg to about 500 μg per dose. Typically, about 400 μg of alum is present for a 120 μg dose of protein nanoparticles.

[0204] Saponin adjuvants Adjuvants containing saponin can also be combined with the immunogens disclosed herein.Saponin is a glycoside derived from the bark of the Quillaja saponaria Molina tree.Typically, saponin is prepared using a multi-step purification process that results in multiple fractions.As used herein, the term "saponin fraction from Quillaja saponaria Molina" is used comprehensively to describe semi-purified or defined saponin fractions of Quillaja saponaria or substantially pure fractions thereof.

[0205] Saponin fraction Several techniques for producing saponin fractions are suitable. Fractions A, B, and C are described in U.S. Patent No. 6,352,697 and can be prepared as follows: A lipophilic fraction from a crude aqueous Quillaja saponaria Molina extract, Quil A, is separated by chromatography and eluted with 70% acetonitrile in water to recover the lipophilic fraction. This lipophilic fraction is then separated by semi-preparative HPLC with elution using a gradient of 25% to 60% acetonitrile in acidic water. The fraction referred to herein as "Fraction A" or "QH-A" corresponds to or is eluted at approximately 39% acetonitrile. The fraction referred to herein as "Fraction B" or "QH-B" corresponds to or is eluted at approximately 47% acetonitrile. The fraction referred to herein as "Fraction C" or "QH-C" corresponds to or is eluted at approximately 49% acetonitrile. Further information regarding fraction purification can be found in U.S. Patent No. 5,057,540. When prepared as described herein, Quillaja saponaria Molina fractions A, B, and C each represent a group or family of closely chemically related molecules with definable properties. The chromatographic conditions under which they are obtained are such that batch-to-batch reproducibility with respect to elution profile and biological activity is highly consistent.

[0206] Other saponin fractions have been described: fractions B3, B4, and B4b are described in EP 0 436 620, and fractions QA1 to QA22 are described in EP 0 3632 279 B2, Q-VAC (Nor-Feed, AS Denmark), Quillaja saponaria Molina Spikoside (Isconova AB, Ultunaallen 2B, 756 51 Uppsala, Sweden). Fractions QA-1, QA-2, QA-3, QA-4, QA-5, QA-6, QA-7, QA-8, QA-9, QA-10, QA-11, QA-12, QA-13, QA-14, QA-15, QA-16, QA-17, QA-18, QA-19, QA-20, QA-21, and QA-22 of EP 03632279 B2, in particular QA-7, QA-17, QA-18, and QA-21, can be used, which can be obtained as described in Example 1 of EP 03632279 B2, in particular pages 6 and 8 and 9.

[0207] The saponin fractions used to form the adjuvants described herein are often substantially pure fractions, i.e., the fractions are substantially free of contamination from other materials. In certain embodiments, a substantially pure saponin fraction may contain up to 40%, up to 30%, up to 25%, up to 20%, up to 15%, up to 10%, up to 7%, up to 5%, up to 2%, up to 1%, up to 0.5%, or up to 0.1% by weight of other compounds, such as other saponins or other adjuvant materials.

[0208] ISCOM Structure The saponin fraction may be administered in the form of cage-like particles called ISCOMs (Immune Stimulating Complexes). ISCOMs may be prepared as described in EP 0109942 B1, EP 0242380 B1 and EP 0180546 B1. In certain embodiments, transport and / or passenger antigens may be used, as described in EP 9600647-3 (PCT / SE97 / 00289).

[0209] Matrix adjuvants In some embodiments, the ISCOM is an ISCOM matrix complex. The ISCOM matrix complex comprises at least one saponin fraction and a lipid. The lipid is at least a sterol, such as cholesterol. In certain aspects, the ISCOM matrix complex also contains a phospholipid. The ISCOM matrix complex may also contain one or more other immunomodulatory (adjuvant-active) substances, not necessarily glycosides, and may be prepared as described in EP0436620B1, the entire contents of which are incorporated herein by reference.

[0210] In another embodiment, the ISCOM is an ISCOM complex. An ISCOM complex contains at least one saponin, at least one lipid, and at least one antigen or epitope. The ISCOM complex contains the antigen associated with the particle through detergent treatment, which incorporates a portion of the antigen into the particle. In contrast, an ISCOM matrix is ​​formulated as an admixture with the antigen, and the association between the ISCOM matrix particle and the antigen is mediated by electrostatic and / or hydrophobic interactions.

[0211] According to one embodiment, the ISCOM matrix complex or the saponin fraction incorporated into the ISCOM complex, or at least one further adjuvant (which is also incorporated into or mixed with the ISCOM or ISCOM matrix complex) is selected from fraction A, fraction B, or fraction C of Quillaja saponaria, a semi-purified preparation of Quillaja saponaria, a purified preparation of Quillaja saponaria, or a portion of any purified fraction, e.g., QA1-21.

[0212] In certain embodiments, each ISCOM particle may contain at least two saponin fractions. Any combination of weight percents of different saponin fractions may be used. Any combination of weight percents of any two fractions may be used. For example, a particle may contain any weight percent of Fraction A and any weight percent of another saponin fraction, such as a crude saponin fraction or Fraction C, respectively. Thus, in certain embodiments, each ISCOM matrix particle or each ISCOM complex particle may contain 0.1-99.9%, 5-95%, 10-90%, 15-85%, 20-80%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, 40-60%, or 50% by weight of one saponin fraction, e.g., fraction A, and the remaining, in each case, up to 100% of another saponin, e.g., any coarse fraction or any other fraction, e.g., fraction C. Weights are calculated as the total weight of the saponin fractions. Examples of ISCOM matrix complex and ISCOM complex adjuvants are disclosed in U.S. Published Application No. 2013 / 0129770, which is incorporated herein by reference in its entirety.

[0213] In certain embodiments, an ISCOM matrix or ISCOM complex comprises 5-99% by weight of one fraction, such as fraction A, and the remaining up to 100% by weight of another fraction, such as a crude saponin fraction or fraction C. Weights are calculated as the total weight of the saponin fractions.

[0214] In another embodiment, the ISCOM matrix or ISCOM complex comprises 40% to 99% by weight of one fraction, such as fraction A, and 1% to 60% by weight of another fraction, such as the crude saponin fraction or fraction C. Weights are calculated as the total weight of the saponin fractions.

[0215] In yet another embodiment, the ISCOM matrix or ISCOM complex comprises 70% to 95% by weight of one fraction, such as Fraction A, and 30% to 5% by weight of another fraction, such as the crude saponin fraction or Fraction C. Weights are calculated as the total weight of the saponin fractions. In another embodiment, the saponin fraction from Quillaja saponaria Molina is selected from any one of QA1-21.

[0216] In addition to particles containing a mixture of saponin fractions, ISCOM matrix particles and ISCOM complex particles may each be formed using only one saponin fraction. The compositions disclosed herein may contain multiple particles, where each particle contains only one saponin fraction. That is, a particular composition may contain one or more different types of ISCOM matrix complex particles and / or one or more different types of ISCOM complex particles, where each individual particle contains one saponin fraction from Quillaja saponaria Molina, and the saponin fraction in one complex particle is different from the saponin fraction in another complex particle.

[0217] In certain embodiments, one type of saponin fraction or crude saponin fraction may be incorporated into one ISCOM matrix complex or particle, and another type of substantially pure saponin fraction or crude saponin fraction may be incorporated into another ISCOM matrix complex or particle. The composition or vaccine may comprise at least two types of complexes or particles, each type having one type of saponin incorporated into a physically distinct particle.

[0218] The composition may also contain mixtures of ISCOM matrix complex particles and / or ISCOM complex particles in which one saponin fraction Quillaja saponaria Molina and another saponin fraction Quillaja saponaria Molina are separately incorporated into different ISCOM matrix complex particles and / or ISCOM complex particles.

[0219] ISCOM matrices or ISCOM complex particles, each containing one saponin fraction, can be present in the composition in any combination of weight percents. In certain embodiments, the composition can contain 0.1% to 99.9%, 5% to 95%, 10% to 90%, 15% to 85%, 20% to 80%, 25% to 75%, 30% to 70%, 35% to 65%, 40% to 60%, 45% to 55%, 40% to 60%, or 50% by weight of ISCOM matrices or complexes containing a first saponin fraction, with the remainder being ISCOM matrices or complexes containing a different saponin fraction. In some embodiments, the remainder is one or more ISCOM matrices or complexes, where each matrix or complex particle contains only one saponin fraction. In other embodiments, the ISCOM matrix or complex particles may contain two or more saponin fractions.

[0220] In certain compositions, the only saponin fraction in the first ISCOM matrix or ISCOM complex particle is fraction A and the only saponin fraction in the second ISCOM matrix or ISCOM complex particle is fraction C.

[0221] In embodiments, Fraction A of Quillaja Saponaria Molina accounts for at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% by weight of the combined weight of Fraction A of Quillaja Saponaria Molina and Fraction C of Quillaja Saponaria Molina in the adjuvant, respectively, and Fraction C of Quillaja Saponaria Molina accounts for the remainder.

[0222] A preferred composition comprises a first ISCOM matrix containing Fraction A and a second ISCOM matrix containing Fraction C, where the Fraction A ISCOM matrix constitutes about 70% by weight of the total saponin adjuvant, and the Fraction C ISCOM matrix constitutes about 30% by weight of the total saponin adjuvant. In another preferred composition, the Fraction A ISCOM matrix constitutes about 85% by weight of the total saponin adjuvant, and the Fraction C ISCOM matrix constitutes about 15% by weight of the total saponin adjuvant. In another preferred composition, the Fraction A ISCOM matrix constitutes about 92% by weight of the total saponin adjuvant, and the Fraction C ISCOM matrix constitutes about 8% by weight of the total saponin adjuvant. Thus, in certain compositions, the Fraction A ISCOM matrix is ​​present in an amount ranging from about 70% to about 85% and the Fraction C ISCOM matrix is ​​present in an amount ranging from about 15% to about 30% by weight of the total saponin adjuvant. In certain compositions, the Fraction A ISCOM matrix is ​​present in an amount ranging from about 70% to about 92% by weight of the total saponin adjuvant in the composition, and the Fraction C ISCOM matrix is ​​present in an amount ranging from about 8% to about 30% by weight. In some embodiments, the Fraction A ISCOM matrix comprises 50% to 96% by weight of the combined weight of the Fraction A ISCOM matrix and Fraction C ISCOMs in the adjuvant, respectively, with the Fraction C ISCOM matrix comprising the remainder. In a particularly preferred composition, referred to herein as MATRIX-M™, the Fraction A ISCOM matrix is ​​present in an amount ranging from about 85% by weight of the total saponin adjuvant in the composition, and the Fraction C ISCOM matrix is ​​present in an amount ranging from about 15% by weight. MATRIX-M™ may also be referred to interchangeably as Matrix-M1.

[0223] Exemplary QS-7 and QS-21 fractions, their production, and their uses are described in U.S. Patent Nos. 5,057,540, 6,231,859, 6,352,697, 6,524,584, 6,846,489, 7,776,343, and 8,173,141, which are incorporated herein by reference.

[0224] In some embodiments, other adjuvants may be used additionally or alternatively. The inclusion of any adjuvant described in Vogel et al., "A Compendium of Vaccine Adjuvants and Excipients (2nd Edition)," incorporated herein by reference in its entirety for all purposes, is contemplated within the scope of this disclosure. Other adjuvants include complete Freund's adjuvant (a nonspecific stimulator of the immune response containing killed Mycobacterium tuberculosis bacteria), incomplete Freund's adjuvant, and aluminum hydroxide adjuvant. Other adjuvants include GMCSP, BCG, MDP compounds such as thur-MDP and nor-MDP, CGP (MTP-PE), lipid A, and monophosphoryl lipid A (MPL), MF-59, and RIBI, which contains three components extracted from bacteria: MPL, trehalose dimycolate (TDM), and cell wall skeleton (CWS) in a 2% squalene / TWEEN® polysorbate 80 emulsion. In some embodiments, the adjuvant can be paucilamellar lipid vesicles, such as NOVASOMES®. NOVASOMES® are paucilamellar non-phospholipid vesicles ranging from about 100 nm to about 500 nm. These contain BRIJ® alcohol ethoxylate 72, cholesterol, oleic acid, and squalene. NOVASOMES® has been shown to be an effective adjuvant (see U.S. Patent Nos. 5,629,021, 6,387,373, and 4,911,928).

[0225] Amount of adjuvant in the composition In embodiments, the immunogenic compositions described herein comprise from about 1 μg to about 400 μg, 1 μg to about 350 μg, 1 μg to about 300 μg, 1 μg to about 250 μg, 1 μg to about 200 μg, 1 μg to about 150 μg, 1 μg to about 100 μg, 1 μg to about 50 μg, or 1 μg to about 25 μg of adjuvant. In embodiments, the immunogenic composition comprises about 1 μg, about 2 μg, about 3 μg, about 4 μg, about 5 μg, about 6 μg, about 7 μg, about 8 μg, about 9 μg, about 10 μg, about 11 μg, about 12 μg, about 13 μg, about 14 μg, about 15 μg, about 16 μg, about 17 μg, about 18 μg, about 19 μg, about 20 μg, about 21 μg, about 22 μg, about 23 μg, about 24 μg, about 25 μg, about 26 μg, about 27 μg, about 28 μg, about 29 μg, about 30 μg, about 31 μg, about 32 μg, about 33 μg, about 34 μg, Approximately 35μg, approximately 36μg, approximately 37μg, approximately 38μg, approximately 39μg, approximately 40μg, approximately 41μg, approximately 42μg, approximately 43μg, approximately 44μg, approximately 45μg, approximately 46μg, approximately 47μg, approximately 48μg, approximately 49μg, approximately 50μg, approximately 51μg, approximately 52μg , about 53μg, about 54μg, about 55μg, about 56μg, about 57μg, about 58μg, about 59μg, about 60μg, about 61μg, about 62μg, about 63μg, about 64μg, about 65μg, about 66μg, about 67μg, about 68μg, about 69μg, about 70μg g, about 71μg, about 72μg, about 73μg, about 74μg, about 75μg, about 76μg, about 77μg, about 78μg, about 79μg, about 80μg, about 81μg, about 82μg, about 83μg, about 84μg, about 85μg, about 86μg, about 87μg, about 88 μg, approximately 89 μg, approximately 90 μg, approximately 91 μg, approximately 92 μg, approximately 93 μg, approximately 94 μg, approximately 95 μg, approximately 96 μg, approximately 97 μg, approximately 98 μg, approximately 99 μg, approximately 100 μg, approximately 101 μg, approximately 102 μg, approximately 103 μg, approximately 104 μg, approximately 10 5μg, about 106μg, about 107μg, about 108μg, about 109μg, about 110μg, about 111μg, about 112μg, about 113μg, about 114μg, about 115μg, about 116μg, about 117μg, about 118μg, about 119μg, about 120μg g, about 121μg, about 122μg, about 123μg, about 124μg, about 125μg, about 126μg, about 127μg, about 128μg, about 129μg, about 130μg, about 131μg, about 132μg, about 133μg, about 134μg, about 135μg,Approximately 136 μg, approximately 137 μg, approximately 138 μg, approximately 139 μg, approximately 140 μg, approximately 141 μg, approximately 142 μg, approximately 143 μg, approximately 144 μg, approximately 145 μg, approximately 146 μg, approximately 147 μg, approximately 148 μg, approximately 149 μg, approximately 150 μg, approximately 151 μg, approximately 152 μg, approximately 153 μg Approximately 154 μg, approximately 155 μg, approximately 156 μg, approximately 157 μg, approximately 158 μg, approximately 159 μg, approximately 160 μg, approximately 161 μg, approximately 162 μg, approximately 163 μg, approximately 164 μg, approximately 165 μg, approximately 166 μg, approximately 167 μg, approximately 168 μg, approximately 169 μg, approximately 170 μg, approximately 171 μg g, approximately 172μg, approximately 173μg, approximately 174μg, approximately 175μg, approximately 176μg, approximately 177μg, approximately 178μg, approximately 179μg, approximately 180μg, approximately 181μg, approximately 182μg, approximately 183μg, approximately 184μg, approximately 185μg, approximately 186μg, approximately 187μg, approximately 188μg, approximately 189μg μg, approximately 190μg, approximately 191μg, approximately 192μg, approximately 193μg, approximately 194μg, approximately 195μg, approximately 196μg, approximately 197μg, approximately 198μg, approximately 199μg, approximately 200μg, approximately 201μg, approximately 202μg, approximately 203μg, approximately 204μg, approximately 205μg, approximately 206μg, approximately 20 7μg, approximately 208μg, approximately 209μg, approximately 210μg, approximately 211μg, approximately 212μg, approximately 213μg, approximately 214μg, approximately 215μg, approximately 216μg, approximately 217μg, approximately 218μg, approximately 219μg, approximately 220μg, approximately 221μg, approximately 222μg, approximately 223μg, approximately 224μg, approximately 2 25μg, approximately 226μg, approximately 227μg, approximately 228μg, approximately 229μg, approximately 230μg, approximately 231μg, approximately 232μg, approximately 233μg, approximately 234μg, approximately 235μg, approximately 236μg, approximately 237μg, approximately 238μg, approximately 239μg, approximately 240μg, approximately 241μg, approximately 242μg, approximately 243μg, approximately 244μg, approximately 245μg, approximately 246μg, approximately 247μg, approximately 248μg, approximately 249μg, approximately 250μg, approximately 251μg, approximately 252μg, approximately 253μg, approximately 254μg, approximately 255μg, approximately 256μg, approximately 257μg, approximately 258μg, approximately 259μg, approximately 260μg Approximately 261 μg, approximately 262 μg, approximately 263 μg, approximately 264 μg, approximately 265 μg, approximately 266 μg, approximately 267 μg, approximately 268 μg, approximately 269 μg, approximately 270 μg, approximately 271 μg, approximately 272 μg, approximately 273 μg, approximately 274 μg, approximately 275 μg, approximately 276 μg, approximately 277 μg, approximately 278 μgApproximately 279μg, approximately 280μg, approximately 281μg, approximately 282μg, approximately 283μg, approximately 284μg, approximately 285μg, approximately 286μg, approximately 287μg, approximately 288μg, approximately 289μg, approximately 290μg, approximately 291μg, approximately 292μg, approximately 293μg, approximately 29 4μg, about 295μg, about 296μg, about 297μg, about 298μg, about 299μg, about 300μg, about 301μg, about 302μg, about 303μg, about 304μg, about 305μg, about 306μg, about 307μg, about 308μg, about 309μg , about 310μg, about 311μg, about 312μg, about 313μg, about 314μg, about 315μg, about 316μg, about 317μg, about 318μg, about 319μg, about 320μg, about 321μg, about 322μg, about 323μg, about 324μg, about 3 25μg, about 326μg, about 327μg, about 328μg, about 329μg, about 330μg, about 331μg, about 332μg, about 333μg, about 334μg, about 335μg, about 336μg, about 337μg, about 338μg, about 339μg, about 340μg , about 341μg, about 342μg, about 343μg, about 344μg, about 345μg, about 346μg, about 347μg, about 348μg, about 349μg, about 350μg, about 351μg, about 352μg, about 353μg, about 354μg, about 355μg, about 3 56μg, about 357μg, about 358μg, about 359μg, about 360μg, about 361μg, about 362μg, about 363μg, about 364μg, about 365μg, about 366μg, about 367μg, about 368μg, about 369μg, about 370μg, about 371μg , about 372 μg, about 373 μg, about 374 μg, about 375 μg, about 376 μg, about 377 μg, about 378 μg, about 379 μg, about 380 μg, about 381 μg, about 382 μg, about 383 μg, about 384 μg, about 385 μg, about 386 μg, about 387 μg, about 388 μg, about 389 μg, about 390 μg, about 391 μg, about 392 μg, about 393 μg, about 394 μg, about 395 μg, about 396 μg, about 397 μg, about 398 μg, about 399 μg, or about 400 μg of adjuvant. In some embodiments, the immunogenic composition comprises about 50 μg of adjuvant. In embodiments, the immunogenic composition comprises about 75 μg of adjuvant.

[0226] Methods for stimulating an immune response against RSV, influenza, SARS-CoV-2 or variants thereof, or combinations thereof In some embodiments, the present disclosure provides a method for eliciting an immune response against one or more of RSV, influenza, and SARS-CoV-2 or variants thereof, comprising administering an immunogenic composition described herein. In some embodiments, the immunogenic composition contains one or more viral glycoproteins. In some embodiments, the one or more viral glycoproteins are selected from any one of the RSV F glycoprotein, CoV S glycoprotein, and influenza HA glycoprotein.

[0227] In some embodiments, the immunogenic composition lacks an adjuvant. In some embodiments, the immunogenic composition comprises an adjuvant. Non-limiting examples of adjuvants are described herein.

[0228] In embodiments, the immunogenic compositions described herein have an efficacy of about 50% to about 99%, about 80% to about 99%, about 75% to about 99%, about 80% to about 95%, about 90% to about 98%, about 75% to about 95%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% against a virus (e.g., RSV, influenza, SARS-CoV-2, a SARS-CoV-2 variant thereof, or a combination thereof).

[0229] The compositions disclosed herein can be administered via systemic, mucosal, or transdermal routes, or directly to specific tissues. As used herein, the term "systemic administration" includes parenteral administration routes. Specifically, parenteral administration includes subcutaneous, intraperitoneal, intravenous, intraarterial, intramuscular, or intrasternal injection, intravenous infusion techniques, or renal dialysis infusion techniques. Typically, systemic parenteral administration is intramuscular injection. As used herein, the term "mucosal administration" includes oral, intranasal, intravaginal, rectal, intratracheal, intestinal, and ophthalmic administration. In some embodiments, the method comprises administering the immunogenic composition described herein intramuscularly. In some embodiments, the method comprises administering the immunogenic composition described herein intranasally.

[0230] The composition can be administered in a single dose schedule or a multiple dose schedule. Multiple doses can be used in a primary immunization schedule or a booster immunization schedule. In multiple embodiments, about 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, or 30 doses are administered. In a multiple dose schedule, the various doses can be administered by the same or different routes, such as parenteral prime and mucosal boost, mucosal prime and parenteral boost, etc. In some embodiments, the boost dose is administered about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months (1 year), about 2 years, about 3 years, about 4 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, or about 10 years after the first dose. In some embodiments, the boost dose is administered annually after the administration of the first dose. In some embodiments, subsequent boost doses are administered 3 or 4 weeks after the administration of the previous dose. In some embodiments, the first dose is administered on day 0 and the boost dose is administered on day 21. In some embodiments, the first dose is administered on day 0 and the boost dose is administered on day 28. In some embodiments, the first dose is administered on day 0, the boost dose is administered on day 21, and the second boost dose is administered about 6 months after administration of the first dose or the second dose. In some embodiments, the first dose is administered on day 0, the boost dose is administered on day 28, and the second boost dose is administered about 6 months after administration of the first dose. In some embodiments, the first dose is administered on day 0, the boost dose is administered on day 21, and the second boost dose is administered about 6 months after administration of the second dose. In some embodiments, the first dose is administered on day 0, the boost dose is administered on day 28, and the second boost dose is administered about 6 months after administration of the second dose.

[0231] In some embodiments, the first dose is administered on day 0, the boost dose is administered on day 21, and the second boost dose is administered about one year after administration of the first dose or the first boost dose. In some embodiments, the first dose is administered on day 0, the first boost dose is administered on day 28, and the second boost dose is administered about one year after administration of the first dose. In some embodiments, the first dose is administered on day 0, the boost dose is administered on day 21, and the second boost dose is administered about one year after administration of the second dose. In some embodiments, the first dose is administered on day 0, the first boost dose is administered on day 28, and the second boost dose is administered about one year after administration of the second dose. In some embodiments, the second boost dose is administered 6 to 24 months or 12 to 24 months after the first boost dose.

[0232] In some embodiments, the dose measured in μg can be the total weight of the dose including solute, or the weight of the RSV F glycoprotein nanoparticles, or the weight of the RSV F glycoprotein. The dose is measured using either the protein concentration assay A280 or ELISA.

[0233] Certain populations may be administered with or without an adjuvant. In certain embodiments, the composition may not include an added adjuvant. In such situations, the dose may be increased by about 10%.

[0234] In some embodiments, the immunogenic compositions described herein are provided in pre-filled syringes. When the immunogenic compositions are prepared in pre-filled syringes, the RSV F glycoprotein and adjuvant are combined prior to administration.

[0235] In some embodiments, the dose is administered in a volume of about 0.1 mL to about 1.5 mL, e.g., about 0.1 mL, about 0.2 mL, about 0.25 mL, about 0.3 mL, about 0.4 mL, about 0.5 mL, about 0.6 mL, about 0.7 mL, about 0.8 mL, about 0.9 mL, about 1.0 mL, about 1.1 mL, about 1.2 mL, about 1.3 mL, about 1.4 mL, or about 1.5 mL. In some embodiments, the dose is administered in a volume of 0.25 mL. In some embodiments, the dose is administered in a volume of 0.5 mL. In some embodiments, the dose is administered in a volume of 0.6 mL.

[0236] In embodiments, the concentration of viral glycoprotein nanoparticles (e.g., RSV F, CoV S, or HA glycoprotein nanoparticles) in the immunogenic or vaccine compositions described herein is from about 1 μg / mL to about 50 μg / mL, 10 μg / mL to about 100 μg / mL, from about 10 μg / mL to about 50 μg / mL, from about 175 μg / mL to about 325 μg / mL, from about 200 μg / mL to about 300 μg / mL, from about 220 μg / mL to about 280 μg / mL, or from about 240 μg / mL to about 260 μg / mL.

[0237] In some embodiments, the method comprises administering about 1 μg to about 300 μg, about 5 μg to about 25 μg, about 1 μg to about 300 μg, about 90 μg to about 270 μg, about 100 μg to about 160 μg, about 110 μg to about 150 μg, about 120 μg to about 140 μg, or about 140 μg to about 160 μg of each RSV F glycoprotein. In some embodiments, the method comprises administering about 5 μg to about 60 μg of each RSV F glycoprotein. In some embodiments, the method comprises administering about 24 μg to about 40 μg of each RSV F glycoprotein. In some embodiments, the method comprises administering about 30 μg to about 40 μg of each RSV F glycoprotein. In some embodiments, the method comprises administering about 30 μg to about 60 μg of each RSV F glycoprotein. In some embodiments, the method comprises administering about 33 μg to about 39 μg of each RSV F glycoprotein. In some embodiments, the method comprises administering about 20 μg to about 240 μg of each RSV F glycoprotein. In some embodiments, the method comprises administering about 30 μg to about 60 μg of each RSV F glycoprotein. In some embodiments, the method comprises administering about 24 μg to about 40 μg of each RSV F glycoprotein. In embodiments, the methods provide for the administration of a medicament containing at least one of the following: about 1 μg, about 2 μg, about 3 μg, about 4 μg, about 5 μg, about 6 μg, about 7 μg, about 8 μg, about 9 μg, about 10 μg, about 11 μg, about 12 μg, about 13 μg, about 14 μg, about 15 μg, about 16 μg, about 17 μg, about 18 μg, about 19 μg, about 20 μg, about 21 μg, about 22 μg, about 23 μg, about 24 μg, about 25 μg, about 26 μg, about 27 μg, about 28 μg, about 29 μg, about 30 μg, about 31 μg, about 32 μg, about 33 μg, about 34 μg, about 35 μg, about 36 μg, about 37 μg, about 38 μg, about 39 μg, about 40 μg, about 41 μg, about 42 μg, about 43 μg, about 44 μg, about 45 μg, about 46 μg, about 47 μg, about 48 μg, about 49 μg, about 50 μg, about 51 μg, about 52 μg, about 53 μg, about 54 μg, about 55 μg, about 56 μg, about 57 μg, about 58 μg, about 59 μg, about 60 μg, about 61 μg, about 62 μg, about 63 μg, about 64 μg, about 65 μg, about 66 μg, about 67 μg, about 68 μg, about 69 μg, about 70 μg, about 71 μg, μg, approx. 39 μg, approx. 40 μg, approx. 41 μg, approx. 42 μg, approx. 43 μg, approx. 44 μg, approx. 45 μg, approx. 46 μg, approx. 47 μg, approx. 48 μg, approx. 49 μg, approx. 50 μg, approx. g, about 58μg, about 59μg, about 60μg, about 61μg, about 62μg, about 63μg, about 64μg, about 65μg, about 66μg, about 67μg, about 68μg, about 69μg, about 70μg, about 71μg, about 72μg, about 73μg, about 74μg, about 75μg, about 76μg,Approximately 77μg, approximately 78μg, approximately 79μg, approximately 80μg, approximately 81μg, approximately 82μg, approximately 83μg, approximately 84μg, approximately 85μg, approximately 86μg, approximately 87μg, approximately 88μg, approximately 89μg, approximately 90μg, approximately 91μg, approximately 92μg, approximately 93μg, approximately 94μg, approximately 95μg, approximately 96μg, approximately 97μg g, approximately 98 μg, approximately 99 μg, approximately 100 μg, approximately 101 μg, approximately 102 μg, approximately 103 μg, approximately 104 μg, approximately 105 μg, approximately 106 μg, approximately 107 μg, approximately 108 μg, approximately 109 μg, approximately 110 μg, approximately 111 μg, approximately 112 μg, approximately 113 μg, approximately 114 μg, approximately 115 μg Approximately 116 μg, approximately 117 μg, approximately 118 μg, approximately 119 μg, approximately 120 μg, approximately 121 μg, approximately 122 μg, approximately 123 μg, approximately 124 μg, approximately 125 μg, approximately 126 μg, approximately 127 μg, approximately 128 μg, approximately 129 μg, approximately 130 μg, approximately 131 μg, approximately 132 μg, approximately 133 μg g, approximately 134μg, approximately 135μg, approximately 136μg, approximately 137μg, approximately 138μg, approximately 139μg, approximately 140μg, approximately 141μg, approximately 142μg, approximately 143μg, approximately 144μg, approximately 145μg, approximately 146μg, approximately 147μg, approximately 148μg, approximately 149μg, approximately 150μg, approximately 151 μg, approximately 152μg, approximately 153μg, approximately 154μg, approximately 155μg, approximately 156μg, approximately 157μg, approximately 158μg, approximately 159μg, approximately 160μg, approximately 161μg, approximately 162μg, approximately 163μg, approximately 164μg, approximately 165μg, approximately 166μg, approximately 167μg, approximately 168μg, approximately 16 9μg, approximately 170μg, approximately 171μg, approximately 172μg, approximately 173μg, approximately 174μg, approximately 175μg, approximately 176μg, approximately 177μg, approximately 178μg, approximately 179μg, approximately 180μg, approximately 181μg, approximately 182μg, approximately 183μg, approximately 184μg, approximately 185μg, approximately 186μg, approximately 1 87μg, approximately 188μg, approximately 189μg, approximately 190μg, approximately 191μg, approximately 192μg, approximately 193μg, approximately 194μg, approximately 195μg, approximately 196μg, approximately 197μg, approximately 198μg, approximately 199μg, approximately 200μg, approximately 201μg, approximately 202μg, approximately 203μg, approximately 204μg, approximately 205μg, approximately 206μg, approximately 207μg, approximately 208μg, approximately 209μg, approximately 210μg, approximately 211μg, approximately 212μg, approximately 213μg, approximately 214μg, approximately 215μg, approximately 216μg, approximately 217μg, approximately 218μg, approximately 219μg, approximately 220μg, approximately 221μg, approximately 222μgApproximately 223μg, approximately 224μg, approximately 225μg, approximately 226μg, approximately 227μg, approximately 228μg, approximately 229μg, approximately 230μg, approximately 231μg, approximately 232μg , about 233 μg, about 234 μg, about 235 μg, about 236 μg, about 237 μg, about 238 μg, about 239 μg, about 240 μg, about 241 μg, about 242 μg g, approximately 243 μg, approximately 244 μg, approximately 245 μg, approximately 246 μg, approximately 247 μg, approximately 248 μg, approximately 249 μg, approximately 250 μg, approximately 251 μg, approximately 252 μg, approx. 253 μg, approx. 254 μg, approx. 255 μg, approx. 256 μg, approx. 257 μg, approx. 258 μg, approx. 259 μg, approx. 260 μg, approx. 261 μg, approx. 26 2μg, about 263μg, about 264μg, about 265μg, about 266μg, about 267μg, about 268μg, about 269μg, about 270μg, about 271μg, about 2 72μg, about 273μg, about 274μg, about 275μg, about 276μg, about 277μg, about 278μg, about 279μg, about 280μg, about 281μg, about In some embodiments, the method comprises administering about 282 μg, about 283 μg, about 284 μg, about 285 μg, about 286 μg, about 287 μg, about 288 μg, about 289 μg, about 290 μg, about 291 μg, about 292 μg, about 293 μg, about 294 μg, about 295 μg, about 296 μg, about 297 μg, about 298 μg, about 299 μg, or about 300 μg of each RSV F glycoprotein (including all values ​​and ranges therebetween). In some embodiments, the method comprises administering about 60 μg of each RSV F glycoprotein. In some embodiments, the method comprises administering about 240 μg of each RSV F glycoprotein.

[0238] In some embodiments, the methods involve administering between about 1 μg and about 1000 μg of total RSV F glycoprotein. In embodiments, the methods provide for the administration of a medicament containing at least one of the following: about 1 μg, about 2 μg, about 3 μg, about 4 μg, about 5 μg, about 6 μg, about 7 μg, about 8 μg, about 9 μg, about 10 μg, about 11 μg, about 12 μg, about 13 μg, about 14 μg, about 15 μg, about 16 μg, about 17 μg, about 18 μg, about 19 μg, about 20 μg, about 21 μg, about 22 μg, about 23 μg, about 24 μg, about 25 μg, about 26 μg, about 27 μg, about 28 μg, about 29 μg, about 30 μg, about 31 μg, about 32 μg, about 33 μg, about 34 μg, about 35 μg, about 36 μg, about 37 μg, about 38 μg, about 39 μg, about 40 μg, about 41 μg, about 42 μg, about 43 μg, about 44 μg, about 45 μg, about 46 μg, about 47 μg, about 48 μg, about 49 μg, about 50 μg, about 51 μg, about 52 μg, about 53 μg, about 54 μg, about 55 μg, about 56 μg, about 57 μg, about 58 μg, about 59 μg, about 60 μg, about 61 μg, about 62 μg, about 63 μg, about 64 μg, about 65 μg, about 66 μg, about 67 μg, about 68 μg, about 69 μg, about 70 μg, about 71 μg, μg, approximately 40 μg, approximately 41 μg, approximately 42 μg, approximately 43 μg, approximately 44 μg, approximately 45 μg, approximately 46 μg, approximately 47 μg, approximately 48 μg, approximately 4 9μg, about 50μg, about 51μg, about 52μg, about 53μg, about 54μg, about 55μg, about 56μg, about 57μg, about 58μg, about 5 9μg, about 60μg, about 61μg, about 62μg, about 63μg, about 64μg, about 65μg, about 66μg, about 67μg, about 68μg, about 69μg, about 70μg, about 71μg, about 72μg, about 73μg, about 74μg, about 75μg, about 76μg, about 77μg, about 78μg, about 79μg, about 80μg, about 81μg, about 82μg, about 83μg, about 84μg, about 85μg, about 86μg, about 87μg, about 88μg, Approximately 89μg, approximately 90μg, approximately 91μg, approximately 92μg, approximately 93μg, approximately 94μg, approximately 95μg, approximately 96μg, approximately 97μg, approximately 98μg , about 99μg, about 100μg, about 101μg, about 102μg, about 103μg, about 104μg, about 105μg, about 106μg, about 10 7μg, about 108μg, about 109μg, about 110μg, about 111μg, about 112μg, about 113μg, about 114μg, about 115μg, Approximately 116μg, approximately 117μg, approximately 118μg, approximately 119μg, approximately 120μg, approximately 121μg, approximately 122μg, approximately 123μg, approximately 12 4μg, about 125μg, about 126μg, about 127μg, about 128μg, about 129μg, about 130μg, about 131μg, about 132μg, Approximately 133μg, approximately 134μg, approximately 135μg, approximately 136μg, approximately 137μg, approximately 138μg, approximately 139μg, approximately 140μg, approximately 14 1μg, about 142μg, about 143μg, about 144μg, about 145μg, about 146μg, about 147μg, about 148μg, about 149μg,Approximately 150 μg, approximately 151 μg, approximately 152 μg, approximately 153 μg, approximately 154 μg, approximately 155 μg, approximately 156 μg, approximately 157 μg, approximately 158 μg, approximately 159 μg, approximately 160 μg, approximately 161 μg, approximately 162 μg, approximately 163 μg, approximately 164 μg, approximately 165 μg, approximately 166 μg, approximately 167 μg Approximately 168 μg, approximately 169 μg, approximately 170 μg, approximately 171 μg, approximately 172 μg, approximately 173 μg, approximately 174 μg, approximately 175 μg, approximately 176 μg, approximately 177 μg, approximately 178 μg, approximately 179 μg, approximately 180 μg, approximately 181 μg, approximately 182 μg, approximately 183 μg, approximately 184 μg, approximately 185 μg g, approximately 186μg, approximately 187μg, approximately 188μg, approximately 189μg, approximately 190μg, approximately 191μg, approximately 192μg, approximately 193μg, approximately 194μg, approximately 195μg, approximately 196μg, approximately 197μg, approximately 198μg, approximately 199μg, approximately 200μg, approximately 201μg, approximately 202μg, approximately 203μg μg, approximately 204μg, approximately 205μg, approximately 206μg, approximately 207μg, approximately 208μg, approximately 209μg, approximately 210μg, approximately 211μg, approximately 212μg, approximately 213μg, approximately 214μg, approximately 215μg, approximately 216μg, approximately 217μg, approximately 218μg, approximately 219μg, approximately 220μg, approximately 22 1 μg, approximately 222 μg, approximately 223 μg, approximately 224 μg, approximately 225 μg, approximately 226 μg, approximately 227 μg, approximately 228 μg, approximately 229 μg, approximately 230 μg, approximately 231 μg, approximately 232 μg, approximately 233 μg, approximately 234 μg, approximately 235 μg, approximately 236 μg, approximately 237 μg, approximately 238 μg, approximately 2 39μg, approximately 240μg, approximately 241μg, approximately 242μg, approximately 243μg, approximately 244μg, approximately 245μg, approximately 246μg, approximately 247μg, approximately 248μg, approximately 249μg, approximately 250μg, approximately 251μg, approximately 252μg, approximately 253μg, approximately 254μg, approximately 255μg, approximately 256μg, approximately 257μg, approximately 258μg, approximately 259μg, approximately 260μg, approximately 261μg, approximately 262μg, approximately 263μg, approximately 264μg, approximately 265μg, approximately 266μg, approximately 267μg, approximately 268μg, approximately 269μg, approximately 270μg, approximately 271μg, approximately 272μg, approximately 273μg, approximately 274μg Approximately 275 μg, approximately 276 μg, approximately 277 μg, approximately 278 μg, approximately 279 μg, approximately 280 μg, approximately 281 μg, approximately 282 μg, approximately 283 μg, approximately 284 μg, approximately 285 μg, approximately 286 μg, approximately 287 μg, approximately 288 μg, approximately 289 μg, approximately 290 μg, approximately 291 μg, approximately 292 μgApproximately 293 μg, approximately 294 μg, approximately 295 μg, approximately 296 μg, approximately 297 μg, approximately 298 μg, approximately 299 μg, approximately 300 μg, approximately 301 μg, approximately 302 μg, approximately 303 μg, approximately 304 μg, approximately 305 μg, approximately 306 μg, approximately 307 μg, approximately 308 μg, approximately 309 μg, approximately 310 μg Approximately 311 μg, approximately 312 μg, approximately 313 μg, approximately 314 μg, approximately 315 μg, approximately 316 μg, approximately 317 μg, approximately 318 μg, approximately 319 μg, approximately 320 μg, approximately 321 μg, approximately 322 μg, approximately 323 μg, approximately 324 μg, approximately 325 μg, approximately 326 μg, approximately 327 μg, approximately 328 μg g, approximately 329 μg, approximately 330 μg, approximately 331 μg, approximately 332 μg, approximately 333 μg, approximately 334 μg, approximately 335 μg, approximately 336 μg, approximately 337 μg, approximately 338 μg, approximately 339 μg, approximately 340 μg, approximately 341 μg, approximately 342 μg, approximately 343 μg, approximately 344 μg, approximately 345 μg, approximately 346 μg μg, approximately 347μg, approximately 348μg, approximately 349μg, approximately 350μg, approximately 351μg, approximately 352μg, approximately 353μg, approximately 354μg, approximately 355μg, approximately 356μg, approximately 357μg, approximately 358μg, approximately 359μg, approximately 360μg, approximately 361μg, approximately 362μg, approximately 363μg, approximately 36 4μg, approximately 365μg, approximately 366μg, approximately 367μg, approximately 368μg, approximately 369μg, approximately 370μg, approximately 371μg, approximately 372μg, approximately 373μg, approximately 374μg, approximately 375μg, approximately 376μg, approximately 377μg, approximately 378μg, approximately 379μg, approximately 380μg, approximately 381μg, approximately 3 82μg, approximately 383μg, approximately 384μg, approximately 385μg, approximately 386μg, approximately 387μg, approximately 388μg, approximately 389μg, approximately 390μg, approximately 391μg, approximately 392μg, approximately 393μg, approximately 394μg, approximately 395μg, approximately 396μg, approximately 397μg, approximately 398μg, approximately 399μg, approximately 400μg, approximately 401μg, approximately 402μg, approximately 403μg, approximately 404μg, approximately 405μg, approximately 406μg, approximately 407μg, approximately 408μg, approximately 409μg, approximately 410μg, approximately 411μg, approximately 412μg, approximately 413μg, approximately 414μg, approximately 415μg, approximately 416μg, approximately 417μg Approximately 418 μg, approximately 419 μg, approximately 420 μg, approximately 421 μg, approximately 422 μg, approximately 423 μg, approximately 424 μg, approximately 425 μg, approximately 426 μg, approximately 427 μg, approximately 428 μg, approximately 429 μg, approximately 430 μg, approximately 431 μg, approximately 432 μg, approximately 433 μg, approximately 434 μg, approximately 435 μgApproximately 436 μg, approximately 437 μg, approximately 438 μg, approximately 439 μg, approximately 440 μg, approximately 441 μg, approximately 442 μg, approximately 443 μg, approximately 444 μg, approximately 445 μg, approximately 446 μg, approximately 447 μg, approximately 448 μg, approximately 449 μg, approximately 450 μg, approximately 451 μg, approximately 452 μg, approximately 453 μg Approximately 454 μg, approximately 455 μg, approximately 456 μg, approximately 457 μg, approximately 458 μg, approximately 459 μg, approximately 460 μg, approximately 461 μg, approximately 462 μg, approximately 463 μg, approximately 464 μg, approximately 465 μg, approximately 466 μg, approximately 467 μg, approximately 468 μg, approximately 469 μg, approximately 470 μg, approximately 471 μg g, approximately 472μg, approximately 473μg, approximately 474μg, approximately 475μg, approximately 476μg, approximately 477μg, approximately 478μg, approximately 479μg, approximately 480μg, approximately 481μg, approximately 482μg, approximately 483μg, approximately 484μg, approximately 485μg, approximately 486μg, approximately 487μg, approximately 488μg, approximately 489μg μg, approximately 490μg, approximately 491μg, approximately 492μg, approximately 493μg, approximately 494μg, approximately 495μg, approximately 496μg, approximately 497μg, approximately 498μg, approximately 499μg, approximately 500μg, approximately 501μg, approximately 502μg, approximately 503μg, approximately 504μg, approximately 505μg, approximately 506μg, approximately 50 7μg, approximately 508μg, approximately 509μg, approximately 510μg, approximately 511μg, approximately 512μg, approximately 513μg, approximately 514μg, approximately 515μg, approximately 516μg, approximately 517μg, approximately 518μg, approximately 519μg, approximately 520μg, approximately 521μg, approximately 522μg, approximately 523μg, approximately 524μg, approximately 5 25μg, approximately 526μg, approximately 527μg, approximately 528μg, approximately 529μg, approximately 530μg, approximately 531μg, approximately 532μg, approximately 533μg, approximately 534μg, approximately 535μg, approximately 536μg, approximately 537μg, approximately 538μg, approximately 539μg, approximately 540μg, approximately 541μg, approximately 542μg, approximately 543μg, approximately 544μg, approximately 545μg, approximately 546μg, approximately 547μg, approximately 548μg, approximately 549μg, approximately 550μg, approximately 551μg, approximately 552μg, approximately 553μg, approximately 554μg, approximately 555μg, approximately 556μg, approximately 557μg, approximately 558μg, approximately 559μg, approximately 560μg Approximately 561 μg, approximately 562 μg, approximately 563 μg, approximately 564 μg, approximately 565 μg, approximately 566 μg, approximately 567 μg, approximately 568 μg, approximately 569 μg, approximately 570 μg, approximately 571 μg, approximately 572 μg, approximately 573 μg, approximately 574 μg, approximately 575 μg, approximately 576 μg, approximately 577 μg, approximately 578 μgApproximately 579 μg, approximately 580 μg, approximately 581 μg, approximately 582 μg, approximately 583 μg, approximately 584 μg, approximately 585 μg, approximately 586 μg, approximately 587 μg, approximately 588 μg, approximately 589 μg, approximately 590 μg, approximately 591 μg, approximately 592 μg, approximately 593 μg, approximately 594 μg, approximately 595 μg, approximately 596 μg Approximately 597 μg, approximately 598 μg, approximately 599 μg, approximately 600 μg, approximately 601 μg, approximately 602 μg, approximately 603 μg, approximately 604 μg, approximately 605 μg, approximately 606 μg, approximately 607 μg, approximately 608 μg, approximately 609 μg, approximately 610 μg, approximately 611 μg, approximately 612 μg, approximately 613 μg, approximately 614 μg g, approximately 615μg, approximately 616μg, approximately 617μg, approximately 618μg, approximately 619μg, approximately 620μg, approximately 621μg, approximately 622μg, approximately 623μg, approximately 624μg, approximately 625μg, approximately 626μg, approximately 627μg, approximately 628μg, approximately 629μg, approximately 630μg, approximately 631μg, approximately 632 μg, approximately 633μg, approximately 634μg, approximately 635μg, approximately 636μg, approximately 637μg, approximately 638μg, approximately 639μg, approximately 640μg, approximately 641μg, approximately 642μg, approximately 643μg, approximately 644μg, approximately 645μg, approximately 646μg, approximately 647μg, approximately 648μg, approximately 649μg, approximately 65μg 0 μg, approximately 651 μg, approximately 652 μg, approximately 653 μg, approximately 654 μg, approximately 655 μg, approximately 656 μg, approximately 657 μg, approximately 658 μg, approximately 659 μg, approximately 660 μg, approximately 661 μg, approximately 662 μg, approximately 663 μg, approximately 664 μg, approximately 665 μg, approximately 666 μg, approximately 667 μg, approximately 6 68μg, approximately 669μg, approximately 670μg, approximately 671μg, approximately 672μg, approximately 673μg, approximately 674μg, approximately 675μg, approximately 676μg, approximately 677μg, approximately 678μg, approximately 679μg, approximately 680μg, approximately 681μg, approximately 682μg, approximately 683μg, approximately 684μg, approximately 685μg, approximately 686μg, approximately 687μg, approximately 688μg, approximately 689μg, approximately 690μg, approximately 691μg, approximately 692μg, approximately 693μg, approximately 694μg, approximately 695μg, approximately 696μg, approximately 697μg, approximately 698μg, approximately 699μg, approximately 700μg, approximately 701μg, approximately 702μg, approximately 703μg Approximately 704 μg, approximately 705 μg, approximately 706 μg, approximately 707 μg, approximately 708 μg, approximately 709 μg, approximately 710 μg, approximately 711 μg, approximately 712 μg, approximately 713 μg, approximately 714 μg, approximately 715 μg, approximately 716 μg, approximately 717 μg, approximately 718 μg, approximately 719 μg, approximately 720 μg, approximately 721 μgApproximately 722 μg, approximately 723 μg, approximately 724 μg, approximately 725 μg, approximately 726 μg, approximately 727 μg, approximately 728 μg, approximately 729 μg, approximately 730 μg, approximately 731 μg, approximately 732 μg, approximately 733 μg, approximately 734 μg, approximately 735 μg, approximately 736 μg, approximately 737 μg, approximately 738 ... μg, approximately 739μg, approximately 740μg, approximately 741μg, approximately 742μg, approximately 743μg, approximately 744μg, approximately 745μg, approximately 746μg, approximately 747μg, approximately 748μg, approximately 749μg, approximately 750μg, approximately 751μg, approximately 752μg, approximately 753μg, approximately 754μg, approximately 755μg, approximately 7 56μg, approximately 757μg, approximately 758μg, approximately 759μg, approximately 760μg, approximately 761μg, approximately 762μg, approximately 763μg, approximately 764μg, approximately 765μg, approximately 766μg, approximately 767μg, approximately 768μg, approximately 769μg, approximately 770μg, approximately 771μg, approximately 772μg, approximately 773μg, approximately 774μg, approximately 775μg, approximately 776μg, approximately 777μg, approximately 778μg, approximately 779μg, approximately 780μg, approximately 781μg, approximately 782μg, approximately 783μg, approximately 784μg, approximately 785μg, approximately 786μg, approximately 787μg, approximately 788μg, approximately 789μg, approximately 790μg, approximately 791μg Approximately 792 μg, approximately 793 μg, approximately 794 μg, approximately 795 μg, approximately 796 μg, approximately 797 μg, approximately 798 μg, approximately 799 μg, approximately 800 μg, approximately 801 μg, approximately 802 μg, approximately 803 μg, approximately 804 μg, approximately 805 μg, approximately 806 μg, approximately 807 μg, approximately 808 μg, approximately 809 μg g, approximately 810 μg, approximately 811 μg, approximately 812 μg, approximately 813 μg, approximately 814 μg, approximately 815 μg, approximately 816 μg, approximately 817 μg, approximately 818 μg, approximately 819 μg, approximately 820 μg, approximately 821 μg, approximately 822 μg, approximately 823 μg, approximately 824 μg, approximately 825 μg, approximately 826 μg, approximately 82 7μg, approximately 828μg, approximately 829μg, approximately 830μg, approximately 831μg, approximately 832μg, approximately 833μg, approximately 834μg, approximately 835μg, approximately 836μg, approximately 837μg, approximately 838μg, approximately 839μg, approximately 840μg, approximately 841μg, approximately 842μg, approximately 843μg, approximately 844μg, approximately 8 45μg, approximately 846μg, approximately 847μg, approximately 848μg, approximately 849μg, approximately 850μg, approximately 851μg, approximately 852μg, approximately 853μg, approximately 854μg, approximately 855μg, approximately 856μg, approximately 857μg, approximately 858μg, approximately 859μg, approximately 860μg, approximately 861μg, approximately 862μg, approximately 863μg, approximately 864μg, approximately 865μg, approximately 866μg, approximately 867μg, approximately 868μg, approximately 869μg, approximately 870μg, approximately 871μg, approximately 872μg, approximately 873μg, approximately 874μg, approximately 875μg, approximately 876μg, approximately 877μg, approximately 878μg, approximately 879μg, approximately 880μgAbout 881 μg, about 882 μg, about 883 μg, about 884 μg, about 885 μg, about 886 μg, about 887 μg, about 888 μg, about 889 μg, about 890 μg, about 891 μg, about 892 μg, about 893 μg, about 894 μg, about 895 μg, about 896 μg, about 897 μg, about 898 μg, about 899 μg, about 900 μg, about 901 μg, about 902 μg, about 903 μg, about 904 μg, about 905 μg, about 906 μg, about 907 μg, about 908 μg, about 909 μg, about 910 μg, about 911μg, about 912μg, about 913μg, about 914μg, about 915μg, about 916μg, about 917μg, about 918μg, about 919μg, about 920μg, about 921μg, about 922μg, about 923μg, about 924μg, about 925μg, about 926μg, about 927μg, about 928μg, about 929μg, about 930μg, about 931μg, about 932μg, about 933μg, about 934μg, about 935μg, about 936μg, about 937μg, about 938μg, about 939μg, about 940μg, about 9 41μg, about 942μg, about 943μg, about 944μg, about 945μg, about 946μg, about 947μg, about 948μg, about 949μg, about 950μg, about 951μg, about 952μg, about 953μg, about 954μg, about 955μg, about 9 56μg, about 957μg, about 958μg, about 959μg, about 960μg, about 961μg, about 962μg, about 963μg, about 964μg, about 965μg, about 966μg, about 967μg, about 968μg, about 969μg, about 970μg, about 97 1 μg, about 972 μg, about 973 μg, about 974 μg, about 975 μg, about 976 μg, about 977 μg, about 978 μg, about 979 μg, about 980 μg, about 981 μg, about 982 μg, about 983 μg, about 984 μg, about 985 μg, about 986 μg, about 987 μg, about 988 μg, about 989 μg, about 990 μg, about 991 μg, about 992 μg, about 993 μg, about 994 μg, about 995 μg, about 996 μg, about 997 μg, about 998 μg, about 999 μg, about 1000 μg of total RSV F glycoprotein (including all values ​​and ranges therebetween).

[0239] In some embodiments, the method comprises administering between about 1 μg and about 100 μg of each CoV S glycoprotein. In some embodiments, the method comprises administering between about 2.5 μg and about 35 μg of each CoV S glycoprotein. In some embodiments, the method comprises administering between about 5 μg and about 25 μg of each CoV S glycoprotein. In some embodiments, the method comprises administering between about 14 μg and about 15 μg of each CoV S glycoprotein. In some embodiments, the method comprises administering between about 15 μg and about 35 μg of each CoV S glycoprotein. In some embodiments, the method comprises administering between about 1 μg and about 100 μg of total CoV S glycoprotein. In embodiments, the methods provide for the administration of about 1 μg, about 2 μg, about 3 μg, about 4 μg, about 5 μg, about 6 μg, about 7 μg, about 8 μg, about 9 μg, about 10 μg, about 11 μg, about 12 μg, about 13 μg, about 14 μg, about 15 μg, about 16 μg, about 17 μg, about 18 μg, about 19 μg, about 20 μg, about 21 μg, about 22 μg, about 23 μg, about 24 μg, about 25 μg, about 26μg, about 27μg, about 28μg, about 29μg, about 30μg, about 31μg, about 32μg, about 33μg, about 34μg, about 35μg, about 36μg, about 37μg, about 38μg, about 3 9μg, about 40μg, about 41μg, about 42μg, about 43μg, about 44μg, about 45μg, about 46μg, about 47μg, about 48μg, about 49μg, about 50μg, about 51μg, about 52 μg, approximately 53 μg, approximately 54 μg, approximately 55 μg, approximately 56 μg, approximately 57 μg, approximately 58 μg, approximately 59 μg, approximately 60 μg, approximately 61 μg, approximately 62 μg, approximately 63 μg, approximately 64 μg, approximately 65 μg, approximately 66 μg, approximately 67 μg, approximately 68 μg, approximately 69 μg, approximately 70 μg, approximately 71 μg, approximately 72 μg, approximately 73 μg, approximately 74 μg, approximately 75 μg, approximately 76 μg, approximately 77 μg, approximately 78 μg The present invention relates to administering about 79 μg, about 80 μg, about 81 μg, about 82 μg, about 83 μg, about 84 μg, about 85 μg, about 86 μg, about 87 μg, about 88 μg, about 89 μg, about 90 μg, about 91 μg, about 92 μg, about 93 μg, about 94 μg, about 95 μg, about 96 μg, about 97 μg, about 98 μg, about 99 μg, about 100 μg (including all values ​​and ranges therebetween) of each CoV S glycoprotein.In embodiments, the methods provide for the administration of a medicament comprising administering to a subject a medicament of at least about 1 μg, at least about 2 μg, at least about 3 μg, at least about 4 μg, at least about 5 μg, at least about 6 μg, at least about 7 μg, at least about 8 μg, at least about 9 μg, at least about 10 μg, at least about 11 μg, at least about 12 μg, at least about 13 μg, at least about 14 μg, at least about 15 μg, at least about 16 μg, at least about 17 μg, at least about 18 μg, at least about 19 μg, at least about 20 μg, at least about 21 μg, at least about 22 μg, at least about 23 μg, at least about 24 μg, at least about 25 μg, at least about 26 μg, at least about 27 μg, at least about 28 μg, at least about 29 μg, at least about 30 μg, at least about 31 μg, at least about 32 μg, at least about 33 μg, at least about 34 μg, at least about 35 μg, at least about 36 μg, at least about 37 μg, at least about 38 μg, at least about 39 μg, at least about 40 μg, at least about 41 μg, at least about 42 μg, at least about 43 μg, at least about 44 μg, at least about 45 μg, at least about 46 μg, at least about 47 μg, at least about 48 μg, at least about 49 μg, at least about 50 μg, at least about 51 μg, at least about 52 μg, at least about 53 μg, at least about 54 μg, at least about 55 μ 5μg, about 26μg, about 27μg, about 28μg, about 29μg, about 30μg, about 31μg, about 32μg, about 33μg, about 34μg, about 35μg, about 36μg, about 37μg, Approximately 38μg, approximately 39μg, approximately 40μg, approximately 41μg, approximately 42μg, approximately 43μg, approximately 44μg, approximately 45μg, approximately 46μg, approximately 47μg, approximately 48μg, approximately 49μg, approximately 50μg , about 51μg, about 52μg, about 53μg, about 54μg, about 55μg, about 56μg, about 57μg, about 58μg, about 59μg, about 60μg, about 61μg, about 62μg, about 63 μg, approximately 64 μg, approximately 65 μg, approximately 66 μg, approximately 67 μg, approximately 68 μg, approximately 69 μg, approximately 70 μg, approximately 71 μg, approximately 72 μg, approximately 73 μg, approximately 74 μg, approximately 75 μg, approximately 7 In some embodiments, the method comprises administering about 6 μg, about 77 μg, about 78 μg, about 79 μg, about 80 μg, about 81 μg, about 82 μg, about 83 μg, about 84 μg, about 85 μg, about 86 μg, about 87 μg, about 88 μg, about 89 μg, about 90 μg, about 91 μg, about 92 μg, about 93 μg, about 94 μg, about 95 μg, about 96 μg, about 97 μg, about 98 μg, about 99 μg, or about 100 μg of total CoV S glycoprotein (including all values ​​and ranges therebetween). In some embodiments, the method comprises administering about 1 μg of CoV S glycoprotein. In some embodiments, the method comprises administering about 13 μg of CoV S glycoprotein. In some embodiments, the immunogenic composition comprises about 5 μg of CoV S glycoprotein. In embodiments, the methods comprise administering about 25 μg of CoV S glycoprotein.

[0240] In some embodiments, the method comprises administering about 1 μg to about 300 μg of each HA glycoprotein. In some embodiments, the method comprises administering about 5 μg to about 60 μg of each HA glycoprotein. In some embodiments, the method comprises administering about 24 μg to about 40 μg of each HA glycoprotein. In some embodiments, the method comprises administering about 30 μg to about 40 μg of each HA glycoprotein. In some embodiments, the method comprises administering about 30 μg to about 60 μg of each HA glycoprotein. In some embodiments, the method comprises administering about 33 μg to about 39 μg of each HA glycoprotein. In some embodiments, the method comprises administering about 20 μg to about 240 μg of each HA glycoprotein. In some embodiments, the method comprises administering about 30 μg to about 60 μg of each HA glycoprotein. In some embodiments, the method comprises administering about 24 μg to about 40 μg of each HA glycoprotein. In embodiments, the methods provide for the administration of about 1 μg, about 2 μg, about 3 μg, about 4 μg, about 5 μg, about 6 μg, about 7 μg, about 8 μg, about 9 μg, about 10 μg, about 11 μg, about 12 μg, about 13 μg, about 14 μg, about 15 μg, about 16 μg, about 17 μg, about 18 μg, about 19 μg, about 20 μg, about 21 μg, about 22 μg, about 23 μg, about 24μg, about 25μg, about 26μg, about 27μg, about 28μg, about 29μg, about 30μg, about 31μg, about 32μg, about 33μg, about 34μg, about 35μg, about 3 6μg, about 37μg, about 38μg, about 39μg, about 40μg, about 41μg, about 42μg, about 43μg, about 44μg, about 45μg, about 46μg, about 47μg, about 48 μg, approximately 49 μg, approximately 50 μg, approximately 51 μg, approximately 52 μg, approximately 53 μg, approximately 54 μg, approximately 55 μg, approximately 56 μg, approximately 57 μg, approximately 58 μg, approximately 59 μg, approximately 60 μg, approximately 61 μg, approximately 62 μg, approximately 63 μg, approximately 64 μg, approximately 65 μg, approximately 66 μg, approximately 67 μg, approximately 68 μg, approximately 69 μg, approximately 70 μg, approximately 71 μg, approximately 72 μg g, about 73μg, about 74μg, about 75μg, about 76μg, about 77μg, about 78μg, about 79μg, about 80μg, about 81μg, about 82μg, about 83μg, about 84μg , about 85μg, about 86μg, about 87μg, about 88μg, about 89μg, about 90μg, about 91μg, about 92μg, about 93μg, about 94μg, about 95μg, about 96μg,Approximately 97 μg, approximately 98 μg, approximately 99 μg, approximately 100 μg, approximately 101 μg, approximately 102 μg, approximately 103 μg, approximately 104 μg, approximately 105 μg, approximately 106 μg, approximately 107 μg, approximately 108 μg, approximately 109 μg, approximately 110 μg, approximately 111 μg, approximately 112 μg, approximately 113 μg, approximately 114 μg, approximately 115μg, approximately 116μg, approximately 117μg, approximately 118μg, approximately 119μg, approximately 120μg, approximately 121μg, approximately 122μg, approximately 123μg, approximately 124μg, approximately 125μg, approximately 126μg, approximately 127μg, approximately 128μg, approximately 129μg, approximately 130μg, approximately 131μg, approximately 132μg Approximately 133 μg, approximately 134 μg, approximately 135 μg, approximately 136 μg, approximately 137 μg, approximately 138 μg, approximately 139 μg, approximately 140 μg, approximately 141 μg, approximately 142 μg, approximately 143 μg, approximately 144 μg, approximately 145 μg, approximately 146 μg, approximately 147 μg, approximately 148 μg, approximately 149 μg, approximately 150 μg Approximately 151 μg, approximately 152 μg, approximately 153 μg, approximately 154 μg, approximately 155 μg, approximately 156 μg, approximately 157 μg, approximately 158 μg, approximately 159 μg, approximately 160 μg, approximately 161 μg, approximately 162 μg, approximately 163 μg, approximately 164 μg, approximately 165 μg, approximately 166 μg, approximately 167 μg, approximately 168 μg g, approximately 169 μg, approximately 170 μg, approximately 171 μg, approximately 172 μg, approximately 173 μg, approximately 174 μg, approximately 175 μg, approximately 176 μg, approximately 177 μg, approximately 178 μg, approximately 179 μg, approximately 180 μg, approximately 181 μg, approximately 182 μg, approximately 183 μg, approximately 184 μg, approximately 185 μg, approximately 18 6μg, approximately 187μg, approximately 188μg, approximately 189μg, approximately 190μg, approximately 191μg, approximately 192μg, approximately 193μg, approximately 194μg, approximately 195μg, approximately 196μg, approximately 197μg, approximately 198μg, approximately 199μg, approximately 200μg, approximately 201μg, approximately 202μg, approximately 203μg, approximately 2 0.4μg, approximately 205μg, approximately 206μg, approximately 207μg, approximately 208μg, approximately 209μg, approximately 210μg, approximately 211μg, approximately 212μg, approximately 213μg, approximately 214μg, approximately 215μg, approximately 216μg, approximately 217μg, approximately 218μg, approximately 219μg, approximately 220μg, approximately 221μg, approximately 222μg, approximately 223μg, approximately 224μg, approximately 225μg, approximately 226μg, approximately 227μg, approximately 228μg, approximately 229μg, approximately 230μg, approximately 231μg, approximately 232μg, approximately 233μg, approximately 234μg, approximately 235μg, approximately 236μg, approximately 237μg, approximately 238μg, approximately 239μgApproximately 240μg, approximately 241μg, approximately 242μg, approximately 243μg, approximately 244μg, approximately 245μg, approximately 246μg, approximately 247μg, approximately 2 48μg, about 249μg, about 250μg, about 251μg, about 252μg, about 253μg, about 254μg, about 255μg, about 256μ g, about 257μg, about 258μg, about 259μg, about 260μg, about 261μg, about 262μg, about 263μg, about 264μg, Approximately 265μg, approximately 266μg, approximately 267μg, approximately 268μg, approximately 269μg, approximately 270μg, approximately 271μg, approximately 272μg, approximately 27 In some embodiments, the method comprises administering about 3 μg, about 274 μg, about 275 μg, about 276 μg, about 277 μg, about 278 μg, about 279 μg, about 280 μg, about 281 μg, about 282 μg, about 283 μg, about 284 μg, about 285 μg, about 286 μg, about 287 μg, about 288 μg, about 289 μg, about 290 μg, about 291 μg, about 292 μg, about 293 μg, about 294 μg, about 295 μg, about 296 μg, about 297 μg, about 298 μg, about 299 μg, or about 300 μg of each HA glycoprotein (including all values ​​and ranges therebetween). In some embodiments, the method comprises administering about 30 μg of each HA glycoprotein. In some embodiments, the method comprises administering about 33 μg of each HA glycoprotein. In some embodiments, the method comprises administering about 39 μg of each HA glycoprotein. In some embodiments, the method comprises administering about 54 μg of each HA glycoprotein. In some embodiments, the method comprises administering about 60 μg of each HA glycoprotein. In some embodiments, the method comprises administering about 240 μg of each HA glycoprotein.

[0241] In embodiments, the methods involve administering between about 1 μg and about 1000 μg of total HA glycoprotein. In embodiments, the methods provide for the administration of a medicament containing at least one of the following: about 1 μg, about 2 μg, about 3 μg, about 4 μg, about 5 μg, about 6 μg, about 7 μg, about 8 μg, about 9 μg, about 10 μg, about 11 μg, about 12 μg, about 13 μg, about 14 μg, about 15 μg, about 16 μg, about 17 μg, about 18 μg, about 19 μg, about 20 μg, about 21 μg, about 22 μg, about 23 μg, about 24 μg, about 25 μg, about 26 μg, about 27 μg, about 28 μg, about 29 μg, about 30 μg, about 31 μg, about 32 μg, about 33 μg, about 34 μg, about 35 μg, about 36 μg, about 37 μg, about 38 μg, about 39 μg, about 40 μg, about 41 μg, about 42 μg, about 43 μg, about 44 μg, about 45 μg, about 46 μg, about 47 μg, about 48 μg, about 49 μg, about 50 μg, about 51 μg, about 52 μg, about 53 μg, about 54 μg, about 55 μg, about 56 μg, about 57 μg, about 58 μg, about 59 μg, about 60 μg, about 61 μg, about 62 μg, about 63 μg, about 64 μg, about 65 μg, about 66 μg, about 67 μg, about 68 μg, about 69 μg, about 70 μg, about 71 μg, μg, approximately 40 μg, approximately 41 μg, approximately 42 μg, approximately 43 μg, approximately 44 μg, approximately 45 μg, approximately 46 μg, approximately 47 μg, approximately 48 μg, approximately 4 9μg, about 50μg, about 51μg, about 52μg, about 53μg, about 54μg, about 55μg, about 56μg, about 57μg, about 58μg, about 5 9μg, about 60μg, about 61μg, about 62μg, about 63μg, about 64μg, about 65μg, about 66μg, about 67μg, about 68μg, about 69μg, about 70μg, about 71μg, about 72μg, about 73μg, about 74μg, about 75μg, about 76μg, about 77μg, about 78μg, about 79μg, about 80μg, about 81μg, about 82μg, about 83μg, about 84μg, about 85μg, about 86μg, about 87μg, about 88μg, Approximately 89μg, approximately 90μg, approximately 91μg, approximately 92μg, approximately 93μg, approximately 94μg, approximately 95μg, approximately 96μg, approximately 97μg, approximately 98μg , about 99μg, about 100μg, about 101μg, about 102μg, about 103μg, about 104μg, about 105μg, about 106μg, about 10 7μg, about 108μg, about 109μg, about 110μg, about 111μg, about 112μg, about 113μg, about 114μg, about 115μg, Approximately 116μg, approximately 117μg, approximately 118μg, approximately 119μg, approximately 120μg, approximately 121μg, approximately 122μg, approximately 123μg, approximately 12 4μg, about 125μg, about 126μg, about 127μg, about 128μg, about 129μg, about 130μg, about 131μg, about 132μg, Approximately 133μg, approximately 134μg, approximately 135μg, approximately 136μg, approximately 137μg, approximately 138μg, approximately 139μg, approximately 140μg, approximately 14 1μg, about 142μg, about 143μg, about 144μg, about 145μg, about 146μg, about 147μg, about 148μg, about 149μg,Approximately 150 μg, approximately 151 μg, approximately 152 μg, approximately 153 μg, approximately 154 μg, approximately 155 μg, approximately 156 μg, approximately 157 μg, approximately 158 μg, approximately 159 μg, approximately 160 μg, approximately 161 μg, approximately 162 μg, approximately 163 μg, approximately 164 μg, approximately 165 μg, approximately 166 μg, approximately 167 μg Approximately 168 μg, approximately 169 μg, approximately 170 μg, approximately 171 μg, approximately 172 μg, approximately 173 μg, approximately 174 μg, approximately 175 μg, approximately 176 μg, approximately 177 μg, approximately 178 μg, approximately 179 μg, approximately 180 μg, approximately 181 μg, approximately 182 μg, approximately 183 μg, approximately 184 μg, approximately 185 μg g, approximately 186μg, approximately 187μg, approximately 188μg, approximately 189μg, approximately 190μg, approximately 191μg, approximately 192μg, approximately 193μg, approximately 194μg, approximately 195μg, approximately 196μg, approximately 197μg, approximately 198μg, approximately 199μg, approximately 200μg, approximately 201μg, approximately 202μg, approximately 203μg μg, approximately 204μg, approximately 205μg, approximately 206μg, approximately 207μg, approximately 208μg, approximately 209μg, approximately 210μg, approximately 211μg, approximately 212μg, approximately 213μg, approximately 214μg, approximately 215μg, approximately 216μg, approximately 217μg, approximately 218μg, approximately 219μg, approximately 220μg, approximately 22 1 μg, approximately 222 μg, approximately 223 μg, approximately 224 μg, approximately 225 μg, approximately 226 μg, approximately 227 μg, approximately 228 μg, approximately 229 μg, approximately 230 μg, approximately 231 μg, approximately 232 μg, approximately 233 μg, approximately 234 μg, approximately 235 μg, approximately 236 μg, approximately 237 μg, approximately 238 μg, approximately 2 39μg, approximately 240μg, approximately 241μg, approximately 242μg, approximately 243μg, approximately 244μg, approximately 245μg, approximately 246μg, approximately 247μg, approximately 248μg, approximately 249μg, approximately 250μg, approximately 251μg, approximately 252μg, approximately 253μg, approximately 254μg, approximately 255μg, approximately 256μg, approximately 257μg, approximately 258μg, approximately 259μg, approximately 260μg, approximately 261μg, approximately 262μg, approximately 263μg, approximately 264μg, approximately 265μg, approximately 266μg, approximately 267μg, approximately 268μg, approximately 269μg, approximately 270μg, approximately 271μg, approximately 272μg, approximately 273μg, approximately 274μg Approximately 275 μg, approximately 276 μg, approximately 277 μg, approximately 278 μg, approximately 279 μg, approximately 280 μg, approximately 281 μg, approximately 282 μg, approximately 283 μg, approximately 284 μg, approximately 285 μg, approximately 286 μg, approximately 287 μg, approximately 288 μg, approximately 289 μg, approximately 290 μg, approximately 291 μg, approximately 292 μgApproximately 293 μg, approximately 294 μg, approximately 295 μg, approximately 296 μg, approximately 297 μg, approximately 298 μg, approximately 299 μg, approximately 300 μg, approximately 301 μg, approximately 302 μg, approximately 303 μg, approximately 304 μg, approximately 305 μg, approximately 306 μg, approximately 307 μg, approximately 308 μg, approximately 309 μg, approximately 310 μg Approximately 311 μg, approximately 312 μg, approximately 313 μg, approximately 314 μg, approximately 315 μg, approximately 316 μg, approximately 317 μg, approximately 318 μg, approximately 319 μg, approximately 320 μg, approximately 321 μg, approximately 322 μg, approximately 323 μg, approximately 324 μg, approximately 325 μg, approximately 326 μg, approximately 327 μg, approximately 328 μg g, approximately 329 μg, approximately 330 μg, approximately 331 μg, approximately 332 μg, approximately 333 μg, approximately 334 μg, approximately 335 μg, approximately 336 μg, approximately 337 μg, approximately 338 μg, approximately 339 μg, approximately 340 μg, approximately 341 μg, approximately 342 μg, approximately 343 μg, approximately 344 μg, approximately 345 μg, approximately 346 μg μg, approximately 347μg, approximately 348μg, approximately 349μg, approximately 350μg, approximately 351μg, approximately 352μg, approximately 353μg, approximately 354μg, approximately 355μg, approximately 356μg, approximately 357μg, approximately 358μg, approximately 359μg, approximately 360μg, approximately 361μg, approximately 362μg, approximately 363μg, approximately 36 4μg, approximately 365μg, approximately 366μg, approximately 367μg, approximately 368μg, approximately 369μg, approximately 370μg, approximately 371μg, approximately 372μg, approximately 373μg, approximately 374μg, approximately 375μg, approximately 376μg, approximately 377μg, approximately 378μg, approximately 379μg, approximately 380μg, approximately 381μg, approximately 3 82μg, approximately 383μg, approximately 384μg, approximately 385μg, approximately 386μg, approximately 387μg, approximately 388μg, approximately 389μg, approximately 390μg, approximately 391μg, approximately 392μg, approximately 393μg, approximately 394μg, approximately 395μg, approximately 396μg, approximately 397μg, approximately 398μg, approximately 399μg, approximately 400μg, approximately 401μg, approximately 402μg, approximately 403μg, approximately 404μg, approximately 405μg, approximately 406μg, approximately 407μg, approximately 408μg, approximately 409μg, approximately 410μg, approximately 411μg, approximately 412μg, approximately 413μg, approximately 414μg, approximately 415μg, approximately 416μg, approximately 417μg Approximately 418 μg, approximately 419 μg, approximately 420 μg, approximately 421 μg, approximately 422 μg, approximately 423 μg, approximately 424 μg, approximately 425 μg, approximately 426 μg, approximately 427 μg, approximately 428 μg, approximately 429 μg, approximately 430 μg, approximately 431 μg, approximately 432 μg, approximately 433 μg, approximately 434 μg, approximately 435 μgApproximately 436 μg, approximately 437 μg, approximately 438 μg, approximately 439 μg, approximately 440 μg, approximately 441 μg, approximately 442 μg, approximately 443 μg, approximately 444 μg, approximately 445 μg, approximately 446 μg, approximately 447 μg, approximately 448 μg, approximately 449 μg, approximately 450 μg, approximately 451 μg, approximately 452 μg, approximately 453 μg Approximately 454 μg, approximately 455 μg, approximately 456 μg, approximately 457 μg, approximately 458 μg, approximately 459 μg, approximately 460 μg, approximately 461 μg, approximately 462 μg, approximately 463 μg, approximately 464 μg, approximately 465 μg, approximately 466 μg, approximately 467 μg, approximately 468 μg, approximately 469 μg, approximately 470 μg, approximately 471 μg g, approximately 472μg, approximately 473μg, approximately 474μg, approximately 475μg, approximately 476μg, approximately 477μg, approximately 478μg, approximately 479μg, approximately 480μg, approximately 481μg, approximately 482μg, approximately 483μg, approximately 484μg, approximately 485μg, approximately 486μg, approximately 487μg, approximately 488μg, approximately 489μg μg, approximately 490μg, approximately 491μg, approximately 492μg, approximately 493μg, approximately 494μg, approximately 495μg, approximately 496μg, approximately 497μg, approximately 498μg, approximately 499μg, approximately 500μg, approximately 501μg, approximately 502μg, approximately 503μg, approximately 504μg, approximately 505μg, approximately 506μg, approximately 50 7μg, approximately 508μg, approximately 509μg, approximately 510μg, approximately 511μg, approximately 512μg, approximately 513μg, approximately 514μg, approximately 515μg, approximately 516μg, approximately 517μg, approximately 518μg, approximately 519μg, approximately 520μg, approximately 521μg, approximately 522μg, approximately 523μg, approximately 524μg, approximately 5 25μg, approximately 526μg, approximately 527μg, approximately 528μg, approximately 529μg, approximately 530μg, approximately 531μg, approximately 532μg, approximately 533μg, approximately 534μg, approximately 535μg, approximately 536μg, approximately 537μg, approximately 538μg, approximately 539μg, approximately 540μg, approximately 541μg, approximately 542μg, approximately 543μg, approximately 544μg, approximately 545μg, approximately 546μg, approximately 547μg, approximately 548μg, approximately 549μg, approximately 550μg, approximately 551μg, approximately 552μg, approximately 553μg, approximately 554μg, approximately 555μg, approximately 556μg, approximately 557μg, approximately 558μg, approximately 559μg, approximately 560μg Approximately 561 μg, approximately 562 μg, approximately 563 μg, approximately 564 μg, approximately 565 μg, approximately 566 μg, approximately 567 μg, approximately 568 μg, approximately 569 μg, approximately 570 μg, approximately 571 μg, approximately 572 μg, approximately 573 μg, approximately 574 μg, approximately 575 μg, approximately 576 μg, approximately 577 μg, approximately 578 μgApproximately 579 μg, approximately 580 μg, approximately 581 μg, approximately 582 μg, approximately 583 μg, approximately 584 μg, approximately 585 μg, approximately 586 μg, approximately 587 μg, approximately 588 μg, approximately 589 μg, approximately 590 μg, approximately 591 μg, approximately 592 μg, approximately 593 μg, approximately 594 μg, approximately 595 μg, approximately 596 μg Approximately 597 μg, approximately 598 μg, approximately 599 μg, approximately 600 μg, approximately 601 μg, approximately 602 μg, approximately 603 μg, approximately 604 μg, approximately 605 μg, approximately 606 μg, approximately 607 μg, approximately 608 μg, approximately 609 μg, approximately 610 μg, approximately 611 μg, approximately 612 μg, approximately 613 μg, approximately 614 μg g, approximately 615μg, approximately 616μg, approximately 617μg, approximately 618μg, approximately 619μg, approximately 620μg, approximately 621μg, approximately 622μg, approximately 623μg, approximately 624μg, approximately 625μg, approximately 626μg, approximately 627μg, approximately 628μg, approximately 629μg, approximately 630μg, approximately 631μg, approximately 632 μg, approximately 633μg, approximately 634μg, approximately 635μg, approximately 636μg, approximately 637μg, approximately 638μg, approximately 639μg, approximately 640μg, approximately 641μg, approximately 642μg, approximately 643μg, approximately 644μg, approximately 645μg, approximately 646μg, approximately 647μg, approximately 648μg, approximately 649μg, approximately 65μg 0 μg, approximately 651 μg, approximately 652 μg, approximately 653 μg, approximately 654 μg, approximately 655 μg, approximately 656 μg, approximately 657 μg, approximately 658 μg, approximately 659 μg, approximately 660 μg, approximately 661 μg, approximately 662 μg, approximately 663 μg, approximately 664 μg, approximately 665 μg, approximately 666 μg, approximately 667 μg, approximately 6 68μg, approximately 669μg, approximately 670μg, approximately 671μg, approximately 672μg, approximately 673μg, approximately 674μg, approximately 675μg, approximately 676μg, approximately 677μg, approximately 678μg, approximately 679μg, approximately 680μg, approximately 681μg, approximately 682μg, approximately 683μg, approximately 684μg, approximately 685μg, approximately 686μg, approximately 687μg, approximately 688μg, approximately 689μg, approximately 690μg, approximately 691μg, approximately 692μg, approximately 693μg, approximately 694μg, approximately 695μg, approximately 696μg, approximately 697μg, approximately 698μg, approximately 699μg, approximately 700μg, approximately 701μg, approximately 702μg, approximately 703μg Approximately 704 μg, approximately 705 μg, approximately 706 μg, approximately 707 μg, approximately 708 μg, approximately 709 μg, approximately 710 μg, approximately 711 μg, approximately 712 μg, approximately 713 μg, approximately 714 μg, approximately 715 μg, approximately 716 μg, approximately 717 μg, approximately 718 μg, approximately 719 μg, approximately 720 μg, approximately 721 μgApproximately 722 μg, approximately 723 μg, approximately 724 μg, approximately 725 μg, approximately 726 μg, approximately 727 μg, approximately 728 μg, approximately 729 μg, approximately 730 μg, approximately 731 μg, approximately 732 μg, approximately 733 μg, approximately 734 μg, approximately 735 μg, approximately 736 μg, approximately 737 μg, approximately 738 μg Approximately 739 μg, approximately 740 μg, approximately 741 μg, approximately 742 μg, approximately 743 μg, approximately 744 μg, approximately 745 μg, approximately 746 μg, approximately 747 μg, approximately 748 μg, approximately 749 μg, approximately 750 μg, approximately 751 μg, approximately 752 μg, approximately 753 μg, approximately 754 μg, approximately 755 μg, approximately 756 μg Approximately 757 μg, approximately 758 μg, approximately 759 μg, approximately 760 μg, approximately 761 μg, approximately 762 μg, approximately 763 μg, approximately 764 μg, approximately 765 μg, approximately 766 μg, approximately 767 μg, approximately 768 μg, approximately 769 μg, approximately 770 μg, approximately 771 μg, approximately 772 μg, approximately 773 μg, approximately 774 μg g, approximately 775μg, approximately 776μg, approximately 777μg, approximately 778μg, approximately 779μg, approximately 780μg, approximately 781μg, approximately 782μg, approximately 783μg, approximately 784μg, approximately 785μg, approximately 786μg, approximately 787μg, approximately 788μg, approximately 789μg, approximately 790μg, approximately 791μg, approximately 792 μg, approximately 793μg, approximately 794μg, approximately 795μg, approximately 796μg, approximately 797μg, approximately 798μg, approximately 799μg, approximately 800μg, approximately 801μg, approximately 802μg, approximately 803μg, approximately 804μg, approximately 805μg, approximately 806μg, approximately 807μg, approximately 808μg, approximately 809μg, approximately 81 0 μg, approximately 811 μg, approximately 812 μg, approximately 813 μg, approximately 814 μg, approximately 815 μg, approximately 816 μg, approximately 817 μg, approximately 818 μg, approximately 819 μg, approximately 820 μg, approximately 821 μg, approximately 822 μg, approximately 823 μg, approximately 824 μg, approximately 825 μg, approximately 826 μg, approximately 827 μg, approximately 8 28μg, approximately 829μg, approximately 830μg, approximately 831μg, approximately 832μg, approximately 833μg, approximately 834μg, approximately 835μg, approximately 836μg, approximately 837μg, approximately 838μg, approximately 839μg, approximately 840μg, approximately 841μg, approximately 842μg, approximately 843μg, approximately 844μg, approximately 845μg, approximately 846μg, approximately 847μg, approximately 848μg, approximately 849μg, approximately 850μg, approximately 851μg, approximately 852μg, approximately 853μg, approximately 854μg, approximately 855μg, approximately 856μg, approximately 857μg, approximately 858μg, approximately 859μg, approximately 860μg, approximately 861μg, approximately 862μg, approximately 863μg Approximately 864 μg, approximately 865 μg, approximately 866 μg, approximately 867 μg, approximately 868 μg, approximately 869 μg, approximately 870 μg, approximately 871 μg, approximately 872 μg, approximately 873 μg, approximately 874 μg, approximately 875 μg, approximately 876 μg, approximately 877 μg, approximately 878 μg, approximately 879 μg, approximately 880 μg, approximately 881 μgAbout 882 μg, about 883 μg, about 884 μg, about 885 μg, about 886 μg, about 887 μg, about 888 μg, about 889 μg, about 890 μg, about 891 μg, about 892 μg, about 893 μg, about 894 μg, about 895 μg, about 896 μg, about 897 μg, about 898 μg, about 899 μg, about 900 μg, about 901 μg, about 902 μg, about 903 μg, about 904 μg, about 905 μg, about 906 μg, about 907 μg, about 908 μg, about 909 μg, about 910 μg, about 911 μg, about 912 μg, 913μg, about 914μg, about 915μg, about 916μg, about 917μg, about 918μg, about 919μg, about 920μg, about 921μg, about 922μg, about 923μg, about 924μg, about 925μg, about 926μg, about 927μg, about 928μg g, about 929μg, about 930μg, about 931μg, about 932μg, about 933μg, about 934μg, about 935μg, about 936μg, about 937μg, about 938μg, about 939μg, about 940μg, about 941μg, about 942μg, about 943μg, about 9 44μg, about 945μg, about 946μg, about 947μg, about 948μg, about 949μg, about 950μg, about 951μg, about 952μg, about 953μg, about 954μg, about 955μg, about 956μg, about 957μg, about 958μg, about 959μg , about 960μg, about 961μg, about 962μg, about 963μg, about 964μg, about 965μg, about 966μg, about 967μg, about 968μg, about 969μg, about 970μg, about 971μg, about 972μg, about 973μg, about 974μg, about 97 and administering about 5 μg, about 976 μg, about 977 μg, about 978 μg, about 979 μg, about 980 μg, about 981 μg, about 982 μg, about 983 μg, about 984 μg, about 985 μg, about 986 μg, about 987 μg, about 988 μg, about 989 μg, about 990 μg, about 991 μg, about 992 μg, about 993 μg, about 994 μg, about 995 μg, about 996 μg, about 997 μg, about 998 μg, about 999 μg, about 1000 μg of total HA glycoprotein (including all values ​​and ranges therebetween).

[0242] In several embodiments, the methods comprise administering about 1 μg to about 400 μg, 1 μg to about 350 μg, 1 μg to about 300 μg, 1 μg to about 250 μg, 1 μg to about 200 μg, 1 μg to about 150 μg, 1 μg to about 100 μg, 1 μg to about 50 μg, or 1 μg to about 25 μg of each viral glycoprotein. In embodiments, the methods provide for the administration of a medicament containing at least one of the following: about 1 μg, about 2 μg, about 3 μg, about 4 μg, about 5 μg, about 6 μg, about 7 μg, about 8 μg, about 9 μg, about 10 μg, about 11 μg, about 12 μg, about 13 μg, about 14 μg, about 15 μg, about 16 μg, about 17 μg, about 18 μg, about 19 μg, about 20 μg, about 21 μg, about 22 μg, about 23 μg, about 24 μg, about 25 μg, about 26 μg, about 27 μg, about 28 μg, about 29 μg, about 30 μg, about 31 μg, about 32 μg, about 33 μg, about 34 μg, about 35 μg g, about 36μg, about 37μg, about 38μg, about 39μg, about 40μg, about 41μg, about 42μg, about 43μg, about 44μg, about 45μg, about 46μg, about 47μg, about 48μg, about 49μg, about 50μg, about 51μg, about 52μg, about 53 54μg, 55μg, 56μg, 57μg, 58μg, 59μg, 60μg, 61μg, 62μg, 63μg, 64μg, 65μg, 66μg, 67μg, 68μg, 69μg, 70μg, 71 μg, approximately 72 μg, approximately 73 μg, approximately 74 μg, approximately 75 μg, approximately 76 μg, approximately 77 μg, approximately 78 μg, approximately 79 μg, approximately 80 μg, approximately 81 μg, approximately 82 μg, approximately 83 μg, approximately 84 μg, approximately 85 μg, approximately 86 μg, approximately 87 μg, approximately 88 μg, approximately 8 9μg, about 90μg, about 91μg, about 92μg, about 93μg, about 94μg, about 95μg, about 96μg, about 97μg, about 98μg, about 99μg, about 100μg, about 101μg, about 102μg, about 103μg, about 104μg, about 105μg, about 1 06μg, about 107μg, about 108μg, about 109μg, about 110μg, about 111μg, about 112μg, about 113μg, about 114μg, about 115μg, about 116μg, about 117μg, about 118μg, about 119μg, about 120μg, about 121 μg, approximately 122 μg, approximately 123 μg, approximately 124 μg, approximately 125 μg, approximately 126 μg, approximately 127 μg, approximately 128 μg, approximately 129 μg, approximately 130 μg, approximately 131 μg, approximately 132 μg, approximately 133 μg, approximately 134 μg, approximately 135 μg, approximately 136 μg,Approximately 137 μg, approximately 138 μg, approximately 139 μg, approximately 140 μg, approximately 141 μg, approximately 142 μg, approximately 143 μg, approximately 144 μg, approximately 145 μg, approximately 146 μg, approximately 147 μg, approximately 148 μg, approximately 149 μg, approximately 150 μg, approximately 151 μg, approximately 152 μg, approximately 153 μg, approximately 154 μg Approximately 155 μg, approximately 156 μg, approximately 157 μg, approximately 158 μg, approximately 159 μg, approximately 160 μg, approximately 161 μg, approximately 162 μg, approximately 163 μg, approximately 164 μg, approximately 165 μg, approximately 166 μg, approximately 167 μg, approximately 168 μg, approximately 169 μg, approximately 170 μg, approximately 171 μg, approximately 172 μg g, approximately 173μg, approximately 174μg, approximately 175μg, approximately 176μg, approximately 177μg, approximately 178μg, approximately 179μg, approximately 180μg, approximately 181μg, approximately 182μg, approximately 183μg, approximately 184μg, approximately 185μg, approximately 186μg, approximately 187μg, approximately 188μg, approximately 189μg, approximately 190 μg, approximately 191μg, approximately 192μg, approximately 193μg, approximately 194μg, approximately 195μg, approximately 196μg, approximately 197μg, approximately 198μg, approximately 199μg, approximately 200μg, approximately 201μg, approximately 202μg, approximately 203μg, approximately 204μg, approximately 205μg, approximately 206μg, approximately 207μg, approximately 20 8μg, approximately 209μg, approximately 210μg, approximately 211μg, approximately 212μg, approximately 213μg, approximately 214μg, approximately 215μg, approximately 216μg, approximately 217μg, approximately 218μg, approximately 219μg, approximately 220μg, approximately 221μg, approximately 222μg, approximately 223μg, approximately 224μg, approximately 225μg, approximately 2 26μg, approximately 227μg, approximately 228μg, approximately 229μg, approximately 230μg, approximately 231μg, approximately 232μg, approximately 233μg, approximately 234μg, approximately 235μg, approximately 236μg, approximately 237μg, approximately 238μg, approximately 239μg, approximately 240μg, approximately 241μg, approximately 242μg, approximately 243μg, approximately 244μg, approximately 245μg, approximately 246μg, approximately 247μg, approximately 248μg, approximately 249μg, approximately 250μg, approximately 251μg, approximately 252μg, approximately 253μg, approximately 254μg, approximately 255μg, approximately 256μg, approximately 257μg, approximately 258μg, approximately 259μg, approximately 260μg, approximately 261μg Approximately 262 μg, approximately 263 μg, approximately 264 μg, approximately 265 μg, approximately 266 μg, approximately 267 μg, approximately 268 μg, approximately 269 μg, approximately 270 μg, approximately 271 μg, approximately 272 μg, approximately 273 μg, approximately 274 μg, approximately 275 μg, approximately 276 μg, approximately 277 μg, approximately 278 μg, approximately 279 μgAbout 280μg, about 281μg, about 282μg, about 283μg, about 284μg, about 285μg, about 286μg, about 287μg, about 288μg, about 289μg, about 290μg, about 291μg, about 292μg, about 293μg, about 294μg, about 295 μg, approx. 296 μg, approx. 297 μg, approx. 298 μg, approx. 299 μg, approx. 300 μg, approx. 301 μg, approx. 302 μg, approx. 303 μg, approx. 304 μg, approx. 311μg, about 312μg, about 313μg, about 314μg, about 315μg, about 316μg, about 317μg, about 318μg, about 319μg, about 320μg, about 321μg, about 322μg, about 323μg, about 324μg, about 325μg, about 326μg g, approx. 327 μg, approx. 328 μg, approx. 329 μg, approx. 330 μg, approx. 331 μg, approx. 332 μg, approx. 333 μg, approx. 334 μg, approx. 335 μg, approx. 336 μg, approx. 42μg, about 343μg, about 344μg, about 345μg, about 346μg, about 347μg, about 348μg, about 349μg, about 350μg, about 351μg, about 352μg, about 353μg, about 354μg, about 355μg, about 356μg, about 357μg , about 358μg, about 359μg, about 360μg, about 361μg, about 362μg, about 363μg, about 364μg, about 365μg, about 366μg, about 367μg, about 368μg, about 369μg, about 370μg, about 371μg, about 372μg, about 37 and administering 3 μg, about 374 μg, about 375 μg, about 376 μg, about 377 μg, about 378 μg, about 379 μg, about 380 μg, about 381 μg, about 382 μg, about 383 μg, about 384 μg, about 385 μg, about 386 μg, about 387 μg, about 388 μg, about 389 μg, about 390 μg, about 391 μg, about 392 μg, about 393 μg, about 394 μg, about 395 μg, about 396 μg, about 397 μg, about 398 μg, about 399 μg, or about 400 μg of each viral glycoprotein.

[0243] In embodiments, the methods include administering about 1 μg to about 400 μg, 1 μg to about 350 μg, 1 μg to about 300 μg, 1 μg to about 250 μg, 1 μg to about 200 μg, 1 μg to about 150 μg, 1 μg to about 100 μg, 1 μg to about 50 μg, or 1 μg to about 25 μg of adjuvant. In embodiments, the immunogenic composition comprises about 1 μg, about 2 μg, about 3 μg, about 4 μg, about 5 μg, about 6 μg, about 7 μg, about 8 μg, about 9 μg, about 10 μg, about 11 μg, about 12 μg, about 13 μg, about 14 μg, about 15 μg, about 16 μg, about 17 μg, about 18 μg, about 19 μg, about 20 μg, about 21 μg, about 22 μg, about 23 μg, about 24 μg, about 25 μg, about 26 μg, about 27 μg, about 28 μg, about 29 μg, about 30 μg, about 31 μg, about 32 μg, about 33 μg, about 34 μg, about 35μg, about 36μg, about 37μg, about 38μg, about 39μg, about 40μg, about 41μg, about 42μg, about 43μg, about 44μg, about 45μg, about 46μg, about 47μg, about 48μg, about 49μg, about 50μg, about 51μg, about 52μg, about 53μg, about 54μg, about 55μg, about 56μg, about 57μg, about 58μg, about 59μg, about 60μg, about 61μg, about 62μg, about 63μg, about 64μg, about 65μg, about 66μg, about 67μg, about 68μg, about 69μg, about 70μg, about 71 μg, about 72 μg, about 73 μg, about 74 μg, about 75 μg, about 76 μg, about 77 μg, about 78 μg, about 79 μg, about 80 μg, about 81 μg, about 82 μg, about 83 μg, about 84 μg, about 85 μg, about 86 μg, about 87 μg, about 88 μg, about 89 μg, about 90 μg, about 91 μg, about 92 μg, about 93 μg, about 94 μg, about 95 μg, about 96 μg, about 97 μg, about 98 μg, about 99 μg, about 100 μg, about 101 μg, about 102 μg, about 103 μg, about 104 μg, about 105 μg, about 106μg, about 107μg, about 108μg, about 109μg, about 110μg, about 111μg, about 112μg, about 113μg, about 114μg, about 115μg, about 116μg, about 117μg, about 118μg, about 119μg, about 120μg, about 121 μg, approximately 122 μg, approximately 123 μg, approximately 124 μg, approximately 125 μg, approximately 126 μg, approximately 127 μg, approximately 128 μg, approximately 129 μg, approximately 130 μg, approximately 131 μg, approximately 132 μg, approximately 133 μg, approximately 134 μg, approximately 135 μg, approximately 136 μg,Approximately 137 μg, approximately 138 μg, approximately 139 μg, approximately 140 μg, approximately 141 μg, approximately 142 μg, approximately 143 μg, approximately 144 μg, approximately 145 μg, approximately 146 μg, approximately 147 μg, approximately 148 μg, approximately 149 μg, approximately 150 μg, approximately 151 μg, approximately 152 μg, approximately 153 μg, approximately 154 μg Approximately 155 μg, approximately 156 μg, approximately 157 μg, approximat...

Claims

1. A respiratory syncytial virus (RSV) fusion (F) glycoprotein comprising an F1 domain, an F2 domain, and p27, said glycoprotein comprising: (a) a deletion of one or more of amino acids 137-146; (b) inactivated primary furin cleavage site; (c) inactivated secondary furin cleavage site; (d) S155C, (e) S290C, (f) S190F, (g) V207L, and (h) N116Q and comprising one or more modifications selected from the group consisting of: The RSV F glycoprotein, wherein the modifications are numbered based on the RSV F glycoprotein of SEQ ID NO:

330.

2. 2. The RSV F glycoprotein of claim 1, comprising: (a) a deletion of one or more of amino acids 137-146; and (b) an inactivated primary furin cleavage site.

3. 3. The RSV F glycoprotein of claim 1 or 2, comprising a deletion of all of amino acids 137-146.

4. 4. The RSV F glycoprotein of any one of claims 1 to 3, wherein the inactivating primary furin cleavage site comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 342-345.

5. 5. The RSV F glycoprotein of any one of claims 1 to 4, wherein the inactivated primary furin cleavage site comprises the amino acid sequence of SEQ ID NO:

342.

6. 6. The RSV F glycoprotein of any one of claims 1 to 5, comprising an S155C and an S290C mutation.

7. 6. The RSV F glycoprotein of any one of claims 1 to 5, comprising an S190F and a V207L ​​mutation.

8. 6. The RSV F glycoprotein of any one of claims 1 to 5, comprising S155C, S290C, S190F, and V207L ​​mutations.

9. 9. The RSV F glycoprotein of any one of claims 1 to 8, comprising an N116Q mutation.

10. 10. The RSV F glycoprotein of any one of claims 1 to 9, comprising an inactivated primary furin cleavage site having the amino acid sequence of KKQKQQ (SEQ ID NO: 342), an S190F mutation, a V207 mutation, and a deletion of amino acids 137 to 146.

11. 10. The RSV F glycoprotein of any one of claims 1 to 9, comprising an inactivated primary furin cleavage site having an amino acid sequence of KKQKQQ (SEQ ID NO: 342), an S190F mutation, a V207 mutation, and a deletion of amino acids 137-146, and having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the RSV F glycoprotein of SEQ ID NO:

337.

12. 12. The RSV F glycoprotein of any one of claims 1 to 11, comprising: (i) an F1 domain having an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the F1 domain of SEQ ID NO: 352; and (ii) an F2 domain having an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the F2 domain of SEQ ID NO:

357.

13. 13. The RSV F glycoprotein of any one of claims 1-12, wherein the F1 domain is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the F1 domain of any one of SEQ ID NOs: 350-355.

14. 14. The RSV F glycoprotein of any one of claims 1 to 13, wherein the F2 domain is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the F2 domain of any one of SEQ ID NOs: 356-357.

15. 15. The RSV F glycoprotein of any one of claims 1-14, wherein the p27 domain is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the p27 domain of any one of SEQ ID NOs: 358-359.

16. 11. The RSV F glycoprotein of any one of claims 1 to 10, which is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the RSV F glycoprotein of any one of SEQ ID NOs: 364-366.

17. 11. The RSV F glycoprotein of any one of claims 1 to 10, which is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the RSV F glycoprotein of SEQ ID NO:

364.

18. 11. The RSV F glycoprotein of any one of claims 1 to 10, which is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 333-340 and 364-366.

19. 19. The RSV F glycoprotein of any one of claims 1 to 18, wherein the p27 and F1 domains form a single subunit designated p27-F1, and p27-F1 and the F2 subunit are connected by a disulfide bond.

20. 20. The RSV F glycoprotein of any one of claims 1 to 19, comprising a signal peptide.

21. 14. The RSV F glycoprotein of claim 13, wherein the signal peptide comprises the sequence of MELLILKANAITTILTAVTFCFASG (SEQ ID NO: 360) or MELLIHRLSAIFLTL (SEQ ID NO: 367).

22. 22. A nucleic acid comprising the RSV F glycoprotein of any one of claims 1 to 21.

23. A vector comprising the nucleic acid of claim 22.

24. 22. A nanoparticle comprising the RSV F glycoprotein of any one of claims 1 to 21 and a non-ionic surfactant core.

25. 25. The nanoparticles of claim 24, wherein the non-ionic surfactant is selected from the group consisting of polysorbate-20 (PS20), polysorbate-40 (PS40), polysorbate-60 (PS60), polysorbate-65 (PS65), and polysorbate-80 (PS80).

26. 25. The nanoparticle of claim 24, wherein the non-ionic surfactant is PS80.

27. 22. A cell expressing the RSV F glycoprotein of any one of claims 1 to 21.

28. 28. The cell of claim 27, which is an insect cell.

29. 27. An immunogenic composition comprising at least one RSV F glycoprotein according to any one of claims 1 to 21 or a nanoparticle according to any one of claims 24 to 26, and a pharmaceutically acceptable buffer.

30. 30. The immunogenic composition of claim 29, comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 different RSV F glycoproteins.

31. (i) a first RSV F glycoprotein having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the RSV F glycoprotein of any one of SEQ ID NOs: 337-340; and (ii) a second RSV F glycoprotein having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the RSV F glycoprotein of any one of SEQ ID NOs: 364-366.

31. The immunogenic composition of claim 29 or 30, comprising:

32. (i) a first RSV F glycoprotein having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the RSV F glycoprotein of SEQ ID NO: 337; and (ii) a second RSV F glycoprotein having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the RSV F glycoprotein of any one of SEQ ID NOs:

364.

31. The immunogenic composition of any one of claims 29 to 30, comprising:

33. 33. The immunogenic composition of any one of claims 29 to 32, comprising 30 μg to about 300 μg of the RSV F glycoprotein.

34. 34. The immunogenic composition of any one of claims 29 to 33, comprising about 60 μg of the RSV F glycoprotein.

35. 35. The immunogenic composition of any one of claims 29 to 34, comprising about 240 μg of the RSV F glycoprotein.

36. 36. The immunogenic composition of any one of claims 29 to 35, comprising at least one viral glycoprotein that is not an RSV F glycoprotein.

37. 1 to about 20, 1 to about 19, 1 to about 18, 1 to about 17, 1 to about 16, 1 to about 15, 1 to about 14, 1 to about 13, 1 to about 12, 1 to about 11, 1 to about 10, 1 to about 9, 1 to about 8, 1 to about 7, 1 to about 6, 1 to about 5, 1 to about 4, 1 to about 3, 1 to about 2, about 2 to about 20, about 2 to about 19, about 2 to about 18, about 2 to about 17, about 2 to about 16, about 2 to about 15, about 2 to about 14, about 2 to about 13, about 2 to about 12, about 2 to about 11, about 2 to about 10, about 2 to about 9, about 2 to about 8, about 2 to about 7, about 2 to about 6, about 2 to about 5, about 2 to about 4, about 2 to about 3, about 3 to about 20, about 3 to about 19, about 3 to about 18, about 3 up to about 17, about 3 to about 16, about 3 to about 15, about 3 to about 14, about 3 to about 13, about 3 to about 12, about 3 to about 11, about 3 to about 10, about 3 to about 9, about 3 to about 8, about 3 to about 7, about 3 to about 6, about 3 to about 5, about 3 to about 4, about 4 to about 20, about 4 to about 19, about 4 to about 18, about 4 to about 17, about 4 to about 16 , about 4 to about 15, about 4 to about 14, about 4 to about 13, about 4 to about 12, about 4 to about 11, about 4 to about 10, about 4 to about 9, about 4 to about 8, about 4 to about 7, about 4 to about 6, about 4 to about 5, about 5 to about 20, about 5 to about 19, about 5 to about 18, about 5 to about 17, about 5 to about 16, about 5 to about 15, about 5 to about 14, about 5 to about 13, about 5 to about 12, about 5 to about 11, about 5 to about 10, about 5 to about 9, about 5 to about 8, about 5 to about 7, about 5 to about 6, about 6 to about 20, about 6 to about 19, about 6 to about 18, about 6 to about 17, about 6 to about 16, about 6 to about 15, about 6 to about 14, about 6 to about 13, about 6 to about 12, about 6 to about 11, about 6 to about 1 0, about 6 to about 9, about 6 to about 8, about 6 to about 7, about 7 to about 20, about 7 to about 19, about 7 to about 18, about 7 to about 17, about 7 to about 16, about 7 to about 15, about 7 to about 14, about 7 to about 13, about 7 to about 12, about 7 to about 11, about 7 to about 10, about 7 to about 9, about 7 to about 8, about 8 to about 20, about 8 to about 19, about 8 to about 18, about 8 to about 17, about 8 to about 16, about 8 to about 15, about 8 to about 14, about 8 to about 13, about 8 to about 12, about 8 to about 11, about 8 to about 10, about 8 to about 9, about 9 to about 20, about 9 to about 19, about 9 to about 18, about 9 to about 17, about 9 to about 16, about 9 to about 15, about 9 to about 14, about 9 to about 13, about 9 to about 12, about 9 to about 11, about 9 to about 10, about 10 to about 20, about 10 to about 19, about 10 to about 18, about 10 to about 17, about 10 to about 16, about 10 to about 15, about 10 to about 14, about 10 to about 13, about 10 to about 12, about 10 to about 11, about 11 to about 20, about 11 to about 19, about 11 to about 18,about 11 to about 17, about 11 to about 16, about 11 to about 15, about 11 to about 14, about 11 to about 13, about 11 to about 12, about 12 to about 20, about 12 to about 19, about 12 to about 18, about 12 to about 17, about 12 to about 16, about 12 to about 15, about 12 to about 14, about 12 to about 13, about 13 to about 20, about 13 to about 19, about 13 to about 18, about 13 to about 17, about 13 to about 16, about 13 to about 15, about 13 to about 14, about 14 to about 20, about 14 to about 19, about 14 to about 18, 37. The immunogenic composition of any one of claims 29 to 36, comprising about 14 to about 17, about 14 to about 16, about 14 to about 15, about 15 to about 20, about 15 to about 19, about 15 to about 18, about 15 to about 17, about 15 to about 16, about 16 to about 20, about 16 to about 19, about 16 to about 18, about 16 to about 17, about 17 to about 20, about 17 to about 19, about 17 to about 18, about 18 to about 20, about 18 to about 19, or about 19 to about 20 different viral glycoproteins.

38. 37. The immunogenic composition of claim 36, wherein the at least one viral glycoprotein that is not an RSV F glycoprotein is an influenza hemagglutinin (HA) glycoprotein or a SARS-CoV-2 spike (CoV S) glycoprotein.

39. 38. The immunogenic composition of claim 37, wherein the at least one viral glycoprotein is an influenza HA glycoprotein.

40. 40. The immunogenic composition of claim 39, wherein the amino acid sequence of the HA glycoprotein has 100% identity to the amino acid sequence of a native HA glycoprotein.

41. 41. The immunogenic composition of claim 39 or 40, wherein the HA glycoprotein has a subtype selected from the group consisting of H1, H3, H4, H5, and H7.

42. 42. The immunogenic composition of any one of claims 39 to 41, comprising a first hemagglutinin (HA) glycoprotein, a second HA glycoprotein, and a third HA glycoprotein, each HA glycoprotein being derived from a different influenza strain.

43. 43. The immunogenic composition of claim 42, comprising a fourth HA glycoprotein, wherein the fourth HA glycoprotein is derived from an influenza strain different from the first HA glycoprotein, the second HA glycoprotein, and the third HA glycoprotein.

44. (i) a surfactant-core nanoparticle comprising the first HA glycoprotein and a non-ionic surfactant; (ii) HaSMaN comprising the second HA glycoprotein; 44. The immunogenic composition of any one of claims 41 to 43, comprising:

45. (i) a first surfactant-core nanoparticle comprising the first HA glycoprotein and a non-ionic surfactant; (ii) a second surfactant-core nanoparticle comprising the second HA glycoprotein and a non-ionic surfactant; (iii) a third surfactant-core nanoparticle comprising the third HA glycoprotein and a non-ionic surfactant; (iv) a fourth surfactant-core nanoparticle comprising the third HA glycoprotein and a non-ionic surfactant; 44. The immunogenic composition of claim 43, comprising:

46. (i) a surfactant-core nanoparticle comprising the first HA glycoprotein and a non-ionic surfactant; (ii) a first HaSMaN comprising the second HA glycoprotein; (iii) a second HaSMaN comprising the third HA glycoprotein; and (iv) a third HaSMaN comprising the fourth HA glycoprotein; and 44. The immunogenic composition of claim 43, comprising:

47. (i) a first surfactant-core nanoparticle comprising the first HA glycoprotein and a non-ionic surfactant; (ii) a second surfactant-core nanoparticle comprising the second HA glycoprotein and a non-ionic surfactant; (iii) a first HaSMaN comprising the third HA glycoprotein; (iv) a second HaSMaN comprising the fourth HA glycoprotein; and 44. The immunogenic composition of claim 43, comprising:

48. (i) a first surfactant-core nanoparticle comprising the first HA glycoprotein and a non-ionic surfactant; (ii) a second surfactant-core nanoparticle comprising the second HA glycoprotein and a non-ionic surfactant; (iii) a third surfactant-core nanoparticle comprising the third HA glycoprotein and a non-ionic surfactant; (iv) HaSMaN comprising the fourth HA glycoprotein; 44. The immunogenic composition of claim 43, comprising:

49. 49. The immunogenic composition of any one of claims 44 to 48, wherein the HA glycoprotein of the surfactant-core nanoparticles is derived from an influenza A strain.

50. 49. The immunogenic composition of any one of claims 44 to 48, wherein the HA glycoprotein of the surfactant-core nanoparticles is derived from an influenza B strain.

51. 49. The immunogenic composition of any one of claims 44 and 46-48, wherein the HA glycoprotein of the HaSMaN is derived from an influenza A strain.

52. 52. The immunogenic composition of any one of claims 39 to 51, comprising about 30 μg to about 300 μg of each HA glycoprotein.

53. 52. The immunogenic composition of any one of claims 39 to 51, comprising about 60 μg of each HA glycoprotein.

54. 52. The immunogenic composition of any one of claims 39 to 51, comprising about 240 μg of each HA glycoprotein.

55. 55. The immunogenic composition of any one of claims 29 to 54, comprising a SARS-CoV-2 spike (CoV S) glycoprotein.

56. 56. The immunogenic composition of claim 55, wherein the SARS-CoV-2 S glycoprotein contains (i) an inactive furin cleavage site, and (ii) prolines at amino acid positions 973 and 974, when the SARS-CoV-2 glycoprotein is numbered based on the polypeptide of SEQ ID NO:

2.

57. 57. The immunogenic composition of claim 56, wherein the inactive furin cleavage site has an amino acid sequence selected from any one of 7-34, 97, and 111.

58. 57. The immunogenic composition of claim 56, wherein the inactive furin cleavage site has the amino acid sequence QQAQ (SEQ ID NO: 7).

59. 59. The immunogenic composition of any one of claims 55-58, wherein the CoV S glycoprotein has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the SARS-CoV-2 S glycoprotein of SEQ ID NO:

87.

60. 59. The immunogenic composition of any one of claims 55-58, wherein the CoV S glycoprotein has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the SARS-CoV-2 S glycoprotein of SEQ ID NO:

274.

61. The CoV S glycoprotein is selected from the group consisting of SEQ ID NOs: 87, 89, 106, 112, 113, 114, 115, 132, 144, 151, 153, 156, 158, 174, 175, 176, 181, 182, 183, 184, 186, 188, 190, 192, 195, 217, 218, 219, 220 , 221, 222, 223, 224, 225, 226, 227, 228, 233, 234, 235, 236, 260, 261, 262, 263, 264, 274, 276, 278, 280, 284, 288, 292, 296, 300, 304, 308, 312, 316, 320, 32 59. The immunogenic composition of any one of claims 55 to 58, having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identity to any one of the polypeptides of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 39, SEQ ID NO: 39, SEQ ID NO: 3

62. (i) a first CoV S glycoprotein comprising (a) an inactive furin cleavage site, and (b) prolines at amino acid positions 973 and 974, when said SARS-CoV-2 glycoprotein is numbered based on the polypeptide of SEQ ID NO:2; (ii) a second CoV S glycoprotein comprising: (a) an inactive furin cleavage site; and (b) prolines at amino acid positions 973 and 974, when said SARS-CoV-2 glycoprotein is numbered based on the polypeptide of SEQ ID NO:2; and (c) 1 to about 2, 1 to about 3, 1 to about 4, 1 to about 5, 2 to about 5, 1 to about 6, 1 to about 7, 1 to about 8, 1 to about 9, 1 to about 10, 1 to about 11, 1 to about 12, 1 to about 13, 1 to about 14, 1 to about 15, 1 to about 16, 1 to about 17, 1 to about 18, 1 to about 18, 1 to about 19, or 1 to about 20, 1 to about 25, 1 to about 30, 1 to about 35, 1 to about 40, 1 to about 45, or 1 to about 50 modifications compared to said first CoV S glycoprotein. S glycoprotein and 62. The immunogenic composition of any one of claims 29 to 61, comprising:

63. (i) a first CoV S glycoprotein comprising (a) an inactive furin cleavage site, and (b) prolines at amino acid positions 973 and 974, when said SARS-CoV-2 glycoprotein is numbered based on the polypeptide of SEQ ID NO:2; (ii) a second CoV S glycoprotein comprising: (a) an inactive furin cleavage site; and (b) prolines at amino acid positions 973 and 974, when said SARS-CoV-2 glycoprotein is numbered based on the polypeptide of SEQ ID NO:2; and (c) 1 to about 2, 1 to about 3, 1 to about 4, 1 to about 5, 2 to about 5, 1 to about 6, 1 to about 7, 1 to about 8, 1 to about 9, 1 to about 10, 1 to about 11, 1 to about 12, 1 to about 13, 1 to about 14, 1 to about 15, 1 to about 16, 1 to about 17, 1 to about 18, 1 to about 18, 1 to about 19, or 1 to about 20, 1 to about 25, 1 to about 30, 1 to about 35, 1 to about 40, 1 to about 45, or 1 to about 50 modifications compared to said first CoV S glycoprotein. S glycoprotein, (iii) a third CoV S glycoprotein comprising: (a) an inactive furin cleavage site; and (b) prolines at amino acid positions 973 and 974, when said SARS-CoV-2 glycoprotein is numbered based on the polypeptide of SEQ ID NO:2; and (c) a third CoV S glycoprotein comprising: the third CoV S glycoprotein comprising 1 to about 2, 1 to about 3, 1 to about 4, 1 to about 5, 2 to about 5, 1 to about 6, 1 to about 7, 1 to about 8, 1 to about 9, 1 to about 10, 1 to about 11, 1 to about 12, 1 to about 13, 1 to about 14, 1 to about 15, 1 to about 16, 1 to about 17, 1 to about 18, 1 to about 18, 1 to about 19, or 1 to about 20, 1 to about 25, 1 to about 30, 1 to about 35, 1 to about 40, 1 to about 45, or 1 to about 50 modifications relative to the S glycoprotein; 62. The immunogenic composition of any one of claims 29 to 61, comprising:

64. (i) a first CoV S glycoprotein comprising (a) an inactive furin cleavage site, and (b) prolines at amino acid positions 973 and 974, when said SARS-CoV-2 glycoprotein is numbered based on the polypeptide of SEQ ID NO:2; (ii) a second CoV S glycoprotein comprising: (a) an inactive furin cleavage site; and (b) prolines at amino acid positions 973 and 974, when said SARS-CoV-2 glycoprotein is numbered based on the polypeptide of SEQ ID NO:2; and (c) 1 to about 2, 1 to about 3, 1 to about 4, 1 to about 5, 2 to about 5, 1 to about 6, 1 to about 7, 1 to about 8, 1 to about 9, 1 to about 10, 1 to about 11, 1 to about 12, 1 to about 13, 1 to about 14, 1 to about 15, 1 to about 16, 1 to about 17, 1 to about 18, 1 to about 18, 1 to about 19, or 1 to about 20, 1 to about 25, 1 to about 30, 1 to about 35, 1 to about 40, 1 to about 45, or 1 to about 50 modifications compared to said first CoV S glycoprotein. S glycoprotein, (iii) a third CoV S glycoprotein comprising: (a) an inactive furin cleavage site; and (b) prolines at amino acid positions 973 and 974, when said SARS-CoV-2 glycoprotein is numbered based on the polypeptide of SEQ ID NO:2; and (c) a third CoV S glycoprotein comprising: the third CoV S glycoprotein comprising 1 to about 2, 1 to about 3, 1 to about 4, 1 to about 5, 2 to about 5, 1 to about 6, 1 to about 7, 1 to about 8, 1 to about 9, 1 to about 10, 1 to about 11, 1 to about 12, 1 to about 13, 1 to about 14, 1 to about 15, 1 to about 16, 1 to about 17, 1 to about 18, 1 to about 18, 1 to about 19, or 1 to about 20, 1 to about 25, 1 to about 30, 1 to about 35, 1 to about 40, 1 to about 45, or 1 to about 50 modifications compared to the S glycoprotein; (iv) a fourth CoV S glycoprotein comprising: (a) an inactive furin cleavage site; and (b) prolines at amino acid positions 973 and 974, when said SARS-CoV-2 glycoprotein is numbered based on the polypeptide of SEQ ID NO:2; and (c) 1 to about 2, 1 to about 3, 1 to about 4, 1 to about 5, 2 to about 5, 1 to about 6, 1 to about 7, 1 to about 8, 1 to about 9, 1 to about 10, 1 to about 11, 1 to about 12, 1 to about 13, 1 to about 14, 1 to about 15, 1 to about 16, 1 to about 17, 1 to about 18, 1 to about 18, 1 to about 19, or 1 to about 20, 1 to about 25, 1 to about 30, 1 to about 35, 1 to about 40, 1 to about 45, or 1 to about 50 modifications compared to said first CoV S glycoprotein. S glycoprotein and 62. The immunogenic composition of any one of claims 29 to 61, comprising:

65. (i) a first CoV S glycoprotein comprising (a) an inactive furin cleavage site, and (b) prolines at amino acid positions 973 and 974, when said SARS-CoV-2 glycoprotein is numbered based on the polypeptide of SEQ ID NO:2; (ii) a second CoV S glycoprotein comprising: (a) an inactive furin cleavage site; and (b) prolines at amino acid positions 973 and 974, when said SARS-CoV-2 glycoprotein is numbered based on the polypeptide of SEQ ID NO:2; and (c) 1 to about 2, 1 to about 3, 1 to about 4, 1 to about 5, 2 to about 5, 1 to about 6, 1 to about 7, 1 to about 8, 1 to about 9, 1 to about 10, 1 to about 11, 1 to about 12, 1 to about 13, 1 to about 14, 1 to about 15, 1 to about 16, 1 to about 17, 1 to about 18, 1 to about 18, 1 to about 19, or 1 to about 20, 1 to about 25, 1 to about 30, 1 to about 35, 1 to about 40, 1 to about 45, or 1 to about 50 modifications compared to said first CoV S glycoprotein. S glycoprotein, (iii) a third CoV S glycoprotein comprising: (a) an inactive furin cleavage site; and (b) prolines at amino acid positions 973 and 974, when said SARS-CoV-2 glycoprotein is numbered based on the polypeptide of SEQ ID NO:2; and (c) a third CoV S glycoprotein comprising: the third CoV S glycoprotein comprising 1 to about 2, 1 to about 3, 1 to about 4, 1 to about 5, 2 to about 5, 1 to about 6, 1 to about 7, 1 to about 8, 1 to about 9, 1 to about 10, 1 to about 11, 1 to about 12, 1 to about 13, 1 to about 14, 1 to about 15, 1 to about 16, 1 to about 17, 1 to about 18, 1 to about 18, 1 to about 19, or 1 to about 20, 1 to about 25, 1 to about 30, 1 to about 35, 1 to about 40, 1 to about 45, or 1 to about 50 modifications compared to the S glycoprotein; (iv) a fourth CoV S glycoprotein comprising: (a) an inactive furin cleavage site; and (b) prolines at amino acid positions 973 and 974, when said SARS-CoV-2 glycoprotein is numbered based on the polypeptide of SEQ ID NO:2; and (c) 1 to about 2, 1 to about 3, 1 to about 4, 1 to about 5, 2 to about 5, 1 to about 6, 1 to about 7, 1 to about 8, 1 to about 9, 1 to about 10, 1 to about 11, 1 to about 12, 1 to about 13, 1 to about 14, 1 to about 15, 1 to about 16, 1 to about 17, 1 to about 18, 1 to about 18, 1 to about 19, or 1 to about 20, 1 to about 25, 1 to about 30, 1 to about 35, 1 to about 40, 1 to about 45, or 1 to about 50 modifications compared to said first CoV S glycoprotein. S glycoprotein, (v) a fifth CoV S glycoprotein comprising: (a) an inactive furin cleavage site; and (b) prolines at amino acid positions 973 and 974, when said SARS-CoV-2 glycoprotein is numbered based on the polypeptide of SEQ ID NO:2; and (c) 1 to about 2, 1 to about 3, 1 to about 4, 1 to about 5, 2 to about 5, 1 to about 6, 1 to about 7, 1 to about 8, 1 to about 9, 1 to about 10, 1 to about 11, 1 to about 12, 1 to about 13, 1 to about 14, 1 to about 15, 1 to about 16, 1 to about 17, 1 to about 18, 1 to about 18, 1 to about 19, or 1 to about 20, 1 to about 25, 1 to about 30, 1 to about 35, 1 to about 40, 1 to about 45, or 1 to about 50 modifications compared to said first CoV S glycoprotein. S glycoprotein and 62. The immunogenic composition of any one of claims 29 to 61, comprising:

66. the first CoV S glycoprotein is selected from the group consisting of SEQ ID NOs: 87, 89, 106, 112, 113, 114, 115, 132, 144, 151, 153, 156, 158, 174, 175, 176, 181, 182, 183, 184, 186, 188, 190, 192, 195, 217, 218, 219, 220 , 221, 222, 223, 224, 225, 226, 227, 228, 233, 234, 235, 236, 260, 261, 262, 263, 264, 274, 276, 278, 280, 284, 288, 292, 296, 300, 304, 308, 312, 316, 320, 32 66. The immunogenic composition of any one of claims 29 to 65, having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identity to any one of the polypeptides of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 39, SEQ ID NO: 3

67. 67. The immunogenic composition of claim 65 or 66, comprising about 5 μg of the first CoV S glycoprotein and 1 ng to about 4.9 μg of each of the second CoV S glycoprotein, the third CoV S glycoprotein, the fourth CoV S glycoprotein, and the fifth CoV S glycoprotein.

68. 68. The immunogenic composition of any one of claims 65 to 67, wherein one or more of the modifications in the second CoV S glycoprotein, the third CoV S glycoprotein, the fourth CoV S glycoprotein, and the fifth CoV S glycoprotein are present in the receptor binding domain (RBD).

69. 68. The immunogenic composition of any one of claims 65 to 67, wherein one or more of the modifications in the second CoV S glycoprotein, the third CoV S glycoprotein, the fourth CoV S glycoprotein, and the fifth CoV S glycoprotein are present in the N-terminal domain (NTD).

70. 68. The immunogenic composition of any one of claims 65 to 67, wherein one or more of the modifications in the second CoV S glycoprotein, the third CoV S glycoprotein, the fourth CoV S glycoprotein, and the fifth CoV S glycoprotein are present in the S1 subunit.

71. 68. The immunogenic composition of any one of claims 65 to 67, wherein one or more of the modifications in the second CoV S glycoprotein, the third CoV S glycoprotein, the fourth CoV S glycoprotein, and the fifth CoV S glycoprotein are present in the S2 subunit.

72. 72. The immunogenic composition of any one of claims 55 to 71, comprising about 1 μg to about 175 μg or about 1 μg to about 10 μg of SARS-CoV-2 S glycoprotein.

73. 72. The immunogenic composition of any one of claims 55 to 71, comprising about 5 μg of the SARS-CoV-2 S glycoprotein.

74. 72. The immunogenic composition of any one of claims 55 to 71, comprising about 25 μg of the SARS-CoV-2 S glycoprotein.

75. 75. The immunogenic composition of any one of claims 29 to 74, comprising an adjuvant.

76. the adjuvant comprises at least two iscom particles; the first iscom particles comprise fraction A of Quillaja Saponaria Molina and do not comprise fraction C of Quillaja Saponaria Molina; the second iscom particles contain fraction C of Quillaja Saponaria Molina and do not contain fraction A of Quillaja Saponaria Molina; The immunogenic composition of claim 75.

77. Fraction A of Quillaja Saponaria Molina and Fraction C of Quillaja Saponaria Molina account for about 85% by weight and about 15% by weight, respectively, based on the total weight of Fraction A of Quillaja Saponaria Molina and Fraction C of Quillaja Saponaria Molina in the adjuvant; The immunogenic composition of claim 76.

78. 77. The immunogenic composition of claim 76, wherein Quillaja Saponaria Molina Fraction A and Quillaja Saponaria Molina Fraction C account for about 92% by weight and about 8% by weight, respectively, of the total weight of Quillaja Saponaria Molina Fraction A and Quillaja Saponaria Molina Fraction C in the adjuvant.

79. 77. The immunogenic composition of claim 76, wherein Fraction A of Quillaja Saponaria Molina and Fraction C of Quillaja Saponaria Molina each account for at least about 85% by weight of the total weight of Fraction A of Quillaja Saponaria Molina and Fraction C of Quillaja Saponaria Molina in the adjuvant, and Fraction C of Quillaja Saponaria Molina accounts for the remainder.

80. The immunogenic composition according to claim 76, wherein Fraction A of Quillaja Saponaria Molina and Fraction C of Quillaja Saponaria Molina account for 50 to 96% by weight, respectively, of the total weight of Fraction A of Quillaja Saponaria Molina and Fraction C of Quillaja Saponaria Molina in the adjuvant, and Fraction C of Quillaja Saponaria Molina accounts for the remainder.

81. 81. The immunogenic composition of any one of claims 75 to 80, comprising about 25 μg to about 100 μg, about 1 μg to about 100 μg, or about 50 μg to about 100 μg of adjuvant.

82. 82. The immunogenic composition of any one of claims 75 to 81, comprising about 20 μg or 50 μg of adjuvant.

83. 82. The immunogenic composition of any one of claims 75 to 81, wherein the adjuvant does not contain 3-O-desacyl-4'-monophosphoryl lipid A from Salmonella Minnesota.

84. 84. The immunogenic composition of any one of claims 29 to 83, which is an intramuscular immunogenic composition.

85. 84. The immunogenic composition of any one of claims 29 to 83, which is an intranasal immunogenic composition.

86. 86. A pre-filled syringe comprising the immunogenic composition of any one of claims 29 to 85.

87. 86. A method of stimulating an immune response in a subject against one or more of respiratory syncytial virus (RSV), influenza, and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), comprising administering to the subject the immunogenic composition of any one of claims 29 to 85.

88. 88. The method of claim 87, comprising administering 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 doses of the immunogenic composition.

89. 89. The method of claim 87 or 88, comprising administering a first dose and a second dose of the immunogenic composition, wherein the second dose is administered 21 days after the first dose.

90. 90. The method of any one of claims 87 to 89, comprising administering 0.5 mL of the immunogenic composition.

91. 91. The method of any one of claims 87 to 90, wherein the immunogenic composition is administered intramuscularly.

92. 91. The method of any one of claims 87 to 90, wherein the immunogenic composition is administered intranasally.

93. 93. The method of any one of claims 87 to 92, comprising administering the immunogenic composition in a pre-filled syringe.

94. at least about 2 months, at least about 2.5 months, at least about 3 months, at least about 3.5 months, at least about 4 months, at least about 4.5 months, at least about 5 months, at least about 5.5 months, at least about 6 months, at least about 6.5 months, at least about 7 months, at least about 7.5 months, at least about 8 months, at least about 8.5 months, at least about 9 months, at least about 9.5 months, at least about 10 months, at least about 10.5 months, at least about 11 months, at least about 11.5 months, at least about 12 months, at least 13 months, at least 14 months, at least 15 months, at least 16 months, at least 17 months, at least 18 months, at least 19 months, at least 20 months, at least 21 months, at least for at least 22 months, at least 23 months, or at least 24 months, about 50% to about 99%, about 50% to about 95%, about 50% to about 90%, about 50% to about 85%, about 50% to about 80%, about 60% to about 99%, about 65% to about 95%, about 65% to about 90%, about 65% to about 85%, about 69% to about 81%, about 60% to about 95%, about 60% to about 90%, about 60% to about 85%, about 60% to about 80%, 94. The method of any one of claims 87-93, wherein the method prevents one or more of RSV, influenza, and SARS-CoV-2 with an effectiveness of about 40% to about 99%, about 40% to about 95%, about 40% to about 90%, about 40% to about 85%, about 40% to about 80%, about 40% to about 75%, about 40% to about 70%, about 40% to about 65%, about 40% to about 55%, or about 40% to about 50%.

95. about 50% to about 99%, about 50% to about 95%, about 50% to about 99% for up to about 2 months, up to about 2.5 months, up to about 3 months, up to about 3.5 months, up to about 4 months, up to about 4.5 months, up to about 5 months, up to about 5.5 months, up to about 6 months, up to about 6.5 months, up to about 7 months, up to about 7.5 months, up to about 8 months, up to about 8.5 months, up to about 9 months, up to about 9.5 months, up to about 10 months, up to about 10.5 months, up to about 11 months, up to about 11.5 months, up to about 12 months, up to about 13 months, up to 14 months, up to 15 months, up to 16 months, up to 17 months, up to 18 months, up to 19 months, up to 20 months, up to 21 months, up to 22 months, up to 23 months, or up to 24 months after administration of the immunogenic composition %, about 50% to about 90%, about 50% to about 85%, about 50% to about 80%, about 60% to about 99%, about 65% to about 95%, about 65% to about 90%, about 65% to about 85%, about 69% to about 81%, about 60% to about 95%, about 60% to about 90%, about 60% to about 85%, about 60% to about 80%, about 40% to about 99%, about 40% to about 95%, about 40% 94. The method of any one of claims 87-93, wherein the method prevents one or more of RSV, influenza, and SARS-CoV-2 with an efficacy of about 90%, about 40% to about 85%, about 40% to about 80%, about 40% to about 75%, about 40% to about 70%, about 40% to about 65%, about 40% to about 55%, or about 40% to about 50%.

96. 96. The method of any one of claims 87 to 95, wherein the subject is a human.

97. 97. The method of any one of claims 87 to 96, wherein the subject is a pregnant woman.

98. 98. The method of any one of claims 87 to 97, wherein the subject is a child.

99. 98. The method of claim 97, comprising administering the immunogenic composition to a pregnant female at about 32 to about 36 weeks of gestation.

100. 98. The method of claim 97, comprising administering the immunogenic composition to a pregnant female at about 28 to about 33 weeks of gestation.

101. 101. The method of any one of claims 87 to 100, comprising administering the immunogenic composition to a human subject who is at least 60 years of age.

102. 99. The method of claim 98, wherein the child is 5 years of age or younger.

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