RSV F vaccine formulations

Abstract

Disclosed herein are RSV F glycoproteins and nanoparticles comprising the glycoproteins suitable for use in vaccines. The nanoparticles present antigens from pathogens surrounding and associated with the detergent core, resulting in enhanced stability and good immunogenicity. Doses, formulations, and methods for preparing the vaccines and nanoparticles are also disclosed.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 63 / 483,119, filed February 3, 2023. The entire contents of that application are incorporated herein for all purposes.

[0003] Description of the text file submitted electronically

[0004] The entire 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.

[0005] field

[0006] The present disclosure generally relates to a RSV F glycoprotein and nanoparticle that can be used to stimulate an immune response. The nanoparticle provides an antigen associated with a detergent core, such as a glycoprotein antigen, and is typically produced using recombinant methods. The nanoparticle has improved stability and enhanced epitope presentation. The disclosure also provides compositions containing the nanoparticles, methods for producing them, and methods for stimulating an immune response. Background of the Invention

[0008] Infectious diseases remain a problem worldwide. While progress has been made in developing vaccines against some pathogens, many still pose a threat to human health. Respiratory syncytial virus (RSV) kills 6,000–10,000 people aged 65 and older and 100–300 children under the age of 5 each year. Developing vaccines to prevent or reduce the severity of life-threatening infectious diseases like RSV is desirable. SUMMARY OF THE INVENTION

[0010] The present disclosure provides non-naturally occurring respiratory syncytial virus (RSV) fusion (F) glycoproteins suitable for inducing an immune response against respiratory syncytial virus (RSV). The present disclosure also provides nanoparticles containing the glycoprotein and methods of stimulating an immune response against RSV.

[0011] Provided herein are RSV F glycoproteins comprising an F1 domain, an F2 domain, and p27; wherein the glycoprotein comprises one or more modifications selected from the group consisting of: (a) a deletion of one or more of amino acids 137-146; (b) an inactivated primary furin cleavage site; (c) an inactivated secondary furin cleavage site; (d) S155C; (e) S290C; (f) S190F; (g) V207L; and (h) N116Q; wherein the modifications are numbered according to the RSV F glycoprotein of SEQ ID NO: 330. In embodiments, the RSV F glycoprotein comprises S155C and S290C mutations. In embodiments, the RSV F glycoprotein comprises S190F and V207L ​​mutations. In embodiments, the RSV F glycoprotein comprises S155C, S290C, S190F, and V207L ​​mutations. In embodiments, the RSV F glycoprotein comprises N116Q mutations. In embodiments, the RSV F glycoprotein comprises an inactivated major 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. In embodiments, the RSV F glycoprotein comprises an inactivated major 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, wherein the RSV F glycoprotein is 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. In embodiments, the RSV F glycoprotein comprises: (i) an F1 domain having an amino acid sequence that is 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 that is 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.In 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 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 DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 Binding of the BV2279 glycoprotein to antibodies recognizing site II (palivizumab), site IV (RSV.42, referred to as "RSV.42.2"), site Φ (D25), and site VIII (hRSV90) epitopes is shown.

[0013] Figure 2 Shown are anti-RSV F IgG titers in cotton rat sera 42 days after immunization with the BV2279 (labeled "prefusion RSV F Cav1") or BV1184 (labeled "prefusogenic RSV F") vaccine compositions of Example 2.

[0014] Figure 3 Shown are RSV neutralization titers against RSV A (Long) and RSV B (18537) strains following immunization with the BV2279 (labeled "prefusion RSV F Cav1") or BV1184 (labeled "prefusion RSV F") vaccine compositions of Example 2.

[0015] Figure 4 Shown are the competing antibody equivalent titers induced by immunization with the BV2279 (labeled "prefusion RSV FCav1") or BV1184 (labeled "prefusion RSV F") vaccine compositions of Example 2.

[0016] Figure 5RSV virus titers in cotton rat tissues four days after viral challenge are shown. The rats were immunized with the vaccine compositions of Example 2, BV2279 (labeled "prefusion RSV F Cav1") or BV1184 (labeled "prefusion RSV F").

[0017] Figure 6 Shown are transmission electron microscopy (TEM) images of influenza HA detergent core nanoparticles alone (left), a saponin adjuvant (i.e., a first iscom particle containing Quillaja Fraction A but not Quillaja Fraction C, and a second iscom particle containing Quillaja Fraction C but not Quillaja Fraction A, wherein Fraction A constitutes 85% of the total weight of Quillaja Fraction A and Quillaja Fraction C in the adjuvant and Quillaja Fraction C constitutes the remainder) (center), and hemagglutinin saponin matrix nanoparticles (HaSMaNs) (right).

[0018] Figure 7A The primary structure of the wild-type SARS-CoV-2 S polypeptide containing the signal peptide is shown, numbered according to SEQ ID NO: 1. Figure 7B The primary structure of the wild-type SARS-CoV-2 S polypeptide without the signal peptide is shown, numbered according to SEQ ID NO: 2. Detailed Description of the Invention

[0020] definition

[0021] As used herein and in 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" may refer to one protein or a mixture of such proteins, and reference to "the method" includes reference to equivalent steps and / or methods known to those skilled in the art, and so forth.

[0022] As used herein, the term "adjuvant" refers to a compound that, when used in combination with an immunogen, enhances or otherwise changes or modifies the immune response induced against the immunogen. Modification of the immune response can include strengthening or amplifying the specificity of one or both of the antibody and cellular immune responses.

[0023] As used herein, the term "about" or "approximately" before a numerical value means a range of plus or minus 10% of the numerical value. For example, "about 100" includes 90 and 110.

[0024] As used herein, the terms "immunogen," "antigen," and "epitope" refer to a substance, such as a protein, including glycoproteins and peptides, that is capable of eliciting an immune response.

[0025] As used herein, an "immunogenic composition" is a composition comprising an antigen, wherein administration of the composition to a subject results in the subject generating a humoral and / or cellular immune response against the antigen.

[0026] As used herein, "substantially" refers to the separation of a substance (e.g., a compound, polynucleotide, or polypeptide) such that the substance constitutes the majority percentage of the sample in which it is contained. For example, in a sample, a substantially purified component comprises 85%, preferably 85%-90%, more preferably at least 95%-99.5%, and most preferably at least 99% of the sample. If a component is replaced in substantial amounts, the amount remaining in the sample is less than or equal to about 0.5% to about 10%, preferably less than about 0.5% to about 1.0%.

[0027] As used herein, the terms "treat," "treatment," and "treating" refer to methods for obtaining a beneficial or desired result (e.g., a clinical result). For the purposes of this disclosure, a beneficial or desired result may include inhibiting or suppressing the development or progression of an infection or disease; ameliorating symptoms of an infection or disease, or reducing the progression of symptoms of an infection or disease; or a combination thereof.

[0028] As used herein, "prevent" and "prevent" are used interchangeably and can mean preventing an infection or disease completely, or preventing the development of symptoms of the infection or disease; delaying the onset of an infection or disease or its symptoms; or lessening the severity of an infection or disease or its symptoms that subsequently develops.

[0029] As used herein, "effective dose" or "effective amount" refers to the amount of an immunogen sufficient to induce an immune response that reduces at least one symptom of a pathogen infection. An effective dose or effective amount can be determined, for example, by measuring the amount of neutralizing secretory and / or serum antibodies, such as by plaque neutralization, complement fixation, enzyme-linked immunosorbent assay (ELISA), or microneutralization assay.

[0030] As used herein, the term "vaccine" refers to an immunogenic composition, such as an immunogen derived from a pathogen, that is used to induce an immune response against the pathogen, thereby providing 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). The protective immune response may include the formation of antibodies and / or cell-mediated responses. Depending on the context, the term "vaccine" may also refer to an immunogen suspension or solution that is administered to a subject to produce protective immunity.

[0031] 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, teenager, child (2 to 14 years old), infant (birth to 2 years old) or newborn (up to 2 months). In certain aspects, the subject is up to 4 months old, or up to 6 months old. In aspects, an adult is about 65 years old or older, or about 60 years old or older. In embodiments, the subject is five years old or younger. In embodiments, the subject is 65 years old or older. In aspects, the subject is a pregnant woman or a woman who intends to become pregnant. In other aspects, the subject is not a human; for example, a non-human primate; for example, a baboon, chimpanzee, gorilla or macaque. In certain aspects, the subject can be a pet, such as a dog or a cat.

[0032] In the context of two or more nucleic acids or polypeptide sequences, the term "percent identity" refers to two or more sequences or subsequences that have a specified percentage of identical nucleotides or amino acid residues when compared. Unless otherwise indicated, percent identity is calculated using the tool EMBOSS Needle over the entire length of the compared sequences, which is available online at ebi.ac.uk / jdispatcher / psa / emboss_needle. When comparing the percent identity of two polypeptides, the following default parameters were used: output format = pair; matrix = BLOSUM62; gap open = 10; gap extension 0.5, end gap = false, end gap open = 10, end gap extension = 0.5. When comparing the percent identity of two nucleic acids, the following default parameters are used: Output format = pairs; Matrix = DNAfull; Gap opening = 10; Gap extension 0.5, End gap = false, End gap opening = 10, End gap extension = 0.5.

[0033] As used herein, the term "vaccine" refers to an immunogenic composition, such as an immunogen derived from a pathogen, that is used to induce an immune response against the pathogen, thereby providing 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). The protective immune response may include the formation of antibodies and / or cell-mediated responses. Depending on the context, the term "vaccine" may also refer to an immunogen suspension or solution that is administered to a subject to produce protective immunity.

[0034] In aspects, the subject's immunodeficiency is impaired. In embodiments, the subject with impaired immunodeficiency is administered an agent causing immunosuppression. The limiting examples of the agent causing immunosuppression include corticosteroids (e.g., prednisone), alkylating agents (e.g., cyclophosphamide), antimetabolites (e.g., azathioprine or 6-mercaptopurine), transplant-related immunosuppressive drugs (e.g., cyclosporine, tacrolimus, sirolimus, or mycophenolate mofetil), mitoxantrone, chemotherapeutics, methotrexate, tumor necrosis factor (TNF) blockers (e.g., etanercept, adalimumab, infliximab). In embodiments, the subject with impaired immunofunction is infected with a virus (e.g., human immunodeficiency virus or Epstein-Barr virus). In embodiments, the virus is a respiratory virus, such as respiratory syncytial virus, influenza virus, parainfluenza virus, adenovirus or picornavirus. In embodiments, the subject with impaired immunofunction suffers from acquired immunodeficiency syndrome (AIDS). In embodiments, the subject with impaired immunofunction is a person coexisting with human immunodeficiency virus (HIV). In embodiments, the subject with impaired immunofunction is immunocompromised due to a treatment regimen designed to prevent inflammation or prevent transplant rejection. In embodiments, the subject with impaired immunofunction is a subject who has received a transplant. In embodiments, the subject with impaired immunofunction has undergone radiotherapy or splenectomy.In embodiments, the immunocompromised subject has been diagnosed with 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 transplantation, sickle cell disease, thalassemia, autoimmune lymphoproliferative syndrome (ALPS), autoimmune polyglandular syndrome type 1 (APS-1), B-cell expansion with NF-κB and T-cell anergy (BENTA) disease, caspase 8 deficiency state (CEDS), chronic granulomatous disease (CGD), common variable immunodeficiency (CVID), congenital neutropenia syndrome, cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) deficiency, DO CK8 deficiency, GATA2 deficiency, glycosylation disorders with immunodeficiency, hyperimmunoglobulin E syndrome (HIES), hyperimmunoglobulin M syndrome, diabetes mellitus type 1, diabetes mellitus type 2, interferon gamma deficiency, interleukin 12 deficiency, interleukin 23 deficiency, leukocyte adhesion deficiency, lipopolysaccharide-reactive beige anchor (LRBA) deficiency, PI3 kinase disorders, PLCG2-associated antibody deficiency and immune dysregulation (PLAID), severe combined immunodeficiency (SCID), STAT3 dominant-negative disease, STAT3 gain-of-function disease, warts, hypogammaglobulinemia, infections, and ineffectively produced chronic myeloid leukopenia (WHIM) syndrome nemia, infections, and myelokathexis (WHIM) syndrome), Wisckott-Aldrich syndrome (WAS), X-linked agammaglobulinemia (XLA), X-linked lymphoproliferative disorder (XLP), uremia, malnutrition, or XMEN disease. In embodiments, the immunocompromised subject is a current or former smoker. In embodiments, the immunocompromised subject has a B cell deficiency, a T cell deficiency, a macrophage deficiency, a cytokine deficiency, a phagocytic deficiency, a phagocytic dysfunction, a complement deficiency, or a combination thereof.

[0035] In an embodiment, the subject is overweight or obese. In an embodiment, the overweight subject has a 2 and <30kg / m 2 In the embodiment, the obese subject has a body mass index (BMI) of ≥30 kg / m 2 In embodiments, the subject suffers from a mental health disorder. In embodiments, the mental health disorder is depression, schizophrenia, or anxiety.

[0036] As used herein, the term "pharmaceutically acceptable" means approved by a U.S. federal regulatory agency or a state government or listed in the U.S. Pharmacopoeia, the European Pharmacopoeia or other generally recognized pharmacopeia for use in mammals, and more particularly in humans. These compositions can be used as vaccines and / or antigenic compositions for inducing a protective immune response in vertebrates.

[0037] As used herein, the term "modification" refers to one or more amino acid mutations, deletions or additions of RSV F glycoprotein when referring to RSV F glycoprotein. The position of modification in RSV F glycoprotein can be determined by comparing the sequence of RSV F glycoprotein with 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 position of modification in SARS-CoV-2 spike (S) glycoprotein (alternatively referred to herein as "CoV S glycoprotein") can be determined by comparing the peptide sequence with 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 sequence of native RSV F glycoprotein with the amino acid sequence of SEQ ID NO: 329 and SEQ ID NO: 330 and the sequence of native CoV S glycoprotein with the amino acid sequence of SEQ ID NO: 1 and SEQ ID NO: 2 are shown in the table below.

[0038]

[0039]

[0040]

[0041]

[0042] The term "efficacy" of an immunogenic composition or vaccine composition described herein refers to the percentage reduction in infection (e.g., RSV virus infection) in a group to which the immunogenic composition is administered compared to a group to which the immunogenic composition is not administered. In embodiments, efficacy (E) is calculated using the following equation: E(%) = (1-RR) x 100, where RR = the relative risk of disease between a group to which the immunogenic composition is administered and a group to which the immunogenic composition is not administered. In embodiments, the immunogenic compositions described herein have 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%, between about 50% and about 99%, between about 50% and about 98%, between about 60% and about 99%, between about 80% and about 85%, between about 90% and about 91%, between about 92%, between about 93%, between about 94%, between about 95%, between about 96%, between about 97%, between about 98%, between about 99%, between about 99%, between about 90% and ... Between about 60% and about 98%, between about 70% and about 98%, between about 70% and about 95%, between about 70% and about 99%, between about 80% and about 99%, between about 80% and about 98%, between about 80% and about 95%, between about 85% and about 99%, between about 85% and about 98%, between about 85% and about 95%, between about 90% and about 95%, between about 90% and about 98%, or between about 90% and about 99% efficacy against RSV virus.

[0043] The term SARS-CoV-2 “variant” is used interchangeably herein with “heterogeneous SARS-CoV-2 strain” to refer to a SARS-CoV-2 virus that comprises a CoV S polypeptide having 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. The S polypeptide may have at least one and at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 21, at most 22, at most 23, at most 24, at most 25, at most 26, at most 27, at most 28, at most 29, at most 30, at most 31, at most 32, at most 33, at most 34, at most 35, at most 36, at most 37, at most 38, at most 39, at most 40, at most 41, at most 42, at most 43, at most 44, at most 45, at most 46, at most 47, at most 48, at most 49, at most 50, at most 51, at most 52, at most 53, at most 54, at most 55, at most 56, at most 57, at most 58, at most 59, at most 60, at most 61 7, 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. In aspects, the SARS-CoV-2 variant can have between about 2 and about 35 modifications, between about 5 and about 10 modifications, between about 5 and about 20 modifications, between about 10 and about 20 modifications, between about 15 and about 25 modifications, between about 20 and 30 modifications, between about 20 and about 40 modifications, between about 25 and about 45 modifications, between about 25 and about 100 modifications, between about 25 and about 45 modifications, or between about 35 and about 100 modifications compared to the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 2.

[0044] As used herein, the terms "intranasal administration" and "nasal administration" refer to the administration of an immunogenic composition as described herein to the nasal cavity of a subject. In embodiments, intranasal administration is achieved using a liquid formulation (e.g., aqueous formulation), an aerosolized formulation, or a dry powder formulation. In embodiments, the liquid formulation, an aerosolized formulation, or a dry powder formulation is administered via an externally propelled nasal delivery device. In embodiments, the liquid formulation, an aerosolized formulation, or a dry powder formulation is administered via a self-propelled (i.e., via inhalation) nasal delivery device. In embodiments, the liquid formulation, an aerosolized formulation, or a dry powder formulation is administered via nasal insufflation (when the immunogenic composition is blown into the nose) or nasal drip (when the immunogenic composition is dripped into the nose). In embodiments, the liquid formulation, an aerosolized formulation, or a dry powder formulation is administered via a gel, cream, ointment, lotion, or paste applied to one or more nasal epithelia (e.g., olfactory epithelium or nasal respiratory epithelium). In embodiments, the immunogenic composition is applied to the mucus in the nasal cavity of a subject.

[0045] As used herein, the term "non-invasive nasal delivery device" refers to an instrument capable of delivering an immunogenic composition to the nasal cavity without piercing the epithelium of a subject. Non-limiting examples of non-invasive nasal delivery devices include aerosol or nebulizer devices of both propellant (e.g., pressurized inhalers) and non-propellant (e.g., pump inhalers) types, particle dispersion devices, sprayers, and pressurized olfactory delivery devices for delivering liquid or powder formulations.

[0046] As used herein, the term "dry powder composition" refers to a lyophilized or spray-dried form of the immunogenic composition described herein. In embodiments, the dry powder composition contains less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less residual water content.

[0047] As used herein, the term "penetration enhancer" refers to a component of an immunogenic composition formulated for intranasal administration that facilitates passage of viral glycoproteins across the nasal epithelium.

[0048] As used herein, the terms "bedside mixture," "bedside formulation," "bedside vaccine composition," "bedside vial," and "bedside vial formulation" refer to a vaccine formulation prepared just before 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., administered as two consecutive injections, or the antigen and adjuvant are combined into a single injection before administration).

[0049] As used herein, the terms "co-formulated mixture," "co-formulation," "co-formulated immunogenic composition," "prefilled syringe," and "premix" refer to vaccine formulations that are 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 an embodiment, the formulation contains a hemagglutinin and an adjuvant (e.g., a saponin adjuvant) and forms HaSMaN (hemagglutinin saponin matrix nanoparticles).

[0050] As used herein, the term "split virions" refers to viruses (e.g., influenza viruses or SARS-CoV-2 viruses) whose viral membranes have been disrupted by a surfactant. Examples of surfactants are described throughout this disclosure. Split virions are not subjected to further purification, so they typically contain a variety of viral proteins.

[0051] As used herein, the term "recombinant" refers to a protein (eg, hemagglutinin) produced in a cell by transcription and translation of a nucleic acid introduced into the cell. The nucleic acid can be introduced via a vector or virus encoding the nucleic acid.

[0052] As used herein, the term "whole influenza virus" refers to a virus including all of its envelope, viral membrane, nucleocapsid, and genetic material. In embodiments, the whole influenza virus is inactivated.

[0053] As used herein, the term "inactivated virus" refers to a virus that has been subjected to a treatment that significantly reduces or eliminates its virulence compared to the wild-type virus.

[0054] The term "bronchoalveolar lavage fluid," also known as "BAL," refers to a fluid sample retrieved from a patient's lungs. In embodiments, BAL is retrieved 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 collected from the lungs.

[0055] The term "mucosal immunity" refers to the 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.

[0056] The term "atomization" refers to the production of fine, inhalable droplets of liquid. The typical size of atomized droplets is in the range of a few microns.

[0057] The term "aerosol" refers to a dispersion of solid or liquid particles in a gas phase. In an embodiment, the gas phase is air.

[0058] Immunogenic compositions and vaccine compositions containing RSV F glycoprotein

[0059] The present disclosure provides non-naturally occurring RSV F glycoproteins, nanoparticles containing RSV F glycoproteins, and immunogenic compositions and vaccine compositions containing non-naturally occurring RSV F glycoproteins or nanoparticles containing RSV F glycoproteins. In embodiments, provided herein are methods of stimulating an immune response against RSV virus using RSV F glycoproteins, nanoparticles containing the same, immunogenic compositions containing RSV F glycoproteins and / or nanoparticles, and vaccine compositions.

[0060] Also provided herein are methods for making the nanoparticles and vaccine compositions. Advantageously, the methods provide nanoparticles that are substantially free from contamination by other proteins, such as proteins associated with recombinant expression of proteins in insect cells. In embodiments, expression occurs in a baculovirus / Sf9 system.

[0061] Non-naturally occurring RSV F glycoprotein antigen

[0062] In embodiments, provided herein are RSV F glycoproteins having one or more modifications compared to native (also referred to as "wild-type") RSV F glycoprotein. Wild-type RSV F glycoprotein is synthesized as a single-chain, inactive precursor called F0 that contains three subunits: F1, F2, and a 27-amino acid glycopeptide called 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 the F1 subunit and the F2 subunit connected by a disulfide bond. Additional sources describing RSV F protein structure can be found at 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.

[0063] In embodiments, the RSV F glycoprotein from wild-type RSV F A2 strain is native RSV F glycoprotein. The RSV F glycoprotein from wild-type RSV F A2 strain comprises the amino acid sequence of SEQ ID NO:330. Sequencing errors (A102P, V379I, V447M) occur in wild-type RSV F A2 strain. These sequencing errors are corrected in the non-naturally occurring RSV F glycoprotein as described herein, i.e., amino acid 102 is alanine, amino acid 379 is valine, and amino acid 447 is valine.

[0064] In embodiments, the RSV F glycoprotein has one or more modifications selected from the group consisting of:

[0065] (a) deletion of 1 to 10 amino acids of the fusion peptide;

[0066] (b) inactivation of the primary furin cleavage site (also known as site II) by mutation of one or more of amino acids 131-136;

[0067] (c) inactivation of the minor furin cleavage site (also known as site 1) by mutation of one or more of amino acids 106-109;

[0068] (d) S155C;

[0069] (e) S290C;

[0070] (f) S190F;

[0071] (g) V207L; and

[0072] (h)N116Q;

[0073] The modifications are numbered according to the RSV F glycoprotein of SEQ ID NO: 330.

[0074] In embodiments, the RSV F glycoprotein has one or more modifications selected from the group consisting of:

[0075] (a) deletion of 1 to 10 amino acids of the fusion peptide;

[0076] (b) inactivation of the primary furin cleavage site (also known as site II) by mutation of one or more of amino acids 131-136;

[0077] (c) inactivation of the minor furin cleavage site (also known as site 1) by mutation of one or more of amino acids 106-109;

[0078] (d) S155C;

[0079] (e) S290C;

[0080] (f) S190F; and

[0081] (g) V207L;

[0082] The modifications are numbered according to the RSV F glycoprotein of SEQ ID NO: 330.

[0083] In embodiments, the RSV F glycoprotein described herein exhibits an antigenic site One or more of II, IV, and V. In embodiments, the RSV F proteins described herein exhibit a mutation that results in the presence of an RSV F antigenic site One or more of II, IV, and V. Potent RSV F neutralizing antibodies (including 5C4, AM22, and D25) target the RSV F antigenic site located at the membrane-distal apex of the prefusion RSV F trimer RSV F antigenic site II (also referred to herein as the palivizumab site) is the target of the antibodies pavizumab 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. RSV F antigenic site IV is located on the F1 subunit of the RSV F glycoprotein and encompasses residues 422-471 of the RSV F glycoprotein of SEQ ID NO: 330. RSV F antigenic site V consists of α2-α3 and β3-β4.

[0084] In embodiments, a specific antigenic site contains an epitope associated with a pre-fusion or post-fusion structure. For example, a RSV F antigenic site In some embodiments, the RSV F glycoprotein contains antigenic sites that induce the formation of neutralizing antibodies against the prefusion conformation of the RSV F glycoprotein. In some embodiments, the RSV F glycoprotein contains antigenic sites that induce the formation of neutralizing antibodies against the postfusion conformation of the RSV F glycoprotein. In some embodiments, the RSV F glycoprotein contains antigenic sites that induce the formation of neutralizing antibodies against the postfusion conformation of the RSV F glycoprotein. In some embodiments, the RSV F glycoprotein described herein contains antigenic sites that induce the production of neutralizing antibodies against the prefusion and postfusion conformations of the RSV F glycoprotein.

[0085] In embodiments, the RSV F glycoprotein comprises a deletion of amino acids 137-146; an inactivated major furin cleavage site; an S155C mutation; and an S290C mutation, wherein the modifications are numbered according to the RSV F glycoprotein of SEQ ID NO: 330. In embodiments, the RSV F glycoprotein comprises a deletion of amino acids 137-146; an inactivated major furin cleavage site; an S155C mutation; an S290C mutation; an S190F mutation; and a V207L ​​mutation, wherein the modifications are numbered according to the RSV F glycoprotein of SEQ ID NO: 330. In embodiments, the RSV F glycoprotein comprises a deletion of amino acids 137-146; an inactivated major furin cleavage site; an S190F mutation; and a V207L ​​mutation, wherein the modifications are numbered according to the RSV F glycoprotein of SEQ ID NO: 330.

[0086] In embodiments, the RSV F glycoprotein comprises a deletion of amino acids 137-146; an inactivated major furin cleavage site; and an N116Q mutation, wherein the modifications are numbered according to the RSV F glycoprotein of SEQ ID NO: 330. In embodiments, the RSV F glycoprotein comprises a deletion of amino acids 137-146; an inactivated major furin cleavage site; an S155C mutation; an S290C mutation; and an N116Q mutation, wherein the modifications are numbered according to the RSV F glycoprotein of SEQ ID NO: 330. In embodiments, the RSV F glycoprotein comprises a deletion of amino acids 137-146; an inactivated major furin cleavage site; an S190F mutation; a V207L ​​mutation; and an N116Q mutation, wherein the modifications are numbered according to the RSV F glycoprotein of SEQ ID NO: 330. In embodiments, the RSV F glycoprotein comprises a deletion of amino acids 137-146; an inactivated major furin cleavage site; an S155C mutation; an S290C mutation; an S190F mutation; a V207L ​​mutation; and an N116Q mutation, wherein the modifications are numbered according to the RSV F glycoprotein of SEQ ID NO: 330.

[0087] Non-naturally occurring RSV F glycoprotein antigen-furin cleavage site mutation

[0088] In embodiments, specific mutations result in the retention of p27 in the mature RSV F protein of the present disclosure 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 embodiments, mutations in the primary furin cleavage site result in the retention of p27.

[0089] The main furin cleavage site (also referred to as site II herein) is located at residues 131 to 136 of SEQ ID NO:330. The wild-type main furin cleavage site has the amino acid sequence of KKRKRR (SEQ ID NO:341). The inactivation of the main fusions cleavage site can be achieved by making the residue mutation in this site, and as a result, furin can no longer recognize the consensus site. For example, the inactivation of the main furin cleavage site can be achieved by introducing at least one, at least two or three amino acid replacements corresponding to the position of arginine 133, arginine 135 and arginine 136 of wild-type RSV F albumen (SEQ ID NO:330). In particular aspects, one, two or all three arginines are mutated to glutamine. In other aspects, inactivation is achieved by mutating the wild-type major 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).

[0090] In embodiments, RSV F glycoprotein as herein described comprises a wild-type minor furin cleavage site. This minor furin cleavage site (also referred to herein as site 1) is located at residues 106 to 109 of SEQ ID NO:330. This wild-type minor furin cleavage site has the amino acid sequence RARR (SEQ ID NO:6). In embodiments, RSV F glycoprotein as herein described comprises an inactive minor furin cleavage site. In embodiments, the inactivation of this minor furin cleavage site is achieved by mutating the wild-type site to one of the following sequences: QQQAQ (SEQ ID NO:7), RAQQ (SEQ ID NO:348) or RANN (SEQ ID NO:349). In embodiments, the minor furin cleavage site of inactivation comprises the amino acid sequence of any one of SEQ ID NO:7-34, 97, 111, 348 and 349. In embodiments, the minor furin cleavage site of inactivation comprises the amino acid sequence of QQAQ (SEQ ID NO:7).

[0091] Non-naturally occurring RSV F glycoprotein antigens—modification of the F1 domain

[0092] In embodiments, the non-naturally occurring RSV F glycoproteins described herein 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 F1 domain of SEQ ID NO: 330. The wild-type F1 domain has the amino acid sequence of SEQ ID NO: 350. In embodiments, the RSV F glycoproteins described herein include an F1 domain that is 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% identical to the F1 domain of SEQ ID NO: 350. The F1 domain of the RSV F glycoprotein herein can 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.

[0093] In 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, wherein the modifications are numbered according to the RSV F glycoprotein of SEQ ID NO: 330. In embodiments, the F1 domain of the RSV F glycoprotein comprises S190F and V207L ​​mutations, wherein the modifications are numbered according to the RSV F glycoprotein of SEQ ID NO: 330. In embodiments, the F1 domain of the RSV F glycoprotein comprises S155C and S290C mutations, wherein the mutations are numbered according to the RSV F glycoprotein of SEQ ID NO: 2. In embodiments, the F1 domain of the RSV F glycoprotein comprises S190F, V207L, S155C, and S290C mutations, wherein the mutations are numbered according to the RSV F glycoprotein of SEQ ID NO: 330.

[0094] In embodiments, the F1 domain of the RSV F glycoprotein comprises an amino acid sequence that is 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% identical to any one of SEQ ID NOS: 350-355.

[0095] Non-naturally occurring RSV F glycoprotein antigens—deletions in the fusion peptide of the F1 domain

[0096] In embodiments, the RSV F glycoprotein of the present disclosure lacks one or more amino acids (amino acids 137-146 of SEQ ID NO: 330) of the RSV F glycoprotein. The fusogenic peptide of the RSV F glycoprotein of SEQ ID NO: 330 has the amino acid sequence of FLGFLLGVGS (SEQ ID NO: 363). In embodiments, provided herein are RSV F glycoproteins lacking this fusogenic peptide. In embodiments, provided herein are RSV F glycoproteins lacking 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3 or 1 to 2 consecutive amino acids of this fusogenic peptide. In embodiments, provided herein are RSV F glycoproteins lacking 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3 or 1 to 2 non-continuous amino acids of this fusogenic peptide. 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 within the fusion peptide may be deleted. 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.

[0097] Non-naturally occurring RSV F glycoprotein antigens—modification of the F2 domain

[0098] In embodiments, the non-naturally occurring RSV F glycoproteins described herein 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 F2 domain of SEQ ID NO: 330. The wild-type F2 domain has the amino acid sequence of SEQ ID NO: 356. In embodiments, the RSV F glycoproteins described herein include an F1 domain that is 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% identical to the F2 domain of SEQ ID NO: 356. The F2 domain of the RSV F glycoprotein herein can have deletions, insertions, or mutations of between about 1 and about 5 amino acids, between about 3 and about 10 amino acids, between about 5 and 10 amino acids, between about 8 and 12 amino acids, between about 10 and 15 amino acids, between about 12 and 17 amino acids, between about 15 and 20 amino acids, between about 18 and 23 amino acids, between about 20 and 25 amino acids, between about 22 and about 27 amino acids, or between about 25 and 30 amino acids compared to the F2 domain of SEQ ID NO: 356.

[0099] In embodiments, the F2 domain of the RSV F glycoprotein can comprise a P102A mutation, where the mutation is numbered according to the RSV F glycoprotein of SEQ ID NO: 330.

[0100] In embodiments, the F2 domain of the RSV F glycoprotein comprises an amino acid sequence that is 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% identical, or 100% identical to any one of SEQ ID NOS: 356-357.

[0101] Non-naturally occurring RSV F glycoprotein antigens—modification of p27

[0102] In embodiments, the non-naturally occurring RSV F glycoproteins described herein 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 p27 of SEQ ID NO: 330. Wild-type p27 has the amino acid sequence of SEQ ID NO: 359. In embodiments, the RSV F glycoproteins described herein comprise p27 that is 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% identical to p27 of SEQ ID NO: 330. The p27 of the RSV F glycoprotein herein can have deletions, insertions, or mutations 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 p27 of SEQ ID NO: 330.

[0103] In embodiments, the p27 domain of the RSV F glycoprotein can comprise an N116Q mutation, where the mutation is numbered according to the RSV F glycoprotein of SEQ ID NO: 330.

[0104] In embodiments, p27 of the RSV F glycoprotein comprises an amino acid sequence that is 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% identical, or 100% identical to any one of SEQ ID NOS: 358-359.

[0105] Non-naturally occurring RSV F glycoprotein antigen – transmembrane domain

[0106] In embodiments, the RSV F glycoprotein described herein comprises a transmembrane domain. In embodiments, the transmembrane domain has a sequence of ITTIIIVIIVILLSLIAVGLLLYCKAR STPVTLSKDQLSGINNIAFSN (SEQ ID NO: 362). In embodiments, the RSV F glycoprotein described herein can 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 embodiments, the RSV F glycoprotein lacks the transmembrane domain.

[0107] Non-naturally occurring RSV F glycoprotein antigen - signal peptide

[0108] In embodiments, the RSV F glycoprotein described herein is expressed using an N-terminal signal peptide. In embodiments, the N-terminal signal peptide has the amino acid sequence of SEQ ID NO: 360 (MELLILK ANAITTILTAVTFCFASG). In embodiments, the signal peptide can be replaced with any signal peptide that enables expression of the RSV F glycoprotein. In embodiments, one or more of the RSV F glycoprotein signal peptide amino acids can be deleted or mutated. The initial methionine residue is maintained to initiate expression. In embodiments, the RSV F glycoprotein is encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NO: 361.

[0109] After RSV F glycoprotein is expressed in host cell, N-terminal signal peptide is cut to provide mature RSV F glycoprotein sequence (SEQ ID NO:331 and 337-340). In embodiments, this signal peptide is cut by host cell protease. In aspect, full-length protein can be separated from host cell, and this signal peptide is cut subsequently.

[0110] Following cleavage of the signal peptide from the RSV F glycoprotein having the 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 and used to produce RSV F nanoparticle vaccines or RSV F nanoparticles.

[0111] Advantageously, the disclosed RSV F glycoproteins can have enhanced protein expression, stability, and immunogenicity relative to native RSV F glycoprotein.

[0112] In embodiments, the RSV F glycoprotein described herein contains further modifications from native RSV F glycoprotein (SEQ ID NO: 330). In 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 native RSV F glycoprotein.

[0113] In embodiments, the RSV F glycoprotein described herein is 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% identical to the RSV F glycoprotein having amino acids selected from the group consisting of SEQ ID NOs: 329-340. In embodiments, provided herein are RSV F glycoproteins that 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%, at least 99.5%, or 100% identical to the RSF glycoprotein of SEQ ID NO: 337. The RSV F glycoprotein 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, up to about 30, up to about 35, up to about 40, up to about 45, or up to about 50 amino acid deletions, insertions, or mutations compared to the amino acid sequence of the RSV F glycoprotein having the amino acid sequence of any one of SEQ ID NOs: 329-340. The RSV F glycoprotein can have deletions, insertions, or mutations 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 the amino acid sequence of any one of SEQ ID NOs: 329-340. In embodiments, the RSV F glycoprotein described herein comprises 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 the amino acid sequence of any one of SEQ ID NOs: 329-340.

[0114] In embodiments, RSV F glycoprotein is at N-terminal, C-terminal, or N-terminal and C-terminal two places extension.In aspect, extension (extension) is to the useful label of the function such as purification or detection.In aspect, this label contains epi-position.For example, this label can be polyglutamate tag, FLAG tag, HA tag, polyHis tag (having about 5-10 histidine) (SEQ ID NO:34), hexahistidine tag (SEQ ID NO:35), 8X-His tag (having eight histidine) (SEQ ID NO:36), Myc tag, glutathione-S-transferase tag, green fluorescent protein tag, maltose binding protein tag, thioredoxin tag or Fc tag.In other aspects, extension can be with protein fusion to enhance the N-terminal signal peptide of expression.Although this type of signal peptide tends to be cut during expression in cell, some nanoparticles may contain antigen with complete signal peptide.Therefore, when nanoparticle comprises antigen, this antigen can contain extension, and therefore can be fusion protein when being incorporated into nanoparticle. In order to calculate the identity with the sequence, the extension is not included. In an embodiment, 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 an embodiment, the protease cleavage site is the HRV3C protease cleavage site.

[0115] In embodiments, RSV F glycoprotein includes fusion protein. In embodiments, RSV F glycoprotein includes N-terminal fusion protein. In embodiments, RSV F glycoprotein includes C-terminal fusion protein. In embodiments, fusion protein encompasses tags that can be used for protein expression, purification or detection. In embodiments, the tag is a polyHis tag (with about 5-10 histidine) (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 embodiments, the tag is an Fc tag. In embodiments, the Fc tag is monomeric, dimeric or trimeric. In embodiments, the tag is a hexahistidine tag, for example, a polyHis tag (SEQ ID NO:35) containing six histidines.

[0116] Immunogenic compositions and / or vaccine compositions containing RSV F glycoprotein and additional viral glycoproteins

[0117] In embodiments, provided herein are immunogenic compositions comprising RSV F glycoproteins. In embodiments, the immunogenic compositions are vaccine compositions. In embodiments, provided herein are immunogenic compositions and vaccine compositions comprising nanoparticles comprising RSV F glycoproteins. In embodiments, the immunogenic compositions comprise 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.

[0118] In another embodiment, the present disclosure provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the components of the vaccine composition.

[0119] The compositions disclosed herein can be used preventively or therapeutically, but are generally preventive. Therefore, the present disclosure includes methods for treating or preventing infection. The method involves administering a therapeutic or preventive amount of the immunogenic composition of the present disclosure to a subject. Preferably, the pharmaceutical composition is a vaccine composition that provides a protective effect. In other aspects, the protective effect may include improving symptoms associated with infection in a certain proportion of exposed people. For example, compared to untreated subjects, the composition can prevent or alleviate one or more viral disease symptoms selected from the following: fever fatigue, muscle pain, headache, sore throat, vomiting, diarrhea, rash, symptoms of impaired renal and liver function, internal bleeding and external bleeding.

[0120] The nanoparticles can be formulated for administration as a vaccine in the presence of various excipients, buffers, etc. For example, a 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, there is about 22 mM sodium phosphate. 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). When sodium chloride is present, its concentration can be about 150 mM. In certain compositions, sodium chloride can be present at a higher concentration (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.

[0121] In embodiments, nanoparticles as herein described have improved stability under certain pH levels. In embodiments, the nanoparticles are stable under weakly acidic pH levels. For example, the nanoparticles are stable under weakly acidic pH (e.g., pH 5.8 to pH 7.0). In embodiments, the nanoparticles and compositions containing the nanoparticles can be stable under a pH in the range of from 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, nanoparticles as herein described and compositions are stable under neutral pH (including about pH 7.0 to about pH 7.4). In embodiments, the nanoparticles and compositions described herein are stable at a weakly alkaline pH, 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.

[0122] In embodiments, the compositions described herein further comprise one or more additional viral glycoproteins in addition to the RSV F glycoprotein. In 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 embodiments, the immunogenic composition comprises the RSV F glycoprotein, an influenza glycoprotein, and a SARS-CoV-2 S glycoprotein as described herein. In embodiments, the immunogenic composition comprises the RSV F glycoprotein and an influenza glycoprotein as described herein. In embodiments, the immunogenic composition comprises the RSV F glycoprotein and the SARS-CoV-2 S glycoprotein as described herein.

[0123] In 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 embodiments, different viral glycoproteins can be from different viruses, different strains, or subtypes of the same virus, or can differ from each other by mutation.

[0124] In embodiments, in addition to the RSV F glycoprotein, the immunogenic composition further comprises 1 to about 50 different influenza glycoproteins (e.g., hemagglutinin and / or neuraminidase). In embodiments, the influenza glycoprotein (e.g., hemagglutinin or neuraminidase) can be from any influenza virus strain.

[0125] 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 influenza glycoproteins, including all values ​​and ranges therebetween.

[0126] In embodiments, the influenza glycoprotein is a hemagglutinin. In 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. From a phylogenetic perspective, influenza can be divided into several groups. In embodiments, the HA glycoprotein is a Group 1 HA glycoprotein (i.e., a glycoprotein from a H1, H2, H5, H6, H8, H9, H11, H12, H13, H16, H17 or H18 subtype). In embodiments, the HA glycoprotein is a Group 2 HA glycoprotein (i.e., a glycoprotein from a H3, H4, H7, H10, H14 or H15 subtype).

[0127] The following patent documents describe various examples of influenza HA glycoproteins, each of which is herein incorporated by reference in its entirety for all purposes: International Publication No. 2017 / 041100 and International Publication No. 2019 / 022930.

[0128] In an embodiment, the immunogenic composition of the present disclosure comprises at least three different HA glycoproteins or at least four different HA glycoproteins. In an embodiment, the immunogenic composition of the present disclosure comprises three or four different HA glycoproteins. In an embodiment, each of the HA glycoproteins is from a different influenza strain. In an embodiment, three HA glycoproteins are from influenza A strains and one HA glycoprotein is from influenza B strains. In an embodiment, two HA glycoproteins are from influenza A strains and two HA glycoproteins are from influenza B strains. In an embodiment, two HA glycoproteins are from influenza A strains and one HA glycoprotein is from influenza B strains.

[0129] In embodiments, each of the at least three different HA glycoproteins is individually isolated using an egg-based manufacturing process.In embodiments, the egg-based manufacturing process comprises (a) propagating influenza virus in eggs and (b) harvesting the influenza virus.

[0130] 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 to grow in eggs. In embodiments, the optimized influenza virus lacks the polybasic cleavage site of hemagglutinin. The following article describes in detail the development of an optimized influenza virus (referred to as a "candidate vaccine virus") and is incorporated herein by reference in its entirety: Belser et al., Virology. 2017 November; 511: 135–141.

[0131] In embodiments, the egg is a chicken egg. In embodiments, the egg is an embryonated chicken egg. In embodiments, the egg is a pathogen-free egg. In embodiments, the influenza virus is propagated by inoculating the virus into the allantoic cavity of an embryonated chicken egg. The following article describes an exemplary inoculation method and is incorporated herein by reference in its entirety: Brauer et al., J Vis Exp. 2015;(97):52421.

[0132] In embodiments, the egg-based manufacturing method comprises purifying 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.

[0133] In embodiments, the egg-based manufacturing method comprises inactivating (also referred to herein as "killing") influenza viruses. In embodiments, influenza viruses are inactivated using low pH (e.g., a pH of about 3.5-5.5), heat, ethanol, ultraviolet light, exposure to detergents (e.g., octylphenol), or exposure to chemicals (e.g., 2-propanol, ethanol, iodopovidone). In embodiments, the purified virus is inactivated with ultraviolet light, β-propiolactone, sodium deoxycholate, formaldehyde, or any combination thereof.

[0134] In embodiments, the egg-based manufacturing method comprises exposing the influenza virus to a surfactant. The surfactant may be sodium taurodeoxycholate, octylphenol ethoxylate ( 100) or cetyltrimethylammonium bromide. Exposure of influenza virus to a surfactant results in the formation of influenza split virions. In embodiments, the at least three HA glycoproteins are in the form of influenza split virions.

[0135] In embodiments, the egg-based manufacturing method comprises purifying the influenza antigen (eg, hemagglutinin) from the virus.

[0136] The following U.S. Food and Drug Administration (FDA)-approved flu vaccines are produced using egg-based manufacturing methods: QUADRIVALENT, QUADRIVALENT, FLULAVAL QUADRIVALENT, QUADRIVALENT, HIGH-DOSEQUADRIVALENT, QUADRIVALENT, and QUADRIVALENT.

[0137] In embodiments, each of the at least three HA glycoproteins is isolated using a cell culture-based method. In embodiments, the cell culture-based method comprises (i) growing the influenza virus in a cell and (ii) harvesting the virus from the cell. In embodiments, the cell culture-based method comprises (i) transfecting the cell with a vector comprising a hemagglutinin and (ii) harvesting the hemagglutinin from the cell. In embodiments, the cell culture-based method comprises (i) transducing the cell with a virus encoding the hemagglutinin and (ii) harvesting the hemagglutinin from the cell. In embodiments, the harvested hemagglutinin is a recombinant hemagglutinin.

[0138] In embodiments, the cell is an animal cell, a bacterial cell, an insect cell, or a fungal cell. In embodiments, the animal is a human, a bird (e.g., chicken), a dog, a reptile, a goat, a pig, a mouse, a rabbit, or a rat.

[0139] In embodiments, the virus encoding the hemagglutinin is a baculovirus, a lentivirus, or an adeno-associated virus.

[0140] 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.

[0141] In an embodiment, hemagglutinin produced using a cell culture-based method is purified. Purification techniques include chromatography, centrifugation, precipitation, and nanofiltration.

[0142] U.S. Food and Drug Administration (FDA)-approved flu vaccines Quadrivalent is produced using a cell culture-based method.

[0143] In an embodiment, each of the at least three HA glycoproteins is a recombinant hemagglutinin. U.S. Food and Drug Administration (FDA) approved influenza vaccines Quadrivalent is a recombinant hemagglutinin.

[0144] In 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 embodiments, the cells that produce the hemagglutinins have been transfected with a vector encoding the hemagglutinins. In embodiments, the cells that produce the hemagglutinins have been transduced with a virus encoding the hemagglutinins.

[0145] In an embodiment, the compositions disclosed herein comprise detergent core nanoparticles comprising hemagglutinin from influenza virus.The aforementioned detergent core nanoparticles are described in detail in US Patent No. 10,426,829, which is incorporated herein by reference in its entirety for all purposes.

[0146] Detergent core nanoparticles include hemagglutinin from influenza virus, which is associated with the detergent core. In embodiments, hemagglutinin is a trimer. Each nanoparticle can contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 trimers. In embodiments, nanoparticles contain hemagglutinin trimers between about 2 to about 9, about 2 to about 6, or about 5. Hemagglutinin is associated with the core of the nanoparticle containing nonionic detergent. In embodiments, detergent is selected from polysorbate 20 (PS20), polysorbate 40 (PS40), polysorbate 60 (PS60), polysorbate 65 (PS65) and polysorbate 80 (PS80). The presence of detergent promotes the formation of nanoparticles by forming a core that organizes and presents antigens. In embodiments, the nanoparticles may contain antigens assembled into poly-oligomeric glycoprotein-PS80 protein-detergent nanoparticles, wherein the head region protrudes outward and the hydrophobic region and PS80 detergent form a central core surrounded by the antigen.

[0147] In an embodiment, the detergent core nanoparticles or HaSMaN are trypsin-resistant nanoparticles produced using neutral pH purification. During the purification and formulation of the HA nanoparticles, trypsin resistance is achieved by being above the neutral pH range of 6.9 to 8.5. Trypsin-resistant influenza glycoproteins and trypsin-resistant influenza nanoparticles and methods for their preparation are described in detail in U.S. Patent No. 10,426,829.

[0148] In embodiments, the hemagglutinin of the detergent core nanoparticles or HaSMaN described herein comprises a full-length wild-type hemagglutinin amino acid sequence. In embodiments, the hemagglutinin is a hemagglutinin variant. In 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 a wild-type hemagglutinin protein.

[0149] HA is a homotrimer in which each monomer consists of approximately 550 amino acid residues. Each monomer of HA is conceptually divided into three domains: an extracellular domain of approximately 515 residues constitutes the extraviral portion of the molecule; a single stretch of 27 residues defines the transmembrane (TM) domain; and approximately 10 residues constitute the cytoplasmic tail (CT). Although some changes may be made to the hemagglutinin, the formation of both detergent core nanoparticles and HaSMaN requires an intact transmembrane domain (TM). Thus, in a particular example, the modified HA protein sequence has 100% identity with the wild-type TM and CT domains, while having some flexibility in the remaining extracellular domain portion, where the identity can be at least 90% or at least 95%. This domain can be modified by comparing it to the cytoplasmic tail of Melikyan et al. (Mol Biol Cell. 1999 Jun; 10(6): 1821–1836) Figure 1 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79

[0150] In embodiments, the immunogenic compositions and vaccine compositions described herein comprise HaSMaN (hemagglutinin saponin matrix nanoparticles). Figure 6 (Right) shows the HaSMaN structure as observed under an electron microscope. The HA glycoprotein is decorated with the matrix cage-like structure. The HaSMaN structure was formed by preparing detergent core nanoparticles containing hemagglutinin from influenza virus and then incubating them with ISCOM matrix adjuvant particles for a certain period of time. The ISCOM matrix particles are Figure 6 It is worth noting that HaSMaN easily forms with influenza A HA protein, but not easily with influenza B HA protein.

[0151] The HaSMaN disclosed herein is produced by incubating detergent core nanoparticles with an ISCOM matrix adjuvant comprising a saponin fraction, cholesterol, and a phospholipid. In embodiments, the HaSMaN is formed by incubating the detergent core nanoparticles with the ISCOM matrix adjuvant for about 24 hours to about 48 hours. For example, the detergent core nanoparticles can be incubated with the ISCOM matrix adjuvant for about 24 hours, about 25 hours, about 26 hours, about 27 hours, about 28 hours, about 29 hours, about 30 hours, about 31 hours, about 32 hours, about 33 hours, about 34 hours, about 35 hours, about 36 hours, about 37 hours, about 38 hours, about 39 hours, about 40 hours, about 41 hours, about 42 hours, about 43 hours, about 44 hours, about 45 hours, about 46 hours, about 47 hours, about 48 hours, or more. In some embodiments, the HaSMaN is formed by incubating detergent core nanoparticles with an 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 embodiments, the HaSMaN is formed by incubating detergent core nanoparticles with an ISCOM matrix adjuvant at a temperature of about 4°C to about 25°C. For example, HaSMaN can be formed by incubating detergent core nanoparticles with an ISCOM matrix adjuvant at a temperature 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 more. In embodiments, HaSMaN is formed by incubating detergent 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., at least about 25° C. Typically, formation requires incubation at 4° C. or 25° C. for about 24 to 48 hours. Higher temperatures promote the formation of HaSMaN.In an embodiment, the formation of HaSMaN occurs by incubating the detergent core nanoparticles with the ISCOM matrix adjuvant at about 25° C. for at least 24 hours. Mixing the detergent core nanoparticles with the ISCOM matrix adjuvant shortly before administration to a subject (i.e., bedside mixing) does not produce HaSMaN. Longer incubation periods do not negatively affect the formation of HaSMaN.

[0152] In embodiments, the immunogenic composition comprises, in addition to the RSV F glycoprotein, 1 to about 50 different SARS-CoV-2 S glycoproteins ("CoV S glycoproteins." In embodiments, the immunogenic composition comprises, in addition to the RSV F glycoprotein, about 5 different SARS-CoV-2 CoV S glycoproteins. The CoV S glycoprotein is synthesized as an inactive precursor (S0) that is proteolytically cleaved at a furin cleavage site into S1 and S2 subunits that remain non-covalently associated to form the prefusion trimer. CoV The S2 domain of the S glycoprotein contains a fusion peptide (FP), two heptad repeat sequences (HR1 and HR2), a transmembrane (TM) domain, and a cytoplasmic tail (CT). The S1 domain of the SARS-CoV-2S protein is folded into four different 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-2S protein trimer undergoes a structural rearrangement from a pre-fusion conformation to a post-fusion conformation after S protein receptor binding and cleavage. In an embodiment, the CoVS 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, in Figure 1 AB, 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 an embodiment, the amino acids of each domain are Figure 7A (shown according to SEQ ID NO: 1), Figure 7B (shown in SEQ ID NO: 2). The SARS-CoV-2 S glycoprotein also contains a furin cleavage site.

[0153] In an embodiment, each of the CoV S glycoproteins described herein contains proline at amino acid positions 973 and 974 and comprises an inactive furin cleavage site, wherein the CoV S glycoprotein is numbered according to the CoV S glycoprotein of SEQ ID NO: 2.

[0154] 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 (MFVFLVLL PLVSS). 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 (MFGFLVLLPLV SS). In embodiments, the signal peptide can be replaced with any signal peptide that enables expression of the CoV S glycoprotein. In embodiments, one or more of the amino acids in the CoV S glycoprotein signal peptide can be deleted or mutated. The 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: 215. NO: 216. In embodiments, the N-terminal signal peptide of the CoV S glycoprotein contains a mutation at Ser-13 relative to the native CoV spike (S) signal polypeptide (SEQ ID NO: 5). In embodiments, Ser-13 is mutated to any natural amino acid. In embodiments, Ser-13 is mutated to alanine, methionine, isoleucine, leucine, threonine, or valine. In embodiments, Ser-13 is mutated to isoleucine.

[0155] After the CoV S glycoprotein is expressed in the host cell, the N-terminal signal peptide is cleaved to obtain 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: 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 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, 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 can be isolated from the host cell and the signal peptide subsequently cleaved.

[0156] During the expression and purification, from SEQ ID NO:88; 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, SEQID NO:205, SEQ ID NO:207, SEQ ID NO:209, SEQ ID NO:211, SEQ ID NO:213, 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 After the CoV spike (S) polypeptide with the amino acid sequence corresponding to any one of NO:327 cleaves the signal peptide,Obtain a product having a 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 IDNO: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: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 and SEQ ID NO:328 consisting of the mature polypeptide of the amino acid sequence and used for producing CoV S nanoparticle vaccine or CoV S nanoparticle.

[0157] Advantageously, the disclosed CoV S polypeptides can have enhanced protein expression and stability relative to native CoV spike (S) protein.

[0158] In embodiments, the CoV S polypeptides described herein contain further modifications from native coronavirus S proteins (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%, at least 99% identity to native CoV S glycoproteins. One skilled in the art will use known techniques to calculate the percent identity of a recombinant coronavirus S protein to the native protein or any CoV S glycoprotein described herein.

[0159] In embodiments, the CoV S polypeptide described herein is 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 NO: 243, SEQ ID NOs: 255-328. The CoV S polypeptide 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, up to about 30, up to about 35, up to about 40, up to about 45, or up to about 50 amino acid deletions, insertions, or mutations compared to the amino acid sequence of the 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 NO: 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, SEQ ID NO: 243, and SEQ ID NOS: 255-328.The CoV S polypeptide has 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, SEQ ID NO:243 and SEQ ID The COVS polypeptide having the amino acid sequence of any one of NOS:255-328 can 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. In embodiments, the CoV S polypeptides described herein are identical to those 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, SEQ ID NO: 243, and SEQ ID NOS: 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 6, 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.

[0160] In embodiments, the coronavirus S polypeptide is extended at the N-terminus, the C-terminus, or both the N-terminus and the C-terminus. In aspects, an extension is a tag useful for functions such as purification or detection. In 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 about 5-10 histidines) (SEQ ID NO: 101), a hexahistidine tag (SEQ ID NO: 100), an 8X-His tag (having eight 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 a protein to enhance expression. Although such signal peptides tend to be cut during expression in cells, some nanoparticles may contain antigens with complete signal peptides. Therefore, when nanoparticles contain antigens, the antigen can contain an extension and can therefore be a fusion protein when incorporated into nanoparticles. For the purpose of calculating the identity to the sequence, extensions are not included. In an embodiment, 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 an embodiment, the protease cleavage site is the HRV3C protease cleavage site. In an embodiment, the protease cleavage site comprises the amino acid sequence of SEQ ID NO: 98.

[0161] In embodiments, CoV S glycoprotein comprises a fusion protein. In embodiments, CoV S glycoprotein comprises an N-terminal fusion protein. In embodiments, Cov S glycoprotein comprises a C-terminal fusion protein. In embodiments, the fusion protein encompasses tags that can be used for protein expression, purification or detection. In embodiments, the tag is a polyHis 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 embodiments, the tag is an Fc tag. In embodiments, the Fc tag is monomeric, dimeric or trimeric. In embodiments, the tag is a hexahistidine tag, for example, a polyHis tag (SEQ ID NO: 100) containing six histidines. In embodiments, the tag is a Twin-Strep tag having the amino acid sequence of SEQ ID NO: 99.

[0162] In an embodiment, the CoV S polypeptide is a fusion protein comprising another coronavirus protein. In an embodiment, the other coronavirus protein is from the same coronavirus. In an embodiment, the other coronavirus protein is from a different coronavirus.

[0163] In aspects, the CoV S glycoprotein can be truncated. For example, the N-terminus can be truncated by about 10 amino acids, about 30 amino acids, about 50 amino acids, about 75 amino acids, about 100 amino acids, or about 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 amino acids, about 30 amino acids, about 50 amino acids, about 75 amino acids, about 100 amino acids, or about 200 amino acids. To calculate identity with a protein having a truncation, identity is measured on the remainder of the protein.

[0164] In an embodiment, the CoV S glycoprotein contains one or more modifications to the S1 subunit having the amino acid sequence of SEQ ID NO: 121.

[0165] The amino acid sequence of the S1 subunit (SEQ ID NO: 121) is shown below.

[0166] QCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPRRAR

[0167] In embodiments, the CoV S polypeptides described herein comprise an S1 subunit that is 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% identical to the S1 subunit of SEQ ID NO: 1 or SEQ ID NO: 2. The S1 subunit 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 amino acids, up to about 35 amino acids, up to about 40 amino acids, up to about 45 amino acids, 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 can have a deletion, insertion or mutation of between about 1 and about 5 amino acids, between about 3 and about 10 amino acids, between about 5 and 10 amino acids, between about 8 and 12 amino acids, between about 10 and 15 amino acids, between about 12 and 17 amino acids, between about 15 and 20 amino acids, between about 18 and 23 amino acids, between about 20 and 25 amino acids, between about 22 and about 27 amino acids, or between about 25 and 30 amino acids compared to the S1 subunit of SEQ ID NO: 1 or SEQ ID NO: 2.

[0168] In embodiments, the S1 subunit may contain any combination of modifications shown in Table 1A.

[0169] Table 1A

[0170]

[0171]

[0172]

[0173]

[0174]

[0175]

[0176]

[0177] In embodiments, the CoV S polypeptide contains one or more modifications to the NTD. In embodiments, the NTD has an 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.

[0178] The amino acid sequence of NTD (SEQ ID NO: 118) is shown below.

[0179] QCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRV YSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKS

[0180] In an embodiment, 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 1-318 of SEQ ID NO: 2. The amino acid sequence of NTD (SEQ ID NO: 45) is shown below.

[0181] QCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVS QPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPN

[0182] 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.

[0183] In embodiments, an NTD as provided herein may extend 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.

[0184] In embodiments, the CoV S polypeptides described herein comprise an NTD that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical 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 can have a deletion, insertion, or mutation of between about 1 and about 5 amino acids, between about 3 and about 10 amino acids, between about 5 and 10 amino acids, between about 8 and 12 amino acids, between about 10 and 15 amino acids, between about 12 and 17 amino acids, between about 15 and 20 amino acids, between about 18 and 23 amino acids, between about 20 and 25 amino acids, between about 22 and about 27 amino acids, or between about 25 and 30 amino acids compared to the NTD of SEQ ID NO: 1 or SEQ ID NO: 2.

[0185] 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.

[0186] 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 NO: 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.

[0187] In embodiments, the NTD may contain any combination of modifications shown in Table 1 B. The modifications are shown with respect to SEQ ID NO: 2 (mature S polypeptide sequence) for reference.

[0188] Table 1B

[0189]

[0190]

[0191]

[0192]

[0193] In an embodiment, the CoV S polypeptide contains one or more modifications to the RBD.

[0194] In an embodiment, the RBD has an 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.

[0195] The amino acid sequence of RBD (SEQ ID NO: 126) is shown below:

[0196] NITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGP

[0197] In an embodiment, the RBD has an 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.

[0198] The amino acid sequence of RBD (SEQ ID NO: 116) is shown below.

[0199] LCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKS

[0200] In embodiments, an RBD as provided herein can be extended at the N-terminus 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.

[0201] In embodiments, the CoV S polypeptides described herein comprise an RBD that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to the RBD of SEQ ID NO: 1 or SEQ ID NO: 2. The RBD 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 RBD of SEQ ID NO: 1 or SEQ ID NO: 2. The RBD can have a deletion, insertion, or mutation 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.

[0202] In an embodiment, the CoV S polypeptide has at least one, at least two, at least three, at least four, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, 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 one aspect, 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 an embodiment, the RBD can contain any combination of modifications as shown in Table 1C.

[0203] Table 1C

[0204]

[0205]

[0206]

[0207] In an embodiment, the CoV S polypeptide contains one or more modifications to SD1 / 2 having the amino acid sequence of SEQ ID NO: 122, which SD1 / 2 corresponds to amino acids 542-681 of SEQ ID NO: 1 or amino acids 529-668 of SEQ ID NO: 2.

[0208] The amino acid sequence of SD1 / 2 (SEQ ID NO: 122) is shown below.

[0209] NFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSP

[0210] In embodiments, the CoV S polypeptide described herein comprises an SD1 / 2 that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to the SD1 / 2 of SEQ ID NO: 1 or SEQ ID NO: 2. The SD1 / 2 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 SD1 / 2 of SEQ ID NO: 1 or SEQ ID NO: 2. The SD1 / 2 can have a deletion, insertion or mutation of between about 1 and about 5 amino acids, between about 3 and about 10 amino acids, between about 5 and 10 amino acids, between about 8 and 12 amino acids, between about 10 and 15 amino acids, between about 12 and 17 amino acids, between about 15 and 20 amino acids, between about 18 and 23 amino acids, between about 20 and 25 amino acids, between about 22 and about 27 amino acids, or between about 25 and 30 amino acids compared to the SD1 / 2 of SEQ ID NO: 1 or SEQ ID NO: 2.

[0211] In embodiments, the CoV S polypeptide has at least one, at least two, at least three, at least four, at least four, at least five, at least six, at least seven, at least eight, at least nine, 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 aspects, 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 as shown in Table ID.

[0212] Table 1D

[0213]

[0214]

[0215] 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, which 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 in a single chain.

[0216] In embodiments, one or more of the amino acid comprising the native furin cleavage site are mutated into any natural amino acid. In embodiments, the amino acid is an L-amino acid. Amino acid whose limiting examples comprises alanine, arginine, glycine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, serine, threonine, histidine, lysine, methionine, proline, valine, isoleucine, leucine, tyrosine, tryptophan and phenylalanine.

[0217] In embodiments, one or more of the amino acids comprising the native furin cleavage site are mutated to glutamine. In embodiments, 1, 2, 3 or 4 amino acids can be mutated to glutamine. In embodiments, one of the arginine comprising the native furin cleavage site is mutated to glutamine. In embodiments, two of the arginine comprising the native furin cleavage site are mutated to glutamine. In embodiments, three of the arginine comprising the native furin cleavage site are mutated to glutamine.

[0218] In embodiments, one or more of the amino acid comprising the native furin cleavage site is mutated to alanine. In embodiments, 1,2,3 or 4 amino acids can be mutated to alanine. In embodiments, one of the arginine comprising the native furin cleavage site is mutated to alanine. In embodiments, two of the arginine comprising the native furin cleavage site are mutated to alanine. In embodiments, three of the arginine comprising the native furin cleavage site are mutated to alanine.

[0219] In embodiments, one or more of the amino acid comprising the native furin cleavage site is mutated to a glycine. In embodiments, 1, 2, 3 or 4 amino acids can be mutated to a glycine. In embodiments, one of the arginine in the native furin cleavage site is mutated to a glycine. In embodiments, two of the arginine comprising the native furin cleavage site are mutated to a glycine. In embodiments, three of the arginine comprising the native furin cleavage site are mutated to a glycine.

[0220] In embodiments, one or more of the amino acid comprising the native furin cleavage site is mutated to asparagine. For example, 1, 2, 3 or 4 amino acids can be mutated to asparagine. In embodiments, one of the arginine comprising the native furin cleavage site is mutated to asparagine. In embodiments, two of the arginine comprising the native furin cleavage site are mutated to asparagine. In embodiments, three of the arginine comprising the native furin cleavage site are mutated to asparagine.

[0221] In embodiments, instead of active furin cleavage site (SEQ ID NO:6), CoV S polypeptides as 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 NO: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).

[0222] Non-limiting examples of amino acid sequences of inactivating furin sites contained within CoV S polypeptides are shown in Table IE.

[0223] Table 1E

[0224]

[0225] 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 686-1273 of SEQ ID NO: 1 or amino acids 673-1260 of SEQ ID NO: 2.

[0226] The amino acid sequence of the S2 subunit (SEQ ID NO: 120) is shown below.

[0227] SVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT

[0228] In embodiments, the CoV S polypeptides described herein comprise an S2 subunit that is 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% identical to the S2 subunit of SEQ ID NO: 1 or SEQ ID NO: 2. The S2 subunit 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 S2 subunit of SEQ ID NO: 1 or SEQ ID NO: 2. The S2 subunit may have a deletion, insertion or mutation 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.

[0229] In embodiments, the CoV S polypeptide comprises a mutation at Lys-973 of the native CoV spike (S) polypeptide (SEQ ID NO: 2). In embodiments, Lys-973 is mutated to any natural amino acid. In embodiments, Lys-973 is mutated to proline. In embodiments, Lys-973 is mutated to glycine. In embodiments, the CoV S polypeptide comprising a mutation at amino acid 973 is selected from the group consisting of SEQ ID NOs: 84-89, 105-106, and 109-110.

[0230] In embodiments, the CoV S polypeptide comprises a mutation at Val-974 of the native CoV spike (S) polypeptide (SEQ ID NO: 2). In embodiments, Val-974 is mutated to any natural amino acid. In embodiments, Val-974 is mutated to proline. In embodiments, Val-974 is mutated to glycine. In embodiments, the CoV S polypeptide comprising a mutation at amino acid 974 is selected from the group consisting of SEQ ID NOs: 84-89, 105-106, and 109-110.

[0231] In an embodiment, the CoV S polypeptide contains mutations at Lys-973 and Val-974 of the native CoV spike (S) polypeptide (SEQ ID NO: 2). In an embodiment, Lys-973 and Val-974 are mutated to any natural amino acid. In an embodiment, Lys-973 and Val-974 are mutated to proline. In an embodiment, 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.

[0232] In embodiments, the S2 subunit may contain any combination of modifications as shown in Table IF.

[0233] Table 1F

[0234]

[0235]

[0236]

[0237] 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.

[0238] Several examples of SARS-CoV-2 S glycoproteins are described in the following patent documents, each of which is incorporated herein by reference in its entirety for all purposes: International Publication No. 2021 / 015220, International Publication No. 2022 / 020974, International Publication No. 2022 / 235662, and International Publication No. 2023 / 102448.

[0239] Nanoparticles containing viral glycoproteins

[0240] In embodiments, the immunogenic compositions and vaccine compositions described herein comprise nanoparticles. The nanoparticles of the present disclosure are non-naturally occurring products whose components do not exist together in nature. In embodiments, the nanoparticles are detergent core nanoparticles comprising viral glycoproteins and non-ionic detergents. In embodiments, provided herein are compositions comprising detergent core nanoparticles comprising RSV F glycoprotein and non-ionic detergents. In embodiments, provided herein are compositions comprising detergent core nanoparticles comprising HA glycoprotein and non-ionic detergents. In embodiments, provided herein are compositions comprising detergent core nanoparticles comprising CoV S glycoprotein and non-ionic detergents.

[0241] In embodiments, a single nanoparticle comprises one or more different viral glycoproteins. In embodiments, provided herein are detergent core nanoparticles comprising RSV F glycoprotein and HA glycoprotein. In embodiments, provided herein are detergent core nanoparticles comprising RSV F glycoprotein and CoV S glycoprotein. In embodiments, provided herein are detergent core nanoparticles comprising RSV F glycoprotein, HA glycoprotein, and CoV S glycoprotein.

[0242] In embodiments, the detergent core nanoparticle comprises a viral glycoprotein having a transmembrane domain, wherein the viral glycoprotein is anchored to the detergent core via the transmembrane domain of the viral glycoprotein.In embodiments, the viral glycoprotein forms a trimer.

[0243] In embodiments, to produce detergent-core nanoparticles, a detergent exchange method is utilized, wherein viral glycoproteins are isolated using a first detergent, and then the first detergent is exchanged for a second detergent to form the nanoparticles.

[0244] The viral glycoproteins contained in the nanoparticles are typically produced by recombinant expression in host cells. Standard recombinant techniques can be used. In embodiments, the viral glycoproteins are expressed in insect host cells using a baculovirus system. In embodiments, 1-50 viral glycoproteins are co-expressed in the host cells. In embodiments, the baculovirus is a cathepsin-L knockout baculovirus or a chitinase knockout baculovirus. Optionally, the baculovirus is a cathepsin-L and chitinase double knockout virus. High-level expression can be obtained in insect cell expression systems. Non-limiting examples of insect cells are Spodoptera frugiperda (Sf) cells, such as Sf9 and Sf21, Trichoplusia ni cells, such as 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.

[0245] Typical transfection and cell growth methods can be used to culture the cells. Vectors, such as vectors containing polynucleotides encoding fusion proteins, can be transfected into host cells according to methods well known in the art. For example, nucleic acids can be introduced into eukaryotic cells by calcium phosphate coprecipitation, electroporation, microinjection, liposome transfection, and transfection using polyamine transfection reagents. In one embodiment, the vector is a recombinant baculovirus.

[0246] Methods for growing host cells include, but are not limited to, batch, fed-batch, continuous, and perfusion cell culture techniques. Cell culture refers to the growth and propagation of cells in a bioreactor (fermentation chamber) where they reproduce and express proteins (e.g., recombinant proteins) for purification and separation. Typically, cell culture is carried out in a bioreactor under sterile, controlled temperature and atmospheric conditions. A bioreactor is a chamber for culturing cells in which 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 pre-sterilized plastic bag (e.g., Wave Biotech, Bridgewater, NJ). In other embodiments, the pre-sterilized plastic bag is about a 50 L to 3500 L bag.

[0247] After the host cells have grown, proteins can be harvested from the host cells using detergents and purification protocols. In one embodiment, multiple viral glycoproteins are purified simultaneously. In one embodiment, host cells expressing multiple viral glycoproteins are pooled together. Once the host cells have grown for 48 to 96 hours, the cells are separated from the culture medium and a detergent-containing solution is added to dissolve the cell membranes, thereby releasing the proteins in the detergent extract. Triton X-100 and Nonylphenol ethoxylate (also known as NP-9) is a preferred detergent for extraction. The detergent can be added to a final concentration of about 0.1% to about 1.0%. For example, the concentration 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 one aspect, the concentration is about 0.5%.

[0248] In other aspects, different first detergents can be used to isolate proteins from host cells. For example, the first detergent can be bis(polyethylene glycol bis[imidazolylcarbonyl]), nonoxynol-9, bis(polyethylene glycol bis[imidazolylcarbonyl]), Polyethylene glycol lauryl ether 35, Polyethylene glycol (3) cetyl ether 56, Alcohol ethoxylate 72, Polyoxyl 2stearyl ether 76, Polyethylene glycol monooleyl ether 92V, Polyoxyethylene (10) oleyl ether 97, Polyethylene glycol cetyl ether 58P, EL Macrogolglycerol ricinoleate, Decaethylene glycol monododecyl ether, N-decanoyl-N-methylglucamine, n-decyl α-D-glucopyranoside, decyl β-D-maltopyranoside, n-dodecanoyl-N-methylglucamide, n-dodecyl α-D-maltoside, n-dodecyl β-D-maltoside, n-dodecyl β-D-maltoside, heptaethylene glycol monodecyl ether, heptaethylene glycol monododecyl ether, heptaethylene glycol monotetradecyl ether, hexadecyl β-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-O-(N-heptylcarbamoyl)-α-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-β-D-glucopyranoside, pentaethylene glycol monodecyl ether, pentaethylene glycol monododecyl ether, pentaethylene glycol monodecyl ether Hexadecyl ether, pentaethylene glycol monohexyl 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(imidazolecarbonyl), polyoxyethylene 25 propylene glycol stearate, saponins from Quillaja bark, 20 Sorbitan laurate, 40 Sorbitan monopalmitate, 60 Sorbitan stearate, 65 Sorbitan tristearate, 80 Sorbitan monooleate, 85 Sorbitan trioleate, Secondary alcohol ethoxylate 15-S-12 type, Secondary alcohol ethoxylate 15-S-30 type, Secondary alcohol ethoxylate 15-S-5 type, Secondary alcohol ethoxylate 15-S-7 type, Secondary alcohol ethoxylate 15-S-9 type, Nonylphenol ethoxylate NP-10 type, Nonylphenol ethoxylate NP-4 type, Nonylphenol ethoxylate NP-40 type, Nonylphenol ethoxylate NP-7 type, Nonylphenol ethoxylate NP-9 type, Branched secondary alcohol ethoxylate TMN-10 type, Branched secondary alcohol ethoxylate TMN-6 type, TRITON™ X-100 polyethylene glycol tert-octylphenyl ether or a combination thereof.

[0249] Nanoparticles can then be separated from the cell debris using centrifugation. In embodiments, gradient centrifugation can be used, such as using cesium chloride, sucrose, and iodixanol. Other techniques can be used as an alternative or supplement, such as standard purification techniques, including, for example, ion exchange chromatography, affinity chromatography, and gel filtration chromatography.

[0250] For example, the first column can be an ion exchange chromatography resin, such as one based on EMD methacrylate 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 a cation exchange column based on EMD methacrylate polymer beads SO3 (EMD Millipore) resin. In other aspects, the cation exchange column can be an MMC column or a Nuvia CPrime column (Bio-Rad Laboratories, Inc.). Preferably, the methods disclosed herein do not utilize detergent extraction columns, such as hydrophobic interaction columns. Such columns are often used to remove detergents during purification, but may negatively impact the methods disclosed herein.

[0251] In order to form detergent core nanoparticles, the first detergent for extracting protein from host cells is substantially replaced with a second detergent to achieve nanoparticle structure. In embodiments, the first detergent is NP-9. Typically, when measured by HPLC, the nanoparticles do not contain detectable NP-9. The second detergent is typically selected from the group consisting of PS20, PS40, PS60, PS65, and PS80. In embodiments, the second detergent is PS80.

[0252] In certain aspects, detergent exchange is performed using affinity chromatography techniques to bind to glycoproteins via their carbohydrate moieties. For example, affinity chromatography can use a legume lectin column. Legume lectins are proteins originally identified in plants and have been 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 Con A, Pisum sativum lectin, Sainfoin, and Lentil lectin. Lentil lectin is a preferred column for detergent exchange due to its binding properties. Lectin columns are commercially available; for example, Capto LentilLectin is available from GE Healthcare. In some aspects, the lentil lectin column can use recombinant lectin. At the molecular level, it is believed that the carbohydrate portion is combined with the lentil lectin, releasing the amino acids of the protein to aggregate around the detergent, resulting in the formation of a detergent core, thereby providing nanoparticles with multiple antigen copies (e.g., glycoprotein oligomers), which can be dimers, trimers, or tetramers anchored in the detergent. In embodiments, viral glycoproteins form trimers. In embodiments, viral glycoprotein trimers are anchored in detergents. In embodiments, each viral glycoprotein nanoparticle contains at least one trimer associated with a nonionic core.

[0253] Detergent, when incubated with protein to form nanoparticles during detergent exchange, can be present at a maximum of about 0.1% (w / v) during the early purification steps, and this amount is reduced to obtain final nanoparticles with optimal stability. For example, a nonionic detergent (e.g., PS80) can be from about 0.005% (v / v) to about 0.1% (v / v), such as 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 0.04% (v / v), about 0.06% (v / v), about 0.08% (v / v), about 0.09% (v / v), about 0.10% (v / v), about 0.11% (v / v), about 0.12% (v / v), about 0.13% (v / v), about 0.14% (v / v), about 0.16% (v / v), about 0.17% (v / v), about 0.18% (v / v), about 0.19% (v / v), about 0.20% (v / v), about 0.21% (v / v), about 0.22% (v / v), about 0.23% (v / v), about 0.24% (v / v), about 0.25% (v / v), about 0.26% (v / v), about 0.27% (v / v), about 0.28% (v / v), about 0.29% (v / v), about In some embodiments, the nanoparticles 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.

[0254] In embodiments, the purified viral glycoprotein is dialyzed. In embodiments, dialysis occurs after purification. In embodiments, the viral glycoprotein is dialyzed in a solution comprising sodium phosphate, NaCl and PS80. In embodiments, the dialysis solution comprising sodium phosphate contains about 5mM to about 100mM sodium phosphate, for example, about 5mM, about 10mM, about 15mM, about 20mM, about 25mM, about 30mM, about 35mM, about 40mM, about 45mM, about 50mM, about 55mM, about 60mM, about 65mM, about 70mM, about 75mM, about 80mM, about 85mM, about 90mM, about 95mM or about 100mM sodium phosphate. In embodiments, the pH of the solution comprising sodium phosphate 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 embodiments, the dialysis solution comprising sodium chloride comprises about 50 mM NaCl to about 750 mM NaCl, for example, 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 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 400mM, about 410mM, about 420mM, about 430mM, about 440mM, about 450mM, about 460mM, about 470mM, about 480mM, about 490mM, about 500mM, about 510mM, about 520mM, about 530mM, about 540mM, about 550mM, about 560mM, about 570mM , about 580mM, about 590mM, about 600mM, about 610mM, about 620mM, about 630mM, about 640mM, about 650mM, about 660mM, about 670mM, about 680mM, about 690, about 700mM, about 710mM, about 720mM, about 730mM, about 740mM, or about 750mM NaCl.In embodiments, the dialysis solution comprising PS80 comprises 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. 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) PS80.

[0255] In an embodiment, 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 an embodiment, the pharmaceutically acceptable buffer comprises 25 mM sodium phosphate, 300 mM NaCl, and 0.03% PS80 at a pH of 7.2. In an embodiment, the pharmaceutically acceptable buffer comprises 25 mM sodium phosphate, 600 mM NaCl, and 0.01% PS80 at a pH of 6.8.

[0256] Detergent exchange can be performed with protein purified as discussed above, purified, stored frozen, and then thawed for detergent exchange.

[0257] The stability of compositions disclosed herein can be measured in a variety of ways. In one method, peptide maps can be made to determine the integrity of the antigenic protein after various treatments designed for applying pressure to nanoparticles by simulating harsh storage conditions. Therefore, the metric of stability is the relative abundance of antigenic peptides in the sample subjected to pressure compared to a control sample. For example, the stability of nanoparticles containing viral glycoproteins can be evaluated by exposing nanoparticles to various pH, proteases, salts, oxidants (including but not limited to hydrogen peroxide), various temperatures, freeze / thaw cycles and stirring. It is believed that the position of the glycoprotein anchored in the detergent core provides enhanced stability by reducing undesirable interactions. For example, improved protection based on protease degradation can be achieved by shielding (shielding) effect, thus causing steric hindrance to block the entry of proteases by anchoring glycoproteins in the core at a molar ratio disclosed herein. Stability can also be measured by monitoring intact proteins.

[0258] In embodiments, provided herein are immunogenic and vaccine compositions containing nanoparticles comprising RSV F glycoprotein, HA glycoprotein, SARS-CoV-2 S glycoprotein, or a combination thereof.

[0259] In embodiments, mature RSV F glycoprotein is used to produce a vaccine comprising RSV F glycoprotein nanoparticles. In embodiments, nanoparticles of the present disclosure comprise RSV F glycoprotein as described herein. In embodiments, immunogenic compositions and vaccine compositions provided herein contain nanoparticles comprising CoV S glycoprotein, influenza HA glycoprotein, RSV F glycoprotein, or any combination thereof.

[0260] In embodiments, nanoparticles of the present disclosure include viral glycoproteins (e.g., RSVF, CoV S, influenza HA glycoproteins, or combinations thereof) associated with a detergent core. The presence of detergent promotes the formation of nanoparticles by forming a core that organizes and presents antigens. In embodiments, nanoparticles can contain viral glycoproteins that are assembled into poly-oligomeric glycoprotein detergents (e.g., PS80) nanoparticles, wherein the head region protrudes outwards and the hydrophobic region and detergent form a central core surrounded by glycoprotein. In embodiments, viral glycoproteins inherently contain or are adapted to contain a transmembrane domain to promote the association of proteins into the detergent core. In embodiments, viral glycoproteins contain a head domain. The transmembrane domains of viral glycoprotein trimers are primarily associated with detergents; however, other parts of the polypeptide may also interact. Advantageously, the nanoparticles have improved resistance to environmental stresses, such that they provide enhanced stability and / or improved presentation to the immune system owing to organizing multiple protein copies around the detergent.

[0261] In embodiments, the detergent core is a nonionic detergent core. In embodiments, the RSV F glycoprotein is associated with a nonionic detergent core. In embodiments, the detergent is selected from the group consisting of polysorbate-20 (PS20), polysorbate-40 (PS40), polysorbate-60 (PS60), polysorbate-65 (PS65), and polysorbate-80 (PS80).

[0262] In embodiments, the second detergent is PS80.

[0263] In embodiments, the RSV F glycoprotein forms trimers. In embodiments, the RSV F glycoprotein nanoparticles are composed of multiple polypeptide trimers surrounding a nonionic detergent core. In embodiments, the nanoparticles contain at least about 1 trimer or more. In embodiments, the nanoparticles contain at least about 5 spike protein trimers to about 30 spike protein trimers. In embodiments, each nanoparticle can 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 can contain nanoparticles with different numbers of trimers. For example, a composition can contain nanoparticles with a trimer number ranging from 2-9; in embodiments, the nanoparticles in the composition can contain 2-6 trimers. In embodiments, the composition contains a heterogeneous population of nanoparticles having 2 to 6 trimers per nanoparticle, or 2 to 9 trimers per nanoparticle. In embodiments, the composition can contain a substantially homogeneous population of nanoparticles. For example, the population may contain about 95% nanoparticles having 5 trimers.

[0264] The particle size range of nano-particles disclosed herein.In embodiments, the particle size from Z average size of nano-particles disclosed herein is in the scope of about 20nm to about 60nm, about 20nm to about 50nm, about 20nm to about 45nm, about 20nm to about 35nm, about 20nm to about 30nm, about 25nm to about 35nm, about 25nm to about 45nm, about 30nm to about 120nm, about 30nm to about 80nm, about 30nm to about 60nm, about 30nm to about 65nm or about 30nm to about 50nm.Unless otherwise stated, particle size (Z-ave) all uses Zetasizer NanoZS (Malvern, UK) to be measured by dynamic light scattering (DLS).

[0265] Several nanoparticle types can be included in the vaccine compositions disclosed herein. In one aspect, the nanoparticle type is in the form of anisotropic rods that can be dimers or monomers. In other aspects, the nanoparticle type is a spherical oligomer. In yet other aspects, the nanoparticle can be described as an intermediate nanoparticle with sedimentation characteristics between the first two types. The formation of nanoparticle types can be regulated by controlling the detergent and protein concentrations during the production process. The type of nanoparticle can be determined by measuring the sedimentation coefficient.

[0266] Amount of viral glycoprotein in immunogenic and vaccine compositions

[0267] In embodiments, the compositions described herein comprise 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 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 35 μg, about 36 μg, about 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 71μg, about 72μg, about 73 μg, 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, about 97μg, about 98μg, about 99μg, about 100μg, about 101μg, about 102μg, about 103μg, about 104μg, about 105μg, about 106μg, about 107μg, about 108 μg, 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 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, about 136μg, about 137μg, about 138μg, about 139μg,about 140 μg, about 141 μg, about 142 μg, about 143 μg, about 144 μg, about 145 μg, about 146 μg, about 147 μg, about 148 μg, about 149 μg, about 150 μg, about 151 μg, about 152 μg, about 153 μg, about 154 μg, about 155 μg, about 156 μg, about 157 μg , about 158μg, about 159μg, about 160μg, about 161μg, about 162μg, about 163μg, about 164μg, about 165μg, about 166μg, about 167μg, about 168μg, about 169μg, about 170μg, about 171μg, about 172μg, about 173μg, about 174μg, about 175μ g, about 176 μg, about 177 μg, about 178 μg, about 179 μg, about 180 μg, about 181 μg, about 182 μg, about 183 μg, about 184 μg, about 185 μg, about 186 μg, about 187 μg, about 188 μg, about 189 μg, about 190 μg, about 191 μg, about 192 μg, about 193 μg, about 194 μg, about 195 μg, about 196 μg, about 197 μg, about 198 μg, about 199 μg, about 200 μg, about 201 μg, about 202 μg, about 203 μg, about 204 μg, about 205 μg, about 206 μg, about 207 μg, about 208 μg, about 209 μg, about 210 μg, about 21 1μg, about 212μg, about 213μg, about 214μg, about 215μg, about 216μg, about 217μg, about 218μg, about 219μg, about 220μg, about 221μg, about 222μg, about 223μg, about 224μg, about 225μg, about 226μg, about 227μg, about 228μg, about 2 29μg, about 230μg, about 231μg, about 232μg, about 233μg, about 234μg, about 235μg, about 236μg, about 237μg, about 238μg, about 239μg, about 240μg, about 241μg, about 242μg, about 243μg, about 244μg, about 245μg, about 246μg, about 247μg, about 248μg, about 249μg, about 250μg, about 251μg, about 252μg, about 253μg, about 254μg, about 255μg, about 256μg, about 257μg, about 258μg, about 259μg, about 260μg, about 261μg, about 262μg, about 263μg, about 264μg, about 265 μg, about 266 μg, about 267 μg, about 268 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, about 300 μg, about 301 μg, about 302 μg, about 303 μg, about 304 μg, about 305 μg, about 306 μg, about 307 μg, about 308 μg, about 309 μg, about 310 μg, about 311 μg, about 312 μg g, about 313 μg, about 314 μg, about 315 μg, about 316 μg, about 317 μg, about 318 μg, about 319 μg, about 320 μg, about 321 μg, about 322 μg, about 323 μg, about 324 μg, about 325 μg, about 326 μg, about 327 μg, about 328 μg, about 329 μg, about 330 μg, about 331 μg, about 332 μg, about 333 μg, about 334 μg, about 335 μg, about 336 μg, about 337 μg, about 338 μg, about 339 μg, about 340 μg, about 341 μg, about 342 μg, 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 373μg, about 374μg or about 400 μg of each viral glycoprotein.

[0268] In an embodiment, the immunogenic composition comprises about 1 μg of each viral glycoprotein. In an embodiment, the immunogenic composition comprises about 5 μg of each viral glycoprotein. In an embodiment, the immunogenic composition comprises about 25 μg of each viral glycoprotein. In an embodiment, the immunogenic composition comprises about 60 μg of each viral glycoprotein. In an embodiment, the immunogenic composition comprises about 240 μg of each viral glycoprotein.

[0269] In embodiments, the immunogenic compositions described herein comprise about 1 μg to about 300 μg of each RSV F glycoprotein. In embodiments, the immunogenic compositions described herein comprise about 5 μg to about 60 μg of each RSV F glycoprotein. In embodiments, the immunogenic compositions described herein comprise about 24 μg to about 40 μg of each RSV F glycoprotein. In embodiments, the immunogenic compositions described herein comprise about 30 μg to about 40 μg of each RSV F glycoprotein. In embodiments, the immunogenic compositions described herein comprise about 30 μg to about 60 μg of each RSV F glycoprotein. In embodiments, the immunogenic compositions described herein comprise about 33 μg to about 39 μg of each RSV F glycoprotein. In embodiments, the immunogenic compositions described herein comprise about 20 μg to about 240 μg of each RSV F glycoprotein. In embodiments, the immunogenic compositions described herein comprise about 30 μg to about 60 μg of each RSV F glycoprotein. In embodiments, the immunogenic compositions described herein comprise about 24 μg to about 40 μg of each RSV F glycoprotein. In 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, 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 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 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, about 89μg, about 90μg, about 91μg, about 92μg, about 93μg, about 94μg, about 95μg, about 96μg, about 97μg, about 98μg, about 99μg,about 100 μg, about 101 μg, about 102 μg, about 103 μg, about 104 μg, about 105 μg, about 106 μg, about 107 μg, about 108 μg, about 109 μg, about 110 μg, about 111 μg, about 112 μg, about 113 μg, about 114 μg, about 115 μg, about 116 μg, about 117 μg , about 118μg, about 119μg, about 120μg, 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 g, about 136 μg, about 137 μg, about 138 μg, about 139 μg, about 140 μg, about 141 μg, about 142 μg, about 143 μg, about 144 μg, about 145 μg, about 146 μg, about 147 μg, about 148 μg, about 149 μg, about 150 μg, about 151 μg, about 152 μg, about 153 μg, about 154 μg, about 155 μg, about 156 μg, about 157 μg, about 158 ​​μg, about 159 μg, about 160 μg, about 161 μg, about 162 μg, about 163 μg, about 164 μg, about 165 μg, about 166 μg, about 167 μg, about 168 μg, about 169 μg, about 170 μg, about 17 1μg, about 172μg, about 173μg, about 174μg, about 175μg, about 176μg, about 177μg, about 178μg, about 179μg, about 180μg, about 181μg, about 182μg, about 183μg, about 184μg, about 185μg, about 186μg, about 187μg, about 188μg, about 1 89μg, about 190μg, about 191μg, about 192μg, about 193μg, about 194μg, about 195μg, about 196μg, about 197μg, about 198μg, about 199μg, about 200μg, about 201μg, about 202μg, about 203μg, about 204μg, about 205μg, about 206μg, about 207μg, about 208μg, about 209μg, about 210μg, about 211μg, about 212μg, about 213μg, about 214μg, about 215μg, about 216μg, about 217μg, about 218μg, about 219μg, about 220μg, about 221μg, about 222μg, about 223μg, about 224μg, about 225 μg, about 226 μg, about 227 μg, about 228 μg, about 229 μg, about 230 μg, about 231 μg, about 232 μg, 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,about 243 μg, about 244 μg, about 245 μg, about 246 μg, about 247 μg, about 248 μg, about 249 μg, about 250 μg, about 251 μg, about 252 μg, about 253 μg, about 254 μg, about 255 μg, about 256 μg, about 257 μg, about 258 μg, about 259 μg, about 260 μg, about 261 μg, about 262 μg, about 263 μg, about 264 μg, about 265 μg, about 266 μg, about 267 μg, about 268 μg, about 269 μg, about 270 μg, about 271 μg, about 272 μg In some embodiments, the immunogenic compositions described herein comprise about 60 μg of each RSV F glycoprotein, including all values ​​and ranges therebetween. In embodiments, the immunogenic compositions described herein comprise about 240 μg of each RSV F glycoprotein.

[0270] In embodiments, the immunogenic compositions described herein comprise about 1 μg to about 1000 μg of total RSV F 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, 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 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 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 71μg, about 72μg, about 73μg, about 74μg, about 75μg, about 76μg, about 77μg, about 78μg, 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 106 μg, about 107 μg, about 108 μg, about 109 μg, about 110 μg, about 111 μg, about 112 μg, about 113 μg, about 114 μg, about 115 μg, about 116μg, about 117μg, about 118μg, about 119μg, about 120μg, 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, about 136μg, about 137μg, about 138μg, about 139μg, about 140μg, about 141μg, about 142μg, about 143μg, about 144μg, about 145μg, about 146μg, about 147μg, about 148μg, about 149μg,about 150 μg, about 151 μg, about 152 μg, about 153 μg, about 154 μg, about 155 μg, about 156 μg, about 157 μg, about 158 ​​μg, about 159 μg, about 160 μg, about 161 μg, about 162 μg, about 163 μg, about 164 μg, about 165 μg, about 166 μg, about 167 μg , about 168μg, about 169μg, about 170μg, about 171μg, about 172μg, about 173μg, about 174μg, about 175μg, about 176μg, about 177μg, about 178μg, about 179μg, about 180μg, about 181μg, about 182μg, about 183μg, about 184μg, about 185μg g, about 186 μg, about 187 μg, about 188 μg, about 189 μg, about 190 μg, about 191 μg, about 192 μg, about 193 μg, about 194 μg, about 195 μg, about 196 μg, about 197 μg, about 198 μg, about 199 μg, about 200 μg, about 201 μg, about 202 μg, about 203 μg, about 204 μg, about 205 μg, about 206 μg, about 207 μg, about 208 μg, about 209 μg, about 210 μg, about 211 μg, about 212 μg, about 213 μg, about 214 μg, about 215 μg, about 216 μg, about 217 μg, about 218 μg, about 219 μg, about 220 μg, about 22 1 μg, about 222 μg, about 223 μg, about 224 μg, about 225 μg, about 226 μg, about 227 μg, about 228 μg, about 229 μg, about 230 μg, about 231 μg, about 232 μg, about 233 μg, about 234 μg, about 235 μg, about 236 μg, about 237 μg, about 238 μg, about 2 39μg, about 240μg, about 241μg, about 242μg, about 243μg, about 244μg, about 245μg, about 246μg, about 247μg, about 248μg, about 249μg, about 250μg, about 251μg, about 252μg, about 253μg, about 254μg, about 255μg, about 256μg, about 257μg, about 258μg, about 259μg, about 260μg, about 261μg, about 262μg, about 263μg, about 264μg, about 265μg, about 266μg, about 267μg, about 268μg, about 269μg, about 270μg, about 271μg, about 272μg, about 273μg, about 274μg, 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, about 300 μg, about 301 μg, about 302 μg, about 303 μg, about 304 μg, about 305 μg, about 306 μg, about 307 μg, about 308 μg, about 309 μg, about 310 μg , about 311 μg, about 312 μg, about 313 μg, about 314 μg, about 315 μg, about 316 μg, about 317 μg, about 318 μg, about 319 μg, about 320 μg, about 321 μg, about 322 μg, about 323 μg, about 324 μg, about 325 μg, about 326 μg, about 327 μg, about 328 μg g, about 329 μg, about 330 μg, about 331 μg, about 332 μg, about 333 μg, about 334 μg, about 335 μg, about 336 μg, about 337 μg, about 338 μg, about 339 μg, about 340 μg, about 341 μg, about 342 μg, about 343 μg, about 344 μg, about 345 μg, about 346 μg, about 347 μg, about 348 μg, about 349 μg, about 350 μg, about 351 μg, about 352 μg, about 353 μg, about 354 μg, about 355 μg, about 356 μg, about 357 μg, about 358 μg, about 359 μg, about 360 μg, about 361 μg, about 362 μg, about 363 μg, about 364 μg 4μg, about 365μg, about 366μg, about 367μg, about 368μg, about 369μg, about 370μg, about 371μg, about 372μg, about 373μg, about 374μg, about 375μg, about 376μg, about 377μg, about 378μg, about 379μg, about 380μg, about 381μg, about 3 82μg, about 383μg, about 384μg, about 385μg, about 386μg, about 387μg, about 388μg, about 389μg, about 390μg, about 391μg, about 392μg, about 393μg, about 394μg, about 395μg, about 396μg, about 397μg, about 398μg, about 399μg, about 400 μg, about 401 μg, about 402 μg, about 403 μg, about 404 μg, about 405 μg, about 406 μg, about 407 μg, about 408 μg, about 409 μg, about 410 μg, about 411 μg, about 412 μg, about 413 μg, about 414 μg, about 415 μg, about 416 μg, about 417 μg, about 418 μg, about 419 μg, about 420 μg, about 421 μg, about 422 μg, about 423 μg, about 424 μg, about 425 μg, about 426 μg, about 427 μg, about 428 μg, about 429 μg, about 430 μg, about 431 μg, about 432 μg, about 433 μg, about 434 μg, about 435 μg,about 436 μg, about 437 μg, about 438 μg, about 439 μg, about 440 μg, about 441 μg, about 442 μg, about 443 μg, about 444 μg, about 445 μg, about 446 μg, about 447 μg, about 448 μg, about 449 μg, about 450 μg, about 451 μg, about 452 μg, about 453 μg , about 454μg, about 455μg, about 456μg, about 457μg, about 458μg, about 459μg, about 460μg, about 461μg, about 462μg, about 463μg, about 464μg, about 465μg, about 466μg, about 467μg, about 468μg, about 469μg, about 470μg, about 471μ g, about 472 μg, about 473 μg, about 474 μg, about 475 μg, about 476 μg, about 477 μg, about 478 μg, about 479 μg, about 480 μg, about 481 μg, about 482 μg, about 483 μg, about 484 μg, about 485 μg, about 486 μg, about 487 μg, about 488 μg, about 489 μg, about 490 μg, about 491 μg, about 492 μg, about 493 μg, about 494 μg, about 495 μg, about 496 μg, about 497 μg, about 498 μg, about 499 μg, about 500 μg, about 501 μg, about 502 μg, about 503 μg, about 504 μg, about 505 μg, about 506 μg, about 507 μg 7μg, about 508μg, about 509μg, about 510μg, about 511μg, about 512μg, about 513μg, about 514μg, about 515μg, about 516μg, about 517μg, about 518μg, about 519μg, about 520μg, about 521μg, about 522μg, about 523μg, about 524μg, about 5 25μg, about 526μg, about 527μg, about 528μg, about 529μg, about 530μg, about 531μg, about 532μg, about 533μg, about 534μg, about 535μg, about 536μg, about 537μg, about 538μg, about 539μg, about 540μg, about 541μg, about 542μg, about 543μg, about 544μg, about 545μg, about 546μg, about 547μg, about 548μg, about 549μg, about 550μg, about 551μg, about 552μg, about 553μg, about 554μg, about 555μg, about 556μg, about 557μg, about 558μg, about 559μg, about 560μg, about 561 μg, about 562 μg, about 563 μg, about 564 μg, about 565 μg, about 566 μg, about 567 μg, about 568 μg, about 569 μg, about 570 μg, about 571 μg, about 572 μg, about 573 μg, about 574 μg, about 575 μg, about 576 μg, about 577 μg, about 578 μg,about 579 μg, about 580 μg, about 581 μg, about 582 μg, about 583 μg, about 584 μg, about 585 μg, about 586 μg, about 587 μg, about 588 μg, about 589 μg, about 590 μg, about 591 μg, about 592 μg, about 593 μg, about 594 μg, about 595 μg, about 596 μg , about 597μg, about 598μg, about 599μg, about 600μg, about 601μg, about 602μg, about 603μg, about 604μg, about 605μg, about 606μg, about 607μg, about 608μg, about 609μg, about 610μg, about 611μg, about 612μg, about 613μg, about 614μg g, about 615 μg, about 616 μg, about 617 μg, about 618 μg, about 619 μg, about 620 μg, about 621 μg, about 622 μg, about 623 μg, about 624 μg, about 625 μg, about 626 μg, about 627 μg, about 628 μg, about 629 μg, about 630 μg, about 631 μg, about 632 μg, about 633 μg, about 634 μg, about 635 μg, about 636 μg, about 637 μg, about 638 μg, about 639 μg, about 640 μg, about 641 μg, about 642 μg, about 643 μg, about 644 μg, about 645 μg, about 646 μg, about 647 μg, about 648 μg, about 649 μg, about 65 0μg, about 651μg, about 652μg, about 653μg, about 654μg, about 655μg, about 656μg, about 657μg, about 658μg, about 659μg, about 660μg, about 661μg, about 662μg, about 663μg, about 664μg, about 665μg, about 666μg, about 667μg, about 6 68μg, about 669μg, about 670μg, about 671μg, about 672μg, about 673μg, about 674μg, about 675μg, about 676μg, about 677μg, about 678μg, about 679μg, about 680μg, about 681μg, about 682μg, about 683μg, about 684μg, about 685μg, about 686μg, about 687μg, about 688μg, about 689μg, about 690μg, about 691μg, about 692μg, about 693μg, about 694μg, about 695μg, about 696μg, about 697μg, about 698μg, about 699μg, about 700μg, about 701μg, about 702μg, about 703μg, about 704 μg, about 705 μg, about 706 μg, about 707 μg, about 708 μg, about 709 μg, about 710 μg, about 711 μg, about 712 μg, about 713 μg, about 714 μg, about 715 μg, about 716 μg, about 717 μg, about 718 μg, about 719 μg, about 720 μg, about 721 μg,about 722 μg, about 723 μg, about 724 μg, about 725 μg, about 726 μg, about 727 μg, about 728 μg, about 729 μg, about 730 μg, about 731 μg, about 732 μg, about 733 μg, about 734 μg, about 735 μg, about 736 μg, about 737 μg, about 738 μg, about 739 μg , about 740μg, about 741μg, about 742μg, about 743μg, about 744μg, about 745μg, about 746μg, about 747μg, about 748μg, about 749μg, about 750μg, about 751μg, about 752μg, about 753μg, about 754μg, about 755μg, about 756μg, about 757μg g, about 758 μg, about 759 μg, about 760 μg, about 761 μg, about 762 μg, about 763 μg, about 764 μg, about 765 μg, about 766 μg, about 767 μg, about 768 μg, about 769 μg, about 770 μg, about 771 μg, about 772 μg, about 773 μg, about 774 μg, about 775 μg, about 776 μg, about 777 μg, about 778 μg, about 779 μg, about 780 μg, about 781 μg, about 782 μg, about 783 μg, about 784 μg, about 785 μg, about 786 μg, about 787 μg, about 788 μg, about 789 μg, about 790 μg, about 791 μg, about 792 μg, about 79 3μg, about 794μg, about 795μg, about 796μg, about 797μg, about 798μg, about 799μg, about 800μg, about 801μg, about 802μg, about 803μg, about 804μg, about 805μg, about 806μg, about 807μg, about 808μg, about 809μg, about 810μg, about 8 11μg, about 812μg, about 813μg, about 814μg, about 815μg, about 816μg, about 817μg, about 818μg, about 819μg, about 820μg, about 821μg, about 822μg, about 823μg, about 824μg, about 825μg, about 826μg, about 827μg, about 828μg, about 829 μg, about 830 μg, about 831 μg, about 832 μg, about 833 μg, about 834 μg, about 835 μg, about 836 μg, about 837 μg, about 838 μg, about 839 μg, about 840 μg, about 841 μg, about 842 μg, about 843 μg, about 844 μg, about 845 μg, about 846 μg, about 847 μg, about 848 μg, about 849 μg, about 850 μg, about 851 μg, about 852 μg, about 853 μg, about 854 μg, about 855 μg, about 856 μg, about 857 μg, about 858 μg, about 859 μg, about 860 μg, about 861 μg, about 862 μg, about 863 μg, about 864 μg,About 865μg, about 866μg, about 867μg, about 868μg, about 869μg, about 870μg, about 871μg, about 872μg, about 873μg, about 874μg, about 875μg, about 876μg, about 877μg, about 878μg, about 879μg, about 880μg, about 881μg, about 882μg, about 883μg, about 884μg, about 885μg, about 886μg, about 887μg, about 888μg, about 889μg, about 890μg, about 891μg, about 892μg, about 893μg, about 894μg, about 895μg, about 896μg, about 897μg, about 898μg, about 899μg, about 900μg, about 901μg, about 902μg, about 903μg, about 904μg, about 905μg, about 906μg, about 907μg, about 908μg, about 909μg, about 910μg, 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 9 33μg, 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 958μg, about 959μg, about 960μg, about 961μg, about 962μg, about 963μg, about 964μg, about 965μg, about 966μg, about 96 7μg, about 968μg, about 969μg, about 970μg, about 971μg, about 972μg, about 973μg, about 974μg, about 975μg, about 976μg, about 977μg, about 978μg, about 979μg, about 980μg, about 981μg, about 982μg, about 983μg, about 984μg, about 985μg, about 986μg, about 987μg, about 988μg, about 989μg, about 990μg, about 991μg, about 992μg, about 993μg, about 994μg, about 995μg, about 996μg, about 997μg, about 998μg, about 999μg, about 1000μg of total RSV F glycoprotein, including all values ​​and ranges therebetween.

[0271] In embodiments, the immunogenic compositions described herein comprise from about 1 μg to about 300 μg of each HA glycoprotein. In embodiments, the immunogenic compositions described herein comprise from about 5 μg to about 60 μg of each HA glycoprotein. In embodiments, the immunogenic compositions described herein comprise from about 24 μg to about 40 μg of each HA glycoprotein. In embodiments, the immunogenic compositions described herein comprise from about 30 μg to about 40 μg of each HA glycoprotein. In embodiments, the immunogenic compositions described herein comprise from about 30 μg to about 60 μg of each HA glycoprotein. In embodiments, the immunogenic compositions described herein comprise from about 33 μg to about 39 μg of each HA glycoprotein. In embodiments, the immunogenic compositions described herein comprise from about 20 μg to about 240 μg of each HA glycoprotein. In embodiments, the immunogenic compositions described herein comprise from about 30 μg to about 60 μg of each HA glycoprotein. In embodiments, the immunogenic compositions described herein comprise from about 24 μg to about 40 μg of each HA 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, 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 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 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 71μg, about 72μg, about 73μg, about 74μg, about 75μg, about 76μg, about 77μg, about 78μg 8μg, 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, about 97μg, about 98μg, about 99μg, about 100μg, about 101μg, about 102μg, about 103μg,about 104 μg, about 105 μg, about 106 μg, about 107 μg, about 108 μg, about 109 μg, about 110 μg, about 111 μg, about 112 μg, about 113 μg, about 114 μg, about 115 μg, about 116 μg, about 117 μg, about 118 μg, about 119 μg, about 120 μg, about 121 μg , 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, about 136μg, about 137μg, about 138μg, about 139μg g, about 140 μg, about 141 μg, about 142 μg, about 143 μg, about 144 μg, about 145 μg, about 146 μg, about 147 μg, about 148 μg, about 149 μg, about 150 μg, about 151 μg, about 152 μg, about 153 μg, about 154 μg, about 155 μg, about 156 μg, about 157 μg, about 158μg, about 159μg, about 160μg, about 161μg, about 162μg, about 163μg, about 164μg, about 165μg, about 166μg, about 167μg, about 168μg, about 169μg, about 170μg, about 171μg, about 172μg, about 173μg, about 174μg, about 176μg, about 177μg 5μg, about 176μg, about 177μg, about 178μg, about 179μg, about 180μg, about 181μg, about 182μg, about 183μg, about 184μg, about 185μg, about 186μg, about 187μg, about 188μg, about 189μg, about 190μg, about 191μg, about 192μg, about 1 93μg, about 194μg, about 195μg, about 196μg, about 197μg, about 198μg, about 199μg, about 200μg, about 201μg, about 202μg, about 203μg, about 204μg, about 205μg, about 206μg, about 207μg, about 208μg, about 209μg, about 210μg, about 211 μg, about 212 μg, about 213 μg, about 214 μg, about 215 μg, about 216 μg, about 217 μg, about 218 μg, about 219 μg, about 220 μg, about 221 μg, about 222 μg, about 223 μg, about 224 μg, about 225 μg, about 226 μg, about 227 μg, about 228 μg, about 229 μg, about 230 μg, about 231 μg, about 232 μg, 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, about 243 μg, about 244 μg, about 245 μg, about 246 μg,about 247 μg, about 248 μg, about 249 μg, about 250 μg, about 251 μg, about 252 μg, about 253 μg, about 254 μg, about 255 μg, about 256 μg, about 257 μg, about 258 μg, about 259 μg, about 260 μg, about 261 μg, about 262 μg, about 263 μg, about 264 μg, about 265 μg, about 266 μg, about 267 μg, about 268 μg, about 269 μg, about 270 μg, about 271 μg, about 272 μg, about 273 μg, about 274 μg, about 275 μg In some embodiments, the immunogenic compositions described herein comprise about 30 μg of each HA glycoprotein. In some 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.

[0272] In embodiments, the immunogenic compositions described herein comprise from about 1 μg to about 1000 μg of total HA 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, 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. g, 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 μg, about 75 μg, about 76 μg, about 77 μg, about 78 μg, about 79 μg g, 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 106 μg, about 107 μg, about 108 μg, about 109 μg, about 110 μg, about 111 μg, about 112 μg, about 113 μg, about 114 μg, about 115 μg, about 116 μg g, about 117 μg, about 118 μg, about 119 μg, about 120 μg, about 121 μg, about 122 μg, about 123 μg, about 124 μg, about 125 μg, about 126 μg, about 127 μg, about 128 μg, about 129 μg, about 130 μg, about 131 μg, about 132 μg, about 133 μg, about 134 μg, about 135 μg, about 136 μg, about 137 μg, about 138 μg, about 139 μg, about 140 μg, about 141 μg, about 142 μg, about 143 μg, about 144 μg, about 145 μg, about 146 μg, about 147 μg, about 148 μg, about 149 μg, about 150 μg,about 151 μg, about 152 μg, about 153 μg, about 154 μg, about 155 μg, about 156 μg, about 157 μg, about 158 ​​μg, about 159 μg, about 160 μg, about 161 μg, about 162 μg, about 163 μg, about 164 μg, about 165 μg, about 166 μg, about 167 μg, about 168 μg , about 169μg, about 170μg, about 171μg, about 172μg, about 173μg, about 174μg, about 175μg, about 176μg, about 177μg, about 178μg, about 179μg, about 180μg, about 181μg, about 182μg, about 183μg, about 184μg, about 185μg, about 186μg g, about 187 μg, about 188 μg, about 189 μg, about 190 μg, about 191 μg, about 192 μg, about 193 μg, about 194 μg, about 195 μg, about 196 μg, about 197 μg, about 198 μg, about 199 μg, about 200 μg, about 201 μg, about 202 μg, about 203 μg, about 204 μg, about 205 μg, about 206 μg, about 207 μg, about 208 μg, about 209 μg, about 210 μg, about 211 μg, about 212 μg, about 213 μg, about 214 μg, about 215 μg, about 216 μg, about 217 μg, about 218 μg, about 219 μg, about 220 μg, about 221 μg, about 222 2μg, about 223μg, about 224μg, about 225μg, about 226μg, about 227μg, about 228μg, about 229μg, about 230μg, about 231μg, about 232μg, about 233μg, about 234μg, about 235μg, about 236μg, about 237μg, about 238μg, about 239μg, about 2 40μg, about 241μg, about 242μg, about 243μg, about 244μg, about 245μg, about 246μg, about 247μg, about 248μg, about 249μg, about 250μg, about 251μg, about 252μg, about 253μg, about 254μg, about 255μg, about 256μg, about 257μg, about 258μg, about 259μg, about 260μg, about 261μg, about 262μg, about 263μg, about 264μg, about 265μg, about 266μg, about 267μg, about 268μg, about 269μg, about 270μg, about 271μg, about 272μg, about 273μg, about 274μg, about 275μg, 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, about 300 μg, about 301 μg, about 302 μg, about 303 μg, about 304 μg, about 305 μg, about 306 μg, about 307 μg, about 308 μg, about 309 μg, about 310 μg, about 311 μg , 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, about 327μg, about 328μg, about 329μg g, about 330 μg, about 331 μg, about 332 μg, about 333 μg, about 334 μg, about 335 μg, about 336 μg, about 337 μg, about 338 μg, about 339 μg, about 340 μg, about 341 μg, about 342 μg, about 343 μg, about 344 μg, about 345 μg, about 346 μg, about 347 μg, about 348 μg, about 349 μg, about 350 μg, about 351 μg, about 352 μg, about 353 μg, about 354 μg, about 355 μg, about 356 μg, about 357 μg, about 358 μg, about 359 μg, about 360 μg, about 361 μg, about 362 μg, about 363 μg, about 364 μg, about 366 μg, about 367 μg, about 368 μg, about 369 μg, about 370 μg, about 371 μg, 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 38 5μg, about 366μg, about 367μg, about 368μg, about 369μg, about 370μg, about 371μg, about 372μg, about 373μg, about 374μg, about 375μg, about 376μg, about 377μg, about 378μg, about 379μg, about 380μg, about 381μg, about 382μg, about 3 83μg, about 384μg, about 385μg, about 386μg, about 387μg, about 388μg, about 389μg, about 390μg, about 391μg, about 392μg, about 393μg, about 394μg, about 395μg, about 396μg, about 397μg, about 398μg, about 399μg, about 400μg, about 401 μg, about 402 μg, about 403 μg, about 404 μg, about 405 μg, about 406 μg, about 407 μg, about 408 μg, about 409 μg, about 410 μg, about 411 μg, about 412 μg, about 413 μg, about 414 μg, about 415 μg, about 416 μg, about 417 μg, about 418 μg, about 419 μg, about 420 μg, about 421 μg, about 422 μg, about 423 μg, about 424 μg, about 425 μg, about 426 μg, about 427 μg, about 428 μg, about 429 μg, about 430 μg, about 431 μg, about 432 μg, about 433 μg, about 434 μg, about 435 μg, about 436 μg,about 437 μg, about 438 μg, about 439 μg, about 440 μg, about 441 μg, about 442 μg, about 443 μg, about 444 μg, about 445 μg, about 446 μg, about 447 μg, about 448 μg, about 449 μg, about 450 μg, about 451 μg, about 452 μg, about 453 μg, about 454 μg , about 455μg, about 456μg, about 457μg, about 458μg, about 459μg, about 460μg, about 461μg, about 462μg, about 463μg, about 464μg, about 465μg, about 466μg, about 467μg, about 468μg, about 469μg, about 470μg, about 471μg, about 472μ g, about 473 μg, about 474 μg, about 475 μg, about 476 μg, about 477 μg, about 478 μg, about 479 μg, about 480 μg, about 481 μg, about 482 μg, about 483 μg, about 484 μg, about 485 μg, about 486 μg, about 487 μg, about 488 μg, about 489 μg, about 490 μg, about 491 μg, about 492 μg, about 493 μg, about 494 μg, about 495 μg, about 496 μg, about 497 μg, about 498 μg, about 499 μg, about 500 μg, about 501 μg, about 502 μg, about 503 μg, about 504 μg, about 505 μg, about 506 μg, about 507 μg, about 50 8μg, about 509μg, about 510μg, about 511μg, about 512μg, about 513μg, about 514μg, about 515μg, about 516μg, about 517μg, about 518μg, about 519μg, about 520μg, about 521μg, about 522μg, about 523μg, about 524μg, about 525μg, about 5 26μg, about 527μg, about 528μg, about 529μg, about 530μg, about 531μg, about 532μg, about 533μg, about 534μg, about 535μg, about 536μg, about 537μg, about 538μg, about 539μg, about 540μg, about 541μg, about 542μg, about 543μg, about 544μg, about 545μg, about 546μg, about 547μg, about 548μg, about 549μg, about 550μg, about 551μg, about 552μg, about 553μg, about 554μg, about 555μg, about 556μg, about 557μg, about 558μg, about 559μg, about 560μg, about 561μg, about 562 μg, about 563 μg, about 564 μg, about 565 μg, about 566 μg, about 567 μg, about 568 μg, about 569 μg, about 570 μg, about 571 μg, about 572 μg, about 573 μg, about 574 μg, about 575 μg, about 576 μg, about 577 μg, about 578 μg, about 579 μg,about 580 μg, about 581 μg, about 582 μg, about 583 μg, about 584 μg, about 585 μg, about 586 μg, about 587 μg, about 588 μg, about 589 μg, about 590 μg, about 591 μg, about 592 μg, about 593 μg, about 594 μg, about 595 μg, about 596 μg, about 597 μg , about 598μg, about 599μg, about 600μg, about 601μg, about 602μg, about 603μg, about 604μg, about 605μg, about 606μg, about 607μg, about 608μg, about 609μg, about 610μg, about 611μg, about 612μg, about 613μg, about 614μg, about 615μg g, about 616 μg, about 617 μg, about 618 μg, about 619 μg, about 620 μg, about 621 μg, about 622 μg, about 623 μg, about 624 μg, about 625 μg, about 626 μg, about 627 μg, about 628 μg, about 629 μg, about 630 μg, about 631 μg, about 632 μg, about 633 μg, about 634 μg, about 635 μg, about 636 μg, about 637 μg, about 638 μg, about 639 μg, about 640 μg, about 641 μg, about 642 μg, about 643 μg, about 644 μg, about 645 μg, about 646 μg, about 647 μg, about 648 μg, about 649 μg, about 650 μg, about 65 1μg, about 652μg, about 653μg, about 654μg, about 655μg, about 656μg, about 657μg, about 658μg, about 659μg, about 660μg, about 661μg, about 662μg, about 663μg, about 664μg, about 665μg, about 666μg, about 667μg, about 668μg, about 6 69μg, about 670μg, about 671μg, about 672μg, about 673μg, about 674μg, about 675μg, about 676μg, about 677μg, about 678μg, about 679μg, about 680μg, about 681μg, about 682μg, about 683μg, about 684μg, about 685μg, about 686μg, about 687μg, about 688μg, about 689μg, about 690μg, about 691μg, about 692μg, about 693μg, about 694μg, about 695μg, about 696μg, about 697μg, about 698μg, about 699μg, about 700μg, about 701μg, about 702μg, about 703μg, about 704μg, about 705 μg, about 706 μg, about 707 μg, about 708 μg, about 709 μg, about 710 μg, about 711 μg, about 712 μg, about 713 μg, about 714 μg, about 715 μg, about 716 μg, about 717 μg, about 718 μg, about 719 μg, about 720 μg, about 721 μg, about 722 μg,about 723 μg, about 724 μg, about 725 μg, about 726 μg, about 727 μg, about 728 μg, about 729 μg, about 730 μg, about 731 μg, about 732 μg, about 733 μg, about 734 μg, about 735 μg, about 736 μg, about 737 μg, about 738 μg, about 739 μg, about 740 μg , about 741 μg, about 742 μg, about 743 μg, about 744 μg, about 745 μg, about 746 μg, about 747 μg, about 748 μg, about 749 μg, about 750 μg, about 751 μg, about 752 μg, about 753 μg, about 754 μg, about 755 μg, about 756 μg, about 757 μg, about 758 μg g, about 759 μg, about 760 μg, about 761 μg, about 762 μg, about 763 μg, about 764 μg, about 765 μg, about 766 μg, about 767 μg, about 768 μg, about 769 μg, about 770 μg, about 771 μg, about 772 μg, about 773 μg, about 774 μg, about 775 μg, about 776 μg, about 777 μg, about 778 μg, about 779 μg, about 780 μg, about 781 μg, about 782 μg, about 783 μg, about 784 μg, about 785 μg, about 786 μg, about 787 μg, about 788 μg, about 789 μg, about 790 μg, about 791 μg, about 792 μg, about 793 μg, about 794 μg, about 795 μg, about 796 μg, about 797 μg, about 798 μg, about 799 μg, about 791 μg, about 792 μg, about 793 μg, about 799 μg, about 791 μg, about 794 μg, about 795 μg, about 796 μg, about 797 μg, about 798 μg, about 799 μg, about 799 μg, about 791 μg, about 792 μg, about 793 ...9 μg, about 799 μg, about 4μg, about 795μg, about 796μg, about 797μg, about 798μg, about 799μg, about 800μg, about 801μg, about 802μg, about 803μg, about 804μg, about 805μg, about 806μg, about 807μg, about 808μg, about 809μg, about 810μg, about 811μg, about 8 12μg, about 813μg, about 814μg, about 815μg, about 816μg, about 817μg, about 818μg, about 819μg, about 820μg, about 821μg, about 822μg, about 823μg, about 824μg, about 825μg, about 826μg, about 827μg, about 828μg, about 829μg, about 830 μg, about 831 μg, about 832 μg, about 833 μg, about 834 μg, about 835 μg, about 836 μg, about 837 μg, about 838 μg, about 839 μg, about 840 μg, about 841 μg, about 842 μg, about 843 μg, about 844 μg, about 845 μg, about 846 μg, about 847 μg, about 848 μg, about 849 μg, about 850 μg, about 851 μg, about 852 μg, about 853 μg, about 854 μg, about 855 μg, about 856 μg, about 857 μg, about 858 μg, about 859 μg, about 860 μg, about 861 μg, about 862 μg, about 863 μg, about 864 μg, about 865 μg,About 866 μg, about 867 μg, about 868 μg, about 869 μg, about 870 μg, about 871 μg, about 872 μg, about 873 μg, about 874 μg, about 875 μg, about 876 μg, about 877 μg, about 878 μg, about 879 μg, about 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 90 0 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, about 913 μg, about 914 μg, about 915 μg, about 916 μg, about 917 μg, about 918 μg, about 919 μg, about 920 μg, about 921 μg, about 922 μg, about 923 μg, about 924 μg, about 925 μg, about 926 μg, about 927 μg, about 928 μg, about 929 μg, about 930 μg, about 931 μg, about 932 μg, about 933 μg, about 934 μg , 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 958μg, about 959μg, about 960μg, about 961μg, about 962μg, about 963μg, about 964μg, about 965μg, about 966μg, about 967μg, about 968μg, about 9 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.

[0273] In embodiments, the immunogenic compositions described herein comprise from about 1 μg to about 100 μg of each CoV S glycoprotein. In embodiments, the immunogenic compositions described herein comprise from about 2.5 μg to about 35 μg of each CoV S glycoprotein. In embodiments, the immunogenic compositions described herein comprise from about 5 μg to about 25 μg of each CoV S glycoprotein. In embodiments, the immunogenic compositions described herein comprise from about 14 μg to about 15 μg of each CoV S glycoprotein. In embodiments, the immunogenic compositions described herein comprise from about 15 μg to about 35 μg of each CoV S glycoprotein. In embodiments, the immunogenic compositions described herein comprise from about 1 μg to 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. , 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 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 71μg, about 72μg, about 73μg, about 74μg, about 75μg, about 76μg, about 77μg, 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 (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 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 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, 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 71μg, about 72μg, about 73μg, about 74μg, about 75μg, about 76μg, about 77μg 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 some embodiments, the immunogenic composition comprises about 25 μg of CoV S glycoprotein.

[0274] Adjuvants in immunogenic and vaccine compositions

[0275] In certain embodiments, the compositions disclosed herein comprise one or more adjuvants. In embodiments, the one or more adjuvants enhance the immune response. In other embodiments, the compositions are prepared without an adjuvant and can therefore be used to administer as a non-adjuvanted composition. Advantageously, the non-adjuvanted compositions disclosed herein can provide a protective immune response when administered in a single dose.

[0276] Aluminum-based adjuvants

[0277] In embodiments, the adjuvant can be alum (e.g., AlPO4 or Al(OH)3). Typically, the nanoparticles are substantially bound to the 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. Typically, in the composition, the nanoparticles are 92% to 97% bound to alum. The amount of alum present per dose is typically in the range of between 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, for a dose of 120 μg of protein nanoparticles, alum is present in about 400 μg.

[0278] Saponin adjuvant

[0279] Adjuvants containing saponins can also be combined with the immunogens disclosed herein. Saponins are glycosides derived from the bark of the soap tree (Quillaja saponaria Molina). Typically, saponins are prepared using a multi-step purification process, resulting in a variety of fractions. As used herein, the term "saponin fraction from Quillaja saponaria" is generally used to describe a semi-purified or defined saponin fraction of the soap tree or its substantially pure fraction.

[0280] Saponin fraction

[0281] Several methods 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. The lipophilic fraction from Quil A (a crude aqueous Quillaja saponaria extract) 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, eluting with a gradient of 25% to 60% acetonitrile in acidic water. The fraction referred to herein as "fraction A" or "QH-A" is or corresponds to the fraction eluted at approximately 39% acetonitrile. The fraction referred to herein as "fraction B" or "QH-B" is or corresponds to the fraction eluted at approximately 47% acetonitrile. The fraction referred to herein as "fraction C" or "QH-C" is or corresponds to the fraction eluted at approximately 49% acetonitrile. Additional information about fraction purification can be found in U.S. Patent No. 5,057,540. When prepared as described herein, fractions A, B, and C of Quillaja saponaria each represent a group or family of chemically closely related molecules with definable properties. The chromatographic conditions under which the fractions were obtained are such that batch-to-batch reproducibility is highly consistent with respect to elution profiles and biological activity.

[0282] Other saponin fractions have been described. Fractions B3, B4 and B4b are described in EP 0436620. Fractions QA1-QA22 are described in EP 03632279 B2, Q-VAC (Nor-Feed, AS Denmark), Quillaja saponaria Molina Spikoside (lsconova AB, Ultunaallén 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, especially QA-7, QA-17, QA-18 and QA-21, from EP 0 3632 279 B2 can be used. The fractions are obtained as described in Example 1 on pages 6, 8 and 9 of EP 0 3632 279 B2.

[0283] The saponin fractions described herein and used to form adjuvants are typically substantially pure fractions; that is, the fractions are substantially free of contamination from other materials. In particular aspects, the substantially pure saponin fractions can 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).

[0284] ISCOM structure

[0285] The saponin fraction can be administered in the form of caged particles known as ISCOMs (immune stimulating complexes). ISCOMs can be prepared as described in EP0109942B1, EP0242380B1, and EP0180546B1. In certain embodiments, transport and / or passenger antigens can be used as described in EP-9600647-3 (PCT / SE97 / 00289).

[0286] Matrix adjuvant

[0287] In an embodiment, 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 further comprises a phospholipid. The ISCOM matrix complex may also contain one or more other immunomodulatory (adjuvant active) substances, not necessarily glycosides, and may be produced as described in EP0436620B1, which is incorporated herein by reference in its entirety.

[0288] In other aspects, the ISCOM is an ISCOM complex. The ISCOM complex contains at least one saponin, at least one lipid, and at least one type of antigen or epitope. The ISCOM complex contains an antigen that is associated by detergent treatment, resulting in a portion of the antigen being incorporated into the particle. In contrast, the ISCOM matrix is ​​formulated as a mixture with the antigen, and the association between the ISCOM matrix particles and the antigen is mediated by electrostatic and / or hydrophobic interactions.

[0289] According to one embodiment, the saponin fraction incorporated into the ISCOM matrix complex or ISCOM complex, or at least one additional adjuvant also incorporated into or admixed 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 any purified subfraction (e.g., QA 1-21).

[0290] In certain aspects, each ISCOM particle can contain at least two saponin fractions. Any combination of weight percents of different saponin fractions can be used. Any combination of weight percents of any two fractions can be used. For example, the particle can 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 aspects, each ISCOM matrix particle or each ISCOM complex particle can contain 0.1 to 99.9 weight percent, 5 to 95 weight percent, 10 to 90 weight percent, 15 to 85 weight percent, 20 to 80 weight percent, 25 to 75 weight percent, 30 to 70 weight percent, 35 to 65 weight percent, 40 to 60 weight percent, 45 to 55 weight percent, 40 to 60 weight percent, or 50 weight percent of one saponin fraction (e.g., Fraction A) and in each case the remainder up to 100% of another saponin (e.g., any crude fraction or any other fraction, such as Fraction C). This weight is calculated as the total weight of the saponin fractions. Examples of ISCOM matrix complexes and ISCOM complex adjuvants are disclosed in US Published Application No. 2013 / 0129770, which is incorporated herein by reference in its entirety.

[0291] In certain embodiments, the ISCOM matrix or ISCOM complex comprises 5-99 wt% of one fraction (e.g., Fraction A) and the remainder up to 100 wt% of another fraction (e.g., crude saponin fraction or Fraction C). This weight is calculated as the total weight of the saponin fraction.

[0292] In another embodiment, the ISCOM matrix or ISCOM complex comprises 40% to 99% by weight of one fraction (e.g., Fraction A) and 1% to 60% by weight of another fraction (e.g., crude saponin fraction or Fraction C). This weight is calculated as the total weight of the saponin fraction.

[0293] In yet another embodiment, the ISCOM matrix or ISCOM complex comprises 70% to 95% by weight of one fraction (e.g., Fraction A) and 30% to 5% by weight of another fraction (e.g., crude saponin fraction or Fraction C). This weight is calculated as the total weight of the saponin fractions. In other embodiments, the saponin fraction from Quillaja saponaria is selected from any one of QA1-21.

[0294] In addition to particles containing a mixture of saponin fractions, ISCOM matrix particles and ISCOM complex particles can each be formed using only one saponin fraction. The compositions disclosed herein can contain a plurality of particles, wherein each particle contains only one saponin fraction. That is, certain compositions can contain one or more different types of ISCOM-matrix complex particles and / or one or more different types of ISCOM complex particles, wherein each individual particle contains a saponin fraction from Quillaja saponaria, wherein the saponin fraction in one complex is different from the saponin fraction in another complex particle.

[0295] In certain aspects, one type of saponin fraction or crude saponin fraction can be incorporated into one ISCOM matrix complex or particle, and another type of substantially pure saponin fraction or crude saponin fraction can be incorporated into another ISCOM matrix complex or particle. A composition or vaccine can comprise at least two types of complexes or particles, each type having one type of saponin incorporated into physically distinct particles.

[0296] In the composition, mixtures of ISCOM matrix complex particles and / or ISCOM complex particles may be used, wherein one saponin fraction Quillaja saponin and another saponin fraction Quillaja saponin are separately incorporated into different ISCOM matrix complex particles and / or ISCOM complex particles.

[0297] The ISCOM matrix or ISCOM complex particles, each having a saponin fraction, can be present in the composition in any combination of weight percent. In particular aspects, 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 an ISCOM matrix or complex containing a first saponin fraction, with the remainder being composed of ISCOM matrices or complexes containing a different saponin fraction. In certain aspects, the remainder is one or more ISCOM matrices or complexes, each matrix or complex particle containing only one saponin fraction. In other aspects, the ISCOM matrix or complex particle can contain more than one saponin fraction.

[0298] In certain compositions, the only saponin fraction in a first ISCOM matrix or ISCOM complex particle is Fraction A, and the only saponin fraction in a second ISCOM matrix or ISCOM complex particle is Fraction C.

[0299] In embodiments, Quillaja Fraction A comprises 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 Quillaja Fraction A and Quillaja Fraction C in the adjuvant and Quillaja Fraction C comprises the remainder of the combined weight of Quillaja Fraction A and Quillaja Fraction C in the adjuvant.

[0300] A preferred composition comprises a first ISCOM matrix comprising Fraction A and a second ISCOM matrix comprising Fraction C, wherein the Fraction A ISCOM matrix comprises approximately 70% by weight of the total saponin adjuvant and the Fraction C ISCOM matrix comprises approximately 30% by weight of the total saponin adjuvant. In another preferred composition, the Fraction A ISCOM matrix comprises approximately 85% by weight of the total saponin adjuvant and the Fraction C ISCOM matrix comprises approximately 15% by weight of the total saponin adjuvant. In another preferred composition, the Fraction A ISCOM matrix comprises approximately 92% by weight of the total saponin adjuvant and the Fraction C ISCOM matrix comprises approximately 8% by weight of the total saponin adjuvant. Thus, in certain compositions, the Fraction A ISCOM matrix is ​​present in a range of about 70% to about 85% of the total weight of the saponin adjuvant in the composition, and the Fraction C ISCOM matrix is ​​present in a range of about 15% to about 30% of the total weight of the saponin adjuvant in the composition. In certain compositions, the Fraction A ISCOM matrix is ​​present in a range of about 70% to about 92% of the total weight of the saponin adjuvant in the composition, and the Fraction C ISCOM matrix is ​​present in a range of about 8% to about 30% of the total weight of the saponin adjuvant in the composition. In embodiments, the Fraction A ISCOM matrix comprises 50-96% by weight of the sum of the weight of the Fraction A ISCOM matrix and the Fraction C ISCOM in the adjuvant, and the Fraction C ISCOM matrix comprises the remainder of the sum of the weight of the Fraction A ISCOM matrix and the Fraction C ISCOM in the adjuvant. In a particularly preferred composition (referred to herein as MATRIX-M TM ), fraction A ISCOM matrix is ​​present at about 85% of the total weight of saponin adjuvant in the composition, and fraction C ISCOM matrix is ​​present at about 15% of the total weight of saponin adjuvant in the composition. MATRIX-M TM Interchangeably referred to as Matrix-M1.

[0301] 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.

[0302] In embodiments, other adjuvants may be used in addition or as alternatives. The inclusion of any adjuvant described in the following document is contemplated to be within the scope of the present disclosure: Vogel et al., "A Compendium of Vaccine Adjuvants and Excipients (2nd Edition)", which is incorporated herein by reference in its entirety. Other adjuvants include complete Freund's adjuvant (a nonspecific immune response stimulator containing killed Mycobacterium tuberculosis), 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, RIBI (which contains three components extracted from bacteria), MPL, trehalose dimycolate (TDM), and in 2% squalene / Cell wall skeleton (CWS) in polysorbate 80 emulsion. In embodiments, the adjuvant may be a paucilamellar lipid vesicle; for example, are oligolamellar, non-phospholipid vesicles ranging from about 100 nm to about 500 nm. They contain Alcohol ethoxylate 72, cholesterol, oleic acid and squalene. It has been shown to be an effective adjuvant (see US Patent Nos. 5,629,021, 6,387,373 and 4,911,928).

[0303] The amount of adjuvant in the composition

[0304] In embodiments, the immunogenic compositions described herein comprise 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 an 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 35 μg, 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 71μg, about 72μg g, 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 106 μg, about 107 μg g, about 108 μg, about 109 μg, about 110 μg, about 111 μg, about 112 μg, about 113 μg, about 114 μg, about 115 μg, about 116 μg, about 117 μg, about 118 μg, about 119 μg, about 120 μg, about 121 μg, about 122 μg, about 123 μg, about 124 μg, about 125 μg, about 126 μg, about 127 μg, about 128 μg, about 129 μg, about 130 μg, about 131 μg, about 132 μg, about 133 μg, about 134 μg, about 135 μg, about 136 μg, about 137 μg, about 138 μg,about 139 μg, about 140 μg, about 141 μg, about 142 μg, about 143 μg, about 144 μg, about 145 μg, about 146 μg, about 147 μg, about 148 μg, about 149 μg, about 150 μg, about 151 μg, about 152 μg, about 153 μg, about 154 μg, about 155 μg, about 156 μg , about 157μg, about 158μg, about 159μg, about 160μg, about 161μg, about 162μg, about 163μg, about 164μg, about 165μg, about 166μg, about 167μg, about 168μg, about 169μg, about 170μg, about 171μg, about 172μg, about 173μg, about 174μ g, about 175 μg, about 176 μg, about 177 μg, about 178 μg, about 179 μg, about 180 μg, about 181 μg, about 182 μg, about 183 μg, about 184 μg, about 185 μg, about 186 μg, about 187 μg, about 188 μg, about 189 μg, about 190 μg, about 191 μg, about 192 μg, about 193 μg, about 194 μg, about 195 μg, about 196 μg, about 197 μg, about 198 μg, about 199 μg, about 200 μg, about 201 μg, about 202 μg, about 203 μg, about 204 μg, about 205 μg, about 206 μg, about 207 μg, about 208 μg, about 209 μg, about 21 0 μg, about 211 μg, about 212 μg, about 213 μg, about 214 μg, about 215 μg, about 216 μg, about 217 μg, about 218 μg, about 219 μg, about 220 μg, about 221 μg, about 222 μg, about 223 μg, about 224 μg, about 225 μg, about 226 μg, about 227 μg, about 2 28μg, about 229μg, about 230μg, about 231μg, about 232μg, about 233μg, about 234μg, about 235μg, about 236μg, about 237μg, about 238μg, about 239μg, about 240μg, about 241μg, about 242μg, about 243μg, about 244μg, about 245μg, about 246μg, about 247μg, about 248μg, about 249μg, about 250μg, about 251μg, about 252μg, about 253μg, about 254μg, about 255μg, about 256μg, about 257μg, about 258μg, about 259μg, about 260μg, about 261μg, about 262μg, about 263μg, about 264 μg, about 265 μg, about 266 μg, about 267 μg, about 268 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, about 300 μg, about 301 μg, about 302 μg, about 303 μg, about 304 μg, about 305 μg, about 306 μg, about 307 μg, about 308 μg, about 309 μg, about 310 μg, about 311 μg , 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, 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, about 342 μg, about 343 μg, about 344 μg, about 345 μg, about 346 μg, about 347 μg, about 348 μg, about 349 μg, about 350 μg, about 351 μg, about 352 μg, about 353 μg, about 354 μg, about 355 μg, about 356 μg, about 357 μg, about 358 μg, about 359 μg, about 360 μg, about 361 μg, about 362 μg, about 363 μg, about 364 μg, about 365 μg, about 366 μg, about 367 μg, about 368 μg, about 369 μg, about 370 μg, about 371 In one embodiment, the immunogenic composition comprises about 50 μg of adjuvant. In one embodiment, the immunogenic composition comprises about 75 μg of adjuvant.

[0305] Methods for stimulating an immune response against RSV, influenza virus, SARS-CoV-2, or variants thereof, or combinations thereof

[0306] In 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, the method comprising administering an immunogenic composition as described herein. In embodiments, the immunogenic composition contains one or more viral glycoproteins. In embodiments, the one or more viral glycoproteins are selected from any one of RSV F glycoprotein, CoV S glycoprotein, and influenza HA glycoprotein.

[0307] In an embodiment, the immunogenic composition lacks an adjuvant. In an embodiment, the immunogenic composition comprises an adjuvant. Non-limiting examples of adjuvants are described herein.

[0308] In embodiments, the immunogenic compositions described herein have an efficacy of between about 50% and about 99%, between about 80% and about 99%, between about 75% and about 99%, between about 80% and about 95%, between about 90% and about 98%, between about 75% and 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).

[0309] The compositions disclosed herein can be administered via a systemic route or a mucosal route or a transdermal route or directly into a specific tissue. As used herein, the term "systemic administration" includes parenteral routes of administration. In particular, parenteral administration includes subcutaneous, intraperitoneal, intravenous, intraarterial, intramuscular or intrasternal injection, intravenous 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 ocular administration. In embodiments, the method includes intramuscular administration of the immunogenic composition described herein. In embodiments, the method includes intranasal administration of the immunogenic composition described herein.

[0310] Composition can be used according to single dose regimen or multiple dose regimen.Multiple doses can be used in the primary immunization scheme or in the booster immunization scheme.In 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 used.In the multiple dose regimen, various dosages can be given by the same or different approaches, such as parenteral first exemption and mucosal reinforcement, mucosal first exemption and parenteral reinforcement, etc. In one aspect, a booster 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 embodiments, a booster dose is administered annually after the initial dose is administered. In embodiments, subsequent booster doses are administered 3 weeks or 4 weeks after the previous dose is administered. In embodiments, the first dose is administered on day 0 and the booster dose is administered on day 21. In embodiments, the first dose is administered on day 0 and the booster dose is administered on day 28. In embodiments, the first dose is administered on day 0, the booster dose is administered on day 21, and the second booster dose is administered about six months after the first dose or the second dose is administered. In embodiments, the first dose is administered on day 0, the booster dose is administered on day 28, and the second booster dose is administered about six months after the first dose is administered. In embodiments, the first dose is administered on day 0, the booster dose is administered on day 21, and the second booster dose is administered about six months after the second dose is administered. In embodiments, the first dose is administered on day 0, the booster dose is administered on day 28, and the second booster dose is administered about six months after the second dose is administered.

[0311] In embodiments, the first dose is administered on day 0, the booster dose is administered on day 21, and the second booster dose is administered about 1 year after the first dose or the first booster dose is administered. In embodiments, the first dose is administered on day 0, the first booster dose is administered on day 28, and the second booster dose is administered about 1 year after the first dose is administered. In embodiments, the first dose is administered on day 0, the booster dose is administered on day 21, and the second booster dose is administered about 1 year after the second dose is administered. In embodiments, the first dose is administered on day 0, the first booster dose is administered on day 28, and the second booster dose is administered about 1 year after the second dose is administered. In embodiments, the second booster dose is administered 6 to 24 months or 12 to 24 months after the first booster dose.

[0312] In embodiments, the dose, as measured in μg, can be the total weight of the dose including solutes, or the weight of RSV F glycoprotein nanoparticles, or the weight of RSV F glycoprotein. The dose is measured using a protein concentration assay (A280 or ELISA).

[0313] Can be administered to certain populations with or without adjuvants. In certain aspects, the composition may contain no added adjuvant. In such cases, the dosage may be increased by about 10%.

[0314] In embodiments, the immunogenic compositions described herein are provided in the form of a prefilled syringe.When the immunogenic composition is prepared in a prefilled syringe, the RSV F glycoprotein and the adjuvant are combined together prior to administration.

[0315] In 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 embodiments, the dose is administered in a volume of 0.25 mL. In embodiments, the dose is administered in a volume of 0.5 mL. In embodiments, the dose is administered in a volume of 0.6 mL.

[0316] 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 about 1 μg / mL to about 50 μg / mL, 10 μg / mL to about 100 μg / mL, about 10 μg / mL to about 50 μg / mL, about 175 μg / mL to about 325 μg / mL, about 200 μg / mL to about 300 μg / mL, about 220 μg / mL to about 280 μg / mL, or about 240 μg / mL to about 260 μg / mL.

[0317] In embodiments, the method includes 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 of each RSV F glycoprotein. In embodiments, the method includes administering about 5 μg to about 60 μg of each RSV F glycoprotein. In embodiments, the method includes administering about 24 μg to about 40 μg of each RSV F glycoprotein. In embodiments, the method includes administering about 30 μg to about 40 μg of each RSV F glycoprotein. In embodiments, the method includes administering about 30 μg to about 60 μg of each RSV F glycoprotein. In embodiments, the method includes administering about 33 μg to about 39 μg of each RSV F glycoprotein. In embodiments, the method comprises administering about 20 μg to about 240 μg of each RSV F glycoprotein. In embodiments, the method comprises administering about 30 μg to about 60 μg of each RSV F glycoprotein. In embodiments, the method comprises administering about 24 μg to about 40 μg of each RSV F glycoprotein. In embodiments, the method comprises administering 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 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 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, about 89μg, about 90μg, about 91μg, about 92μg, about 93μg, about 94μg, about 95μg,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 106 μg, about 107 μg, about 108 μg, about 109 μg, about 110 μg, about 111 μg, about 112 μg, about 113 μg, about 1 14μg, about 115μg, about 116μg, about 117μg, about 118μg, about 119μg, about 120μg, 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, about 136μg, about 137μg, about 138μg, about 139μg, about 140μg, about 141μg, about 142μg, about 143μg, about 144μg, about 145μg, about 146μg, about 147μg, about 148μg, about 149μg , about 150μg, about 151μg, about 152μg, about 153μg, about 154μg, about 155μg, about 156μg, about 157μg, about 158μg, about 159μg, about 160μg, about 161μg, about 162μg, about 163μg, about 164μg, about 165μg, about 166μg, about 167μg g, about 168 μg, about 169 μg, about 170 μg, about 171 μg, about 172 μg, about 173 μg, about 174 μg, about 175 μg, about 176 μg, about 177 μg, about 178 μg, about 179 μg, about 180 μg, about 181 μg, about 182 μg, about 183 μg, about 184 μg, about 186 μg, about 187 μg, about 188 μg, about 189 μg, about 190 μg, about 191 μg, about 192 μg, about 193 μg, about 194 μg, about 195 μg, about 196 μg, about 197 μg, about 198 μg, about 199 μg, about 200 5μg, about 186μg, about 187μg, about 188μg, about 189μg, about 190μg, about 191μg, about 192μg, about 193μg, about 194μg, about 195μg, about 196μg, about 197μg, about 198μg, about 199μg, about 200μg, about 201μg, about 202μg, about 2 03μg, about 204μg, about 205μg, about 206μg, about 207μg, about 208μg, about 209μg, about 210μg, about 211μg, about 212μg, about 213μg, about 214μg, about 215μg, about 216μg, about 217μg, about 218μg, about 219μg, about 220μg, about 221 μg, about 222 μg, about 223 μg, about 224 μg, about 225 μg, about 226 μg, about 227 μg, about 228 μg, about 229 μg, about 230 μg, about 231 μg, about 232 μg, about 233 μg, about 234 μg, about 235 μg, about 236 μg, about 237 μg, about 238 μg,about 239μg, about 240μg, about 241μg, about 242μg, about 243μg, about 244μg, about 245μg, about 246μg, about 247μg, about 248μg, about 249μg, about 250μg, about 251μg, about 252μg, about 253μg, about 254μg, about 255μg, about 256μg, about 257μg, about 258μg, about 259μg, about 260μg, about 261μg, about 262μg, about 263μg, about 264μg, about 265μg, about 266μg, about 267μg, about 268μg, about 269μg, about 270μg or 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 embodiments, the method comprises administering about 60 μg of each RSV F glycoprotein. In embodiments, the method comprises administering about 240 μg of each RSV F glycoprotein.

[0318] In embodiments, the method comprises administering about 1 μg to about 1000 μg of total RSV F glycoprotein. In embodiments, the method comprises administering 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 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 71μg, about 72μg, about 73μg, about 74μg, about 75μg, about 76μg, about 77μg, about 78μg, about 79μg, about 80μg 0μg, 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, about 97μg, about 98μg, about 99μg, about 100μg, about 101μg, about 102μg, about 103μg, about 104μg, about 105μg, about 106μg, about 107μg, about 108μg, about 109μg, about 110μg, about 111μg, about 112μg, about 113μg, about 114μg, about 115μg, about 116μg, about 117 μg, about 118μg, about 119μg, about 120μg, 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, about 136μg, about 137μg, about 138μg, about 139μg, about 140μg, about 141μg, about 142μg, about 143μg, about 144μg, about 145μg, about 146μg, about 147μg, about 148μg, about 149μg, about 150μg, about 151μg,about 152 μg, about 153 μg, about 154 μg, about 155 μg, about 156 μg, about 157 μg, about 158 ​​μg, about 159 μg, about 160 μg, about 161 μg, about 162 μg, about 163 μg, about 164 μg, about 165 μg, about 166 μg, about 167 μg, about 168 μg, about 169 μg , about 170μg, about 171μg, about 172μg, about 173μg, about 174μg, about 175μg, about 176μg, about 177μg, about 178μg, about 179μg, about 180μg, about 181μg, about 182μg, about 183μg, about 184μg, about 185μg, about 186μg, about 187μg g, about 188 μg, about 189 μg, about 190 μg, about 191 μg, about 192 μg, about 193 μg, about 194 μg, about 195 μg, about 196 μg, about 197 μg, about 198 μg, about 199 μg, about 200 μg, about 201 μg, about 202 μg, about 203 μg, about 204 μg, about 205 μg, about 206 μg, about 207 μg, about 208 μg, about 209 μg, about 210 μg, about 211 μg, about 212 μg, about 213 μg, about 214 μg, about 215 μg, about 216 μg, about 217 μg, about 218 μg, about 219 μg, about 220 μg, about 221 μg, about 222 μg, about 223 μg, about 224 μg 3μg, about 224μg, about 225μg, about 226μg, about 227μg, about 228μg, about 229μg, about 230μg, about 231μg, about 232μg, about 233μg, about 234μg, about 235μg, about 236μg, about 237μg, about 238μg, about 239μg, about 240μg, about 2 41μg, about 242μg, about 243μg, about 244μg, about 245μg, about 246μg, about 247μg, about 248μg, about 249μg, about 250μg, about 251μg, about 252μg, about 253μg, about 254μg, about 255μg, about 256μg, about 257μg, about 258μg, about 259μg, about 260μg, about 261μg, about 262μg, about 263μg, about 264μg, about 265μg, about 266μg, about 267μg, about 268μg, about 269μg, about 270μg, about 271μg, about 272μg, about 273μg, about 274μg, about 275μg, about 276μg, 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, about 300 μg, about 301 μg, about 302 μg, about 303 μg, about 304 μg, about 305 μg, about 306 μg, about 307 μg, about 308 μg, about 309 μg, about 310 μg, about 311 μg, about 312 μg , 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, about 327μg, about 328μg, about 329μg, about 330μg g, about 331 μg, about 332 μg, about 333 μg, about 334 μg, about 335 μg, about 336 μg, about 337 μg, about 338 μg, about 339 μg, about 340 μg, about 341 μg, about 342 μg, about 343 μg, about 344 μg, about 345 μg, about 346 μg, about 347 μg, about 348 μg, about 349 μg, about 350 μg, about 351 μg, about 352 μg, about 353 μg, about 354 μg, about 355 μg, about 356 μg, about 357 μg, about 358 μg, about 359 μg, about 360 μg, about 361 μg, about 362 μg, about 363 μg, about 364 μg, about 365 μg, about 366 μg, about 367 μg, about 368 μg, about 369 μg, about 370 μg, about 371 μg, 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 38 6μg, about 367μg, about 368μg, about 369μg, about 370μg, about 371μg, about 372μg, about 373μg, about 374μg, about 375μg, about 376μg, about 377μg, about 378μg, about 379μg, about 380μg, about 381μg, about 382μg, about 383μg, about 3 84μg, about 385μg, about 386μg, about 387μg, about 388μg, about 389μg, about 390μg, about 391μg, about 392μg, about 393μg, about 394μg, about 395μg, about 396μg, about 397μg, about 398μg, about 399μg, about 400μg, about 401μg, about 402 μg, about 403 μg, about 404 μg, about 405 μg, about 406 μg, about 407 μg, about 408 μg, about 409 μg, about 410 μg, about 411 μg, about 412 μg, about 413 μg, about 414 μg, about 415 μg, about 416 μg, about 417 μg, about 418 μg, about 419 μg, about 420 μg, about 421 μg, about 422 μg, about 423 μg, about 424 μg, about 425 μg, about 426 μg, about 427 μg, about 428 μg, about 429 μg, about 430 μg, about 431 μg, about 432 μg, about 433 μg, about 434 μg, about 435 μg, about 436 μg, about 437 μg,about 438 μg, about 439 μg, about 440 μg, about 441 μg, about 442 μg, about 443 μg, about 444 μg, about 445 μg, about 446 μg, about 447 μg, about 448 μg, about 449 μg, about 450 μg, about 451 μg, about 452 μg, about 453 μg, about 454 μg, about 455 μg , about 456μg, about 457μg, about 458μg, about 459μg, about 460μg, about 461μg, about 462μg, about 463μg, about 464μg, about 465μg, about 466μg, about 467μg, about 468μg, about 469μg, about 470μg, about 471μg, about 472μg, about 473μg g, about 474 μg, about 475 μg, about 476 μg, about 477 μg, about 478 μg, about 479 μg, about 480 μg, about 481 μg, about 482 μg, about 483 μg, about 484 μg, about 485 μg, about 486 μg, about 487 μg, about 488 μg, about 489 μg, about 490 μg, about 491 μg, about 492 μg, about 493 μg, about 494 μg, about 495 μg, about 496 μg, about 497 μg, about 498 μg, about 499 μg, about 500 μg, about 501 μg, about 502 μg, about 503 μg, about 504 μg, about 505 μg, about 506 μg, about 507 μg, about 508 μg, about 50 9μg, about 510μg, about 511μg, about 512μg, about 513μg, about 514μg, about 515μg, about 516μg, about 517μg, about 518μg, about 519μg, about 520μg, about 521μg, about 522μg, about 523μg, about 524μg, about 525μg, about 526μg, about 5 27μg, about 528μg, about 529μg, about 530μg, about 531μg, about 532μg, about 533μg, about 534μg, about 535μg, about 536μg, about 537μg, about 538μg, about 539μg, about 540μg, about 541μg, about 542μg, about 543μg, about 544μg, about 545μg, about 546μg, about 547μg, about 548μg, about 549μg, about 550μg, about 551μg, about 552μg, about 553μg, about 554μg, about 555μg, about 556μg, about 557μg, about 558μg, about 559μg, about 560μg, about 561μg, about 562μg, about 563 μg, about 564 μg, about 565 μg, about 566 μg, about 567 μg, about 568 μg, about 569 μg, about 570 μg, about 571 μg, about 572 μg, about 573 μg, about 574 μg, about 575 μg, about 576 μg, about 577 μg, about 578 μg, about 579 μg, about 580 μg,about 581 μg, about 582 μg, about 583 μg, about 584 μg, about 585 μg, about 586 μg, about 587 μg, about 588 μg, about 589 μg, about 590 μg, about 591 μg, about 592 μg, about 593 μg, about 594 μg, about 595 μg, about 596 μg, about 597 μg, about 598 μg , about 599 μg, about 600 μg, about 601 μg, about 602 μg, about 603 μg, about 604 μg, about 605 μg, about 606 μg, about 607 μg, about 608 μg, about 609 μg, about 610 μg, about 611 μg, about 612 μg, about 613 μg, about 614 μg, about 615 μg, about 616 μg g, about 617 μg, about 618 μg, about 619 μg, about 620 μg, about 621 μg, about 622 μg, about 623 μg, about 624 μg, about 625 μg, about 626 μg, about 627 μg, about 628 μg, about 629 μg, about 630 μg, about 631 μg, about 632 μg, about 633 μg, about 634 μg, about 635 μg, about 636 μg, about 637 μg, about 638 μg, about 639 μg, about 640 μg, about 641 μg, about 642 μg, about 643 μg, about 644 μg, about 645 μg, about 646 μg, about 647 μg, about 648 μg, about 649 μg, about 650 μg, about 651 μg, about 65 2μg, about 653μg, about 654μg, about 655μg, about 656μg, about 657μg, about 658μg, about 659μg, about 660μg, about 661μg, about 662μg, about 663μg, about 664μg, about 665μg, about 666μg, about 667μg, about 668μg, about 669μg, about 6 70μg, about 671μg, about 672μg, about 673μg, about 674μg, about 675μg, about 676μg, about 677μg, about 678μg, about 679μg, about 680μg, about 681μg, about 682μg, about 683μg, about 684μg, about 685μg, about 686μg, about 687μg, about 688μg, about 689μg, about 690μg, about 691μg, about 692μg, about 693μg, about 694μg, about 695μg, about 696μg, about 697μg, about 698μg, about 699μg, about 700μg, about 701μg, about 702μg, about 703μg, about 704μg, about 705μg, about 706 μg, about 707 μg, about 708 μg, about 709 μg, about 710 μg, about 711 μg, about 712 μg, about 713 μg, about 714 μg, about 715 μg, about 716 μg, about 717 μg, about 718 μg, about 719 μg, about 720 μg, about 721 μg, about 722 μg, about 723 μg,about 724 μg, about 725 μg, about 726 μg, about 727 μg, about 728 μg, about 729 μg, about 730 μg, about 731 μg, about 732 μg, about 733 μg, about 734 μg, about 735 μg, about 736 μg, about 737 μg, about 738 μg, about 739 μg, about 740 μg, about 741 μg , about 742μg, about 743μg, about 744μg, about 745μg, about 746μg, about 747μg, about 748μg, about 749μg, about 750μg, about 751μg, about 752μg, about 753μg, about 754μg, about 755μg, about 756μg, about 757μg, about 758μg, about 759μ g, about 760 μg, about 761 μg, about 762 μg, about 763 μg, about 764 μg, about 765 μg, about 766 μg, about 767 μg, about 768 μg, about 769 μg, about 770 μg, about 771 μg, about 772 μg, about 773 μg, about 774 μg, about 775 μg, about 776 μg, about 777 μg, about 778 μg, about 779 μg, about 780 μg, about 781 μg, about 782 μg, about 783 μg, about 784 μg, about 785 μg, about 786 μg, about 787 μg, about 788 μg, about 789 μg, about 790 μg, about 791 μg, about 792 μg, about 793 μg, about 794 μg, about 796 μg, about 797 μg, about 798 μg, about 799 μg, about 791 μg, about 792 μg, about 793 μg, about 794 ... 5μg, about 796μg, about 797μg, about 798μg, about 799μg, about 800μg, about 801μg, about 802μg, about 803μg, about 804μg, about 805μg, about 806μg, about 807μg, about 808μg, about 809μg, about 810μg, about 811μg, about 812μg, about 8 13μg, about 814μg, about 815μg, about 816μg, about 817μg, about 818μg, about 819μg, about 820μg, about 821μg, about 822μg, about 823μg, about 824μg, about 825μg, about 826μg, about 827μg, about 828μg, about 829μg, about 830μg, about 831 μg, about 832 μg, about 833 μg, about 834 μg, about 835 μg, about 836 μg, about 837 μg, about 838 μg, about 839 μg, about 840 μg, about 841 μg, about 842 μg, about 843 μg, about 844 μg, about 845 μg, about 846 μg, about 847 μg, about 848 μg, about 849 μg, about 850 μg, about 851 μg, about 852 μg, about 853 μg, about 854 μg, about 855 μg, about 856 μg, about 857 μg, about 858 μg, about 859 μg, about 860 μg, about 861 μg, about 862 μg, about 863 μg, about 864 μg, about 865 μg, about 866 μg,About 867μg, about 868μg, about 869μg, about 870μg, about 871μg, about 872μg, about 873μg, about 874μg, about 875μg, about 876μg, about 877μg, about 878μg, about 879μg, about 880μg, about 881μg, about 882μg, about 883μg, about 884μg, about 885μg, about 886μg, about 887μg, about 888μg, about 889μg, about 890μg, about 891μg, about 892μg, about 893μg, about 894μg, about 895μg, about 896μg, about 897μg, about 898μg, about 899μg, 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, about 913 μg, about 914 μg, about 915 μg, about 916 μg, about 917 μg, about 918 μg, about 919 μg, about 920 μg, about 921 μg, about 922 μg, about 923 μg, about 924 μg, about 925 μg, about 926 μg, about 927 μg, about 928 μg, about 929 μg, about 930 μg, about 931 μg, about 932 μg, about 933 μg, about 9 34μg, 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 958μg, about 959μg, about 960μg, about 961μg, about 962μg, about 963μg, about 964μg, about 965μg, about 966μg, about 967μg, About 968 μg, about 969 μg, about 970 μg, about 971 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.

[0319] In embodiments, the method comprises administering from about 1 μg to about 100 μg of each CoV S glycoprotein. In embodiments, the method comprises administering from about 2.5 μg to about 35 μg of each CoV S glycoprotein. In embodiments, the method comprises administering from about 5 μg to about 25 μg of each CoV S glycoprotein. In embodiments, the method comprises administering from about 14 μg to about 15 μg of each CoV S glycoprotein. In embodiments, the method comprises administering from about 15 μg to about 35 μg of each CoV S glycoprotein. In embodiments, the method comprises administering from about 1 μg to about 100 μg of total CoV S glycoprotein. In embodiments, the method comprises administering 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 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 71μg, about 72μg, about 73μg, about 74μg, about 75μg, about 76μg, about 77μg, about 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 (including all values ​​and ranges therebetween) of each CoV S glycoprotein.In embodiments, the method comprises administering 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 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 5 2μg, 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 71μg, about 72μg, about 73μg, about 74μg, about 75μg, about 76μg, about 77μg, about 78 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 some embodiments, the method comprises administering about 25 μg of CoV S glycoprotein.

[0320] In embodiments, the method comprises administering from about 1 μg to about 300 μg of each HA glycoprotein. In embodiments, the method comprises administering from about 5 μg to about 60 μg of each HA glycoprotein. In embodiments, the method comprises administering from about 24 μg to about 40 μg of each HA glycoprotein. In embodiments, the method comprises administering from about 30 μg to about 40 μg of each HA glycoprotein. In embodiments, the method comprises administering from about 30 μg to about 60 μg of each HA glycoprotein. In embodiments, the method comprises administering from about 33 μg to about 39 μg of each HA glycoprotein. In embodiments, the method comprises administering from about 20 μg to about 240 μg of each HA glycoprotein. In embodiments, the method comprises administering from about 30 μg to about 60 μg of each HA glycoprotein. In embodiments, the method comprises administering from about 24 μg to about 40 μg of each HA glycoprotein. In embodiments, the method comprises administering 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 29μg, about 30μg, about 31μg, about 32μg, about 33μg, about 34μg, 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 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, about 77μg, about 78μg, about 79μg, about 80μg, about 81μg, about 82μg, about 83μg, about 84μg, about 85μg, about 86μg, about 87μg 6μg, about 87μg, 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, about 106μg, about 107μg, about 108μg, about 109μg, about 110μg, about 111μg, about 112μg,about 113 μg, about 114 μg, about 115 μg, about 116 μg, about 117 μg, about 118 μg, about 119 μg, about 120 μg, about 121 μg, about 122 μg, about 123 μg, about 124 μg, about 125 μg, about 126 μg, about 127 μg, about 128 μg, about 129 μg, about 130 μg , about 131 μg, about 132 μg, about 133 μg, about 134 μg, about 135 μg, about 136 μg, about 137 μg, about 138 μg, about 139 μg, about 140 μg, about 141 μg, about 142 μg, about 143 μg, about 144 μg, about 145 μg, about 146 μg, about 147 μg, about 148 μg g, about 149 μg, about 150 μg, about 151 μg, about 152 μg, about 153 μg, about 154 μg, about 155 μg, about 156 μg, about 157 μg, about 158 ​​μg, about 159 μg, about 160 μg, about 161 μg, about 162 μg, about 163 μg, about 164 μg, about 165 μg, about 166 μg, about 167 μg, about 168 μg, about 169 μg, about 170 μg, about 171 μg, about 172 μg, about 173 μg, about 174 μg, about 175 μg, about 176 μg, about 177 μg, about 178 μg, about 179 μg, about 180 μg, about 181 μg, about 182 μg, about 183 μg, about 184 μg 4μg, about 185μg, about 186μg, about 187μg, about 188μg, about 189μg, about 190μg, about 191μg, about 192μg, about 193μg, about 194μg, about 195μg, about 196μg, about 197μg, about 198μg, about 199μg, about 200μg, about 201μg, about 2 02μg, about 203μg, about 204μg, about 205μg, about 206μg, about 207μg, about 208μg, about 209μg, about 210μg, about 211μg, about 212μg, about 213μg, about 214μg, about 215μg, about 216μg, about 217μg, about 218μg, about 219μg, about 220 μg, about 221 μg, about 222 μg, about 223 μg, about 224 μg, about 225 μg, about 226 μg, about 227 μg, about 228 μg, about 229 μg, about 230 μg, about 231 μg, about 232 μg, 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, about 243 μg, about 244 μg, about 245 μg, about 246 μg, about 247 μg, about 248 μg, about 249 μg, about 250 μg, about 251 μg, about 252 μg, about 253 μg, about 254 μg, about 255 μg,about 256 μg, about 257 μg, about 258 μg, about 259 μg, about 260 μg, about 261 μg, about 262 μg, about 263 μg, about 264 μg, about 265 μg, about 266 μg, about 267 μg, about 268 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 In some embodiments, the method comprises administering 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 method comprises administering about 30 μg of each HA glycoprotein. In embodiments, the method comprises administering about 33 μg of each HA glycoprotein. In embodiments, the method comprises administering about 39 μg of each HA glycoprotein. In embodiments, the method comprises administering about 54 μg of each HA glycoprotein. In embodiments, the method comprises administering about 60 μg of each HA glycoprotein. In embodiments, the method comprises administering about 240 μg of each HA glycoprotein.

[0321] In embodiments, the method comprises administering about 1 μg to about 1000 μg of total HA glycoprotein. In embodiments, the method comprises administering 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 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 71μg, about 72μg, about 73μg, about 74μg, about 75μg, about 76μg, about 77μg, about 78μg, about 79μg, about 80μg 0μg, 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, about 97μg, about 98μg, about 99μg, about 100μg, about 101μg, about 102μg, about 103μg, about 104μg, about 105μg, about 106μg, about 107μg, about 108μg, about 109μg, about 110μg, about 111μg, about 112μg, about 113μg, about 114μg, about 115μg, about 116μg, about 117 μg, about 118μg, about 119μg, about 120μg, 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, about 136μg, about 137μg, about 138μg, about 139μg, about 140μg, about 141μg, about 142μg, about 143μg, about 144μg, about 145μg, about 146μg, about 147μg, about 148μg, about 149μg, about 150μg, about 151μg,about 152 μg, about 153 μg, about 154 μg, about 155 μg, about 156 μg, about 157 μg, about 158 ​​μg, about 159 μg, about 160 μg, about 161 μg, about 162 μg, about 163 μg, about 164 μg, about 165 μg, about 166 μg, about 167 μg, about 168 μg, about 169 μg , about 170μg, about 171μg, about 172μg, about 173μg, about 174μg, about 175μg, about 176μg, about 177μg, about 178μg, about 179μg, about 180μg, about 181μg, about 182μg, about 183μg, about 184μg, about 185μg, about 186μg, about 187μg g, about 188 μg, about 189 μg, about 190 μg, about 191 μg, about 192 μg, about 193 μg, about 194 μg, about 195 μg, about 196 μg, about 197 μg, about 198 μg, about 199 μg, about 200 μg, about 201 μg, about 202 μg, about 203 μg, about 204 μg, about 205 μg, about 206 μg, about 207 μg, about 208 μg, about 209 μg, about 210 μg, about 211 μg, about 212 μg, about 213 μg, about 214 μg, about 215 μg, about 216 μg, about 217 μg, about 218 μg, about 219 μg, about 220 μg, about 221 μg, about 222 μg, about 223 μg, about 224 μg 3μg, about 224μg, about 225μg, about 226μg, about 227μg, about 228μg, about 229μg, about 230μg, about 231μg, about 232μg, about 233μg, about 234μg, about 235μg, about 236μg, about 237μg, about 238μg, about 239μg, about 240μg, about 2 41μg, about 242μg, about 243μg, about 244μg, about 245μg, about 246μg, about 247μg, about 248μg, about 249μg, about 250μg, about 251μg, about 252μg, about 253μg, about 254μg, about 255μg, about 256μg, about 257μg, about 258μg, about 259μg, about 260μg, about 261μg, about 262μg, about 263μg, about 264μg, about 265μg, about 266μg, about 267μg, about 268μg, about 269μg, about 270μg, about 271μg, about 272μg, about 273μg, about 274μg, about 275μg, about 276μg, 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, about 300 μg, about 301 μg, about 302 μg, about 303 μg, about 304 μg, about 305 μg, about 306 μg, about 307 μg, about 308 μg, about 309 μg, about 310 μg, about 311 μg, about 312 μg , 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, about 327μg, about 328μg, about 329μg, about 330μg g, about 331 μg, about 332 μg, about 333 μg, about 334 μg, about 335 μg, about 336 μg, about 337 μg, about 338 μg, about 339 μg, about 340 μg, about 341 μg, about 342 μg, about 343 μg, about 344 μg, about 345 μg, about 346 μg, about 347 μg, about 348 μg, about 349 μg, about 350 μg, about 351 μg, about 352 μg, about 353 μg, about 354 μg, about 355 μg, about 356 μg, about 357 μg, about 358 μg, about 359 μg, about 360 μg, about 361 μg, about 362 μg, about 363 μg, about 364 μg, about 365 μg, about 366 μg, about 367 μg, about 368 μg, about 369 μg, about 370 μg, about 371 μg, 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 38 6μg, about 367μg, about 368μg, about 369μg, about 370μg, about 371μg, about 372μg, about 373μg, about 374μg, about 375μg, about 376μg, about 377μg, about 378μg, about 379μg, about 380μg, about 381μg, about 382μg, about 383μg, about 3 84μg, about 385μg, about 386μg, about 387μg, about 388μg, about 389μg, about 390μg, about 391μg, about 392μg, about 393μg, about 394μg, about 395μg, about 396μg, about 397μg, about 398μg, about 399μg, about 400μg, about 401μg, about 402 μg, about 403 μg, about 404 μg, about 405 μg, about 406 μg, about 407 μg, about 408 μg, about 409 μg, about 410 μg, about 411 μg, about 412 μg, about 413 μg, about 414 μg, about 415 μg, about 416 μg, about 417 μg, about 418 μg, about 419 μg, about 420 μg, about 421 μg, about 422 μg, about 423 μg, about 424 μg, about 425 μg, about 426 μg, about 427 μg, about 428 μg, about 429 μg, about 430 μg, about 431 μg, about 432 μg, about 433 μg, about 434 μg, about 435 μg, about 436 μg, about 437 μg,about 438 μg, about 439 μg, about 440 μg, about 441 μg, about 442 μg, about 443 μg, about 444 μg, about 445 μg, about 446 μg, about 447 μg, about 448 μg, about 449 μg, about 450 μg, about 451 μg, about 452 μg, about 453 μg, about 454 μg, about 455 μg , about 456μg, about 457μg, about 458μg, about 459μg, about 460μg, about 461μg, about 462μg, about 463μg, about 464μg, about 465μg, about 466μg, about 467μg, about 468μg, about 469μg, about 470μg, about 471μg, about 472μg, about 473μg g, about 474 μg, about 475 μg, about 476 μg, about 477 μg, about 478 μg, about 479 μg, about 480 μg, about 481 μg, about 482 μg, about 483 μg, about 484 μg, about 485 μg, about 486 μg, about 487 μg, about 488 μg, about 489 μg, about 490 μg, about 491 μg, about 492 μg, about 493 μg, about 494 μg, about 495 μg, about 496 μg, about 497 μg, about 498 μg, about 499 μg, about 500 μg, about 501 μg, about 502 μg, about 503 μg, about 504 μg, about 505 μg, about 506 μg, about 507 μg, about 508 μg, about 50 9μg, about 510μg, about 511μg, about 512μg, about 513μg, about 514μg, about 515μg, about 516μg, about 517μg, about 518μg, about 519μg, about 520μg, about 521μg, about 522μg, about 523μg, about 524μg, about 525μg, about 526μg, about 5 27μg, about 528μg, about 529μg, about 530μg, about 531μg, about 532μg, about 533μg, about 534μg, about 535μg, about 536μg, about 537μg, about 538μg, about 539μg, about 540μg, about 541μg, about 542μg, about 543μg, about 544μg, about 545μg, about 546μg, about 547μg, about 548μg, about 549μg, about 550μg, about 551μg, about 552μg, about 553μg, about 554μg, about 555μg, about 556μg, about 557μg, about 558μg, about 559μg, about 560μg, about 561μg, about 562μg, about 563 μg, about 564 μg, about 565 μg, about 566 μg, about 567 μg, about 568 μg, about 569 μg, about 570 μg, about 571 μg, about 572 μg, about 573 μg, about 574 μg, about 575 μg, about 576 μg, about 577 μg, about 578 μg, about 579 μg, about 580 μg,about 581 μg, about 582 μg, about 583 μg, about 584 μg, about 585 μg, about 586 μg, about 587 μg, about 588 μg, about 589 μg, about 590 μg, about 591 μg, about 592 μg, about 593 μg, about 594 μg, about 595 μg, about 596 μg, about 597 μg, about 598 μg , about 599 μg, about 600 μg, about 601 μg, about 602 μg, about 603 μg, about 604 μg, about 605 μg, about 606 μg, about 607 μg, about 608 μg, about 609 μg, about 610 μg, about 611 μg, about 612 μg, about 613 μg, about 614 μg, about 615 μg, about 616 μg g, about 617 μg, about 618 μg, about 619 μg, about 620 μg, about 621 μg, about 622 μg, about 623 μg, about 624 μg, about 625 μg, about 626 μg, about 627 μg, about 628 μg, about 629 μg, about 630 μg, about 631 μg, about 632 μg, about 633 μg, about 634 μg, about 635 μg, about 636 μg, about 637 μg, about 638 μg, about 639 μg, about 640 μg, about 641 μg, about 642 μg, about 643 μg, about 644 μg, about 645 μg, about 646 μg, about 647 μg, about 648 μg, about 649 μg, about 650 μg, about 651 μg, about 65 2μg, about 653μg, about 654μg, about 655μg, about 656μg, about 657μg, about 658μg, about 659μg, about 660μg, about 661μg, about 662μg, about 663μg, about 664μg, about 665μg, about 666μg, about 667μg, about 668μg, about 669μg, about 6 70μg, about 671μg, about 672μg, about 673μg, about 674μg, about 675μg, about 676μg, about 677μg, about 678μg, about 679μg, about 680μg, about 681μg, about 682μg, about 683μg, about 684μg, about 685μg, about 686μg, about 687μg, about 688μg, about 689μg, about 690μg, about 691μg, about 692μg, about 693μg, about 694μg, about 695μg, about 696μg, about 697μg, about 698μg, about 699μg, about 700μg, about 701μg, about 702μg, about 703μg, about 704μg, about 705μg, about 706 μg, about 707 μg, about 708 μg, about 709 μg, about 710 μg, about 711 μg, about 712 μg, about 713 μg, about 714 μg, about 715 μg, about 716 μg, about 717 μg, about 718 μg, about 719 μg, about 720 μg, about 721 μg, about 722 μg, about 723 μg,about 724 μg, about 725 μg, about 726 μg, about 727 μg, about 728 μg, about 729 μg, about 730 μg, about 731 μg, about 732 μg, about 733 μg, about 734 μg, about 735 μg, about 736 μg, about 737 μg, about 738 μg, about 739 μg, about 740 μg, about 741 μg , about 742μg, about 743μg, about 744μg, about 745μg, about 746μg, about 747μg, about 748μg, about 749μg, about 750μg, about 751μg, about 752μg, about 753μg, about 754μg, about 755μg, about 756μg, about 757μg, about 758μg, about 759μ g, about 760 μg, about 761 μg, about 762 μg, about 763 μg, about 764 μg, about 765 μg, about 766 μg, about 767 μg, about 768 μg, about 769 μg, about 770 μg, about 771 μg, about 772 μg, about 773 μg, about 774 μg, about 775 μg, about 776 μg, about 777 μg, about 778 μg, about 779 μg, about 780 μg, about 781 μg, about 782 μg, about 783 μg, about 784 μg, about 785 μg, about 786 μg, about 787 μg, about 788 μg, about 789 μg, about 790 μg, about 791 μg, about 792 μg, about 793 μg, about 794 μg, about 796 μg, about 797 μg, about 798 μg, about 799 μg, about 791 μg, about 792 μg, about 793 μg, about 794 ... 5μg, about 796μg, about 797μg, about 798μg, about 799μg, about 800μg, about 801μg, about 802μg, about 803μg, about 804μg, about 805μg, about 806μg, about 807μg, about 808μg, about 809μg, about 810μg, about 811μg, about 812μg, about 8 13μg, about 814μg, about 815μg, about 816μg, about 817μg, about 818μg, about 819μg, about 820μg, about 821μg, about 822μg, about 823μg, about 824μg, about 825μg, about 826μg, about 827μg, about 828μg, about 829μg, about 830μg, about 831 μg, about 832 μg, about 833 μg, about 834 μg, about 835 μg, about 836 μg, about 837 μg, about 838 μg, about 839 μg, about 840 μg, about 841 μg, about 842 μg, about 843 μg, about 844 μg, about 845 μg, about 846 μg, about 847 μg, about 848 μg, about 849 μg, about 850 μg, about 851 μg, about 852 μg, about 853 μg, about 854 μg, about 855 μg, about 856 μg, about 857 μg, about 858 μg, about 859 μg, about 860 μg, about 861 μg, about 862 μg, about 863 μg, about 864 μg, about 865 μg, about 866 μg,About 867μg, about 868μg, about 869μg, about 870μg, about 871μg, about 872μg, about 873μg, about 874μg, about 875μg, about 876μg, about 877μg, about 878μg, about 879μg, about 880μg, about 881μg, about 882μg, about 883μg, about 884μg, about 885μg, about 886μg, about 887μg, about 888μg, about 889μg, about 890μg, about 891μg, about 892μg, about 893μg, about 894μg, about 895μg, about 896μg, about 897μg, about 898μg, about 899μg, about 900μg, about 901μg, about 902μg, about 903μg, about 904μg, about 905μg, about 906μg, about 907μg, about 908μg, about 909μg, about 910μg, 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 936μg 5μg, 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 958μg, about 959μg, about 960μg, about 961μg, about 962μg, about 963μg, about 964μg, about 965μg, about 966μg, about 967μg, about 968μg, about 969 , about 970 μg, about 971 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 HA glycoprotein, including all values ​​and ranges therebetween.

[0322] In embodiments, the method comprises 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 method comprises administering 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 g, 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 7 3μg, 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, about 97μg, about 98μg, about 99μg, about 100μg, about 101μg, about 102μg, about 103μg, about 104μg, about 105μg, about 106μg, about 107μg, about 108 μg, 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 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, about 136μg, about 137μg, about 138μg, about 139μg,about 140 μg, about 141 μg, about 142 μg, about 143 μg, about 144 μg, about 145 μg, about 146 μg, about 147 μg, about 148 μg, about 149 μg, about 150 μg, about 151 μg, about 152 μg, about 153 μg, about 154 μg, about 155 μg, about 156 μg, about 157 μg , about 158μg, about 159μg, about 160μg, about 161μg, about 162μg, about 163μg, about 164μg, about 165μg, about 166μg, about 167μg, about 168μg, about 169μg, about 170μg, about 171μg, about 172μg, about 173μg, about 174μg, about 175μ g, about 176 μg, about 177 μg, about 178 μg, about 179 μg, about 180 μg, about 181 μg, about 182 μg, about 183 μg, about 184 μg, about 185 μg, about 186 μg, about 187 μg, about 188 μg, about 189 μg, about 190 μg, about 191 μg, about 192 μg, about 193 μg, about 194 μg, about 195 μg, about 196 μg, about 197 μg, about 198 μg, about 199 μg, about 200 μg, about 201 μg, about 202 μg, about 203 μg, about 204 μg, about 205 μg, about 206 μg, about 207 μg, about 208 μg, about 209 μg, about 210 μg, about 21 1μg, about 212μg, about 213μg, about 214μg, about 215μg, about 216μg, about 217μg, about 218μg, about 219μg, about 220μg, about 221μg, about 222μg, about 223μg, about 224μg, about 225μg, about 226μg, about 227μg, about 228μg, about 2 29μg, about 230μg, about 231μg, about 232μg, about 233μg, about 234μg, about 235μg, about 236μg, about 237μg, about 238μg, about 239μg, about 240μg, about 241μg, about 242μg, about 243μg, about 244μg, about 245μg, about 246μg, about 247μg, about 248μg, about 249μg, about 250μg, about 251μg, about 252μg, about 253μg, about 254μg, about 255μg, about 256μg, about 257μg, about 258μg, about 259μg, about 260μg, about 261μg, about 262μg, about 263μg, about 264μg, about 265 μg, about 266 μg, about 267 μg, about 268 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, about 300 μg, about 301 μg, about 302 μg, about 303 μg, about 304 μg, about 305 μg, about 306 μg, about 307 μg, about 308 μg, about 309 μg, about 310 μg, about 311 μg, about 312 μg g, about 313 μg, about 314 μg, about 315 μg, about 316 μg, about 317 μg, about 318 μg, about 319 μg, about 320 μg, about 321 μg, about 322 μg, about 323 μg, about 324 μg, about 325 μg, about 326 μg, about 327 μg, about 328 μg, about 329 μg, about 330 μg, about 331 μg, about 332 μg, about 333 μg, about 334 μg, about 335 μg, about 336 μg, about 337 μg, about 338 μg, about 339 μg, about 340 μg, about 341 μg, about 342 μg, 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 373μg, about 374μg or about 400 μg of each viral glycoprotein.

[0323] In embodiments, the method comprises 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 an embodiment, 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 35 μg, about 36 μg, 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 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, 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, about 106μg, about 107μg, about 108μg 8μg, 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 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, about 136μg, about 137μg, about 138μg, about 139μg,about 140 μg, about 141 μg, about 142 μg, about 143 μg, about 144 μg, about 145 μg, about 146 μg, about 147 μg, about 148 μg, about 149 μg, about 150 μg, about 151 μg, about 152 μg, about 153 μg, about 154 μg, about 155 μg, about 156 μg, about 157 μg , about 158μg, about 159μg, about 160μg, about 161μg, about 162μg, about 163μg, about 164μg, about 165μg, about 166μg, about 167μg, about 168μg, about 169μg, about 170μg, about 171μg, about 172μg, about 173μg, about 174μg, about 175μ g, about 176 μg, about 177 μg, about 178 μg, about 179 μg, about 180 μg, about 181 μg, about 182 μg, about 183 μg, about 184 μg, about 185 μg, about 186 μg, about 187 μg, about 188 μg, about 189 μg, about 190 μg, about 191 μg, about 192 μg, about 193 μg, about 194 μg, about 195 μg, about 196 μg, about 197 μg, about 198 μg, about 199 μg, about 200 μg, about 201 μg, about 202 μg, about 203 μg, about 204 μg, about 205 μg, about 206 μg, about 207 μg, about 208 μg, about 209 μg, about 210 μg, about 21 1μg, about 212μg, about 213μg, about 214μg, about 215μg, about 216μg, about 217μg, about 218μg, about 219μg, about 220μg, about 221μg, about 222μg, about 223μg, about 224μg, about 225μg, about 226μg, about 227μg, about 228μg, about 2 29μg, about 230μg, about 231μg, about 232μg, about 233μg, about 234μg, about 235μg, about 236μg, about 237μg, about 238μg, about 239μg, about 240μg, about 241μg, about 242μg, about 243μg, about 244μg, about 245μg, about 246μg, about 247μg, about 248μg, about 249μg, about 250μg, about 251μg, about 252μg, about 253μg, about 254μg, about 255μg, about 256μg, about 257μg, about 258μg, about 259μg, about 260μg, about 261μg, about 262μg, about 263μg, about 264μg, about 265 μg, about 266 μg, about 267 μg, about 268 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, about 300 μg, about 301 μg, about 302 μg, about 303 μg, about 304 μg, about 305 μg, about 306 μg, about 307 μg, about 308 μg, about 309 μg, about 310 μg, about 311 μg, about 312 μg, about 313 μg, about 314 μg, about 315 μg, about 316 μg, about 317 μg, about 318 μg, about 319 μg, about 320 μg, about 321 μg, about 322 μg, about 323 μg, about 324 μg, about 325 μg, about 326 μg, about 327 μg, about 328 μg, about 329 μg, about 330 μg, about 331 μg, about 332 μg, about 333 μg, about 334 μg, about 335 μg, about 336 μg, about 337 μg, about 338 μg, about 339 μg, about 340 μg, about 341 μg, 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 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 In some embodiments, the method comprises administering about 50 μg of adjuvant. In some embodiments, the method comprises administering about 75 μg of adjuvant.

[0324] In another embodiment, the present disclosure provides a method for formulating a vaccine composition that induces immunity to an infection or at least one disease symptom thereof in a mammal, the method comprising adding an effective dose of RSV F glycoprotein or RSV F glycoprotein nanoparticles to the composition. The disclosed RSV F glycoprotein and RSV F glycoprotein nanoparticles can be used to prepare compositions that stimulate an immune response that confers immunity or substantial immunity to an infectious agent. Therefore, in one embodiment, the present disclosure provides a method for inducing immunity to an infection or at least one disease symptom thereof in a subject, the method comprising administering at least one effective dose of RSV F glycoprotein or RSV F glycoprotein nanoparticles.

[0325] In an embodiment, the present disclosure provides a method for producing one or more high-affinity anti-RSV antibodies. The high-affinity antibodies produced by immunizing with the nanoparticles disclosed herein are produced by administering an immunogenic composition comprising RSV F glycoprotein or RSV F glycoprotein nanoparticles to an animal, collecting serum and / or plasma from the animal, and purifying the antibodies from the serum and / or plasma. In one embodiment, the animal is a human. In an embodiment, the animal is a chicken, mouse, guinea pig, rat, rabbit, goat, human, horse, sheep or cow. In one embodiment, the animal is a cow or horse. In another embodiment, the cow or horse animal is transgenic. In another embodiment, the transgenic cow or horse animal produces human antibodies. In an embodiment, the animal produces monoclonal antibodies. In an embodiment, the animal produces polyclonal antibodies. In one embodiment, the method further comprises administering an adjuvant or an immunostimulatory compound. In another embodiment, the purified high-affinity antibodies are administered to a human subject. In one embodiment, the human subject is at risk of infection with RSV.

[0326] In embodiments, the composition is administered intranasally. In embodiments, intranasal administration of the composition comprises administering drops of the composition directly to the nasal epithelium. In embodiments, the method comprises administering 1 to 100, 1 to 90, 1 to 80, 1 to 70, 1 to 60, 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 10, or 1 to 5 drops of the immunogenic composition intranasally. In embodiments, the drops are delivered via an intranasal delivery device described herein.

[0327] In embodiments, the method comprises administering an aerosol of an immunogenic composition as described herein to the nasal epithelium. In embodiments, the aerosol is a liquid aerosol. In embodiments, the aerosol is a powder aerosol. The powder aerosol comprises a dry powder composition. In embodiments, the method comprises administering 1 to 100, 1 to 90, 1 to 80, 1 to 70, 1 to 60, 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 10, or 1 to 5 puffs of an aerosol intranasally. In embodiments, the aerosol is delivered by an intranasal delivery device. Intranasal delivery devices are described in International Publication No. 2012 / 105236 and U.S. Patent No. 10,441,436, which are incorporated herein by reference in their entirety for all purposes. The intranasal delivery device can also be a prefilled syringe, as described herein.

[0328] In embodiments, the method comprises administering the intranasal immunogenic composition in the form of a solution. In embodiments, the method comprises administering the intranasal immunogenic composition in the form of a suspension.

[0329] In embodiments, the method comprises intranasally administering a spray or puff of the intranasal immunogenic composition to the nostrils of the patient. In embodiments, the method comprises intranasally administering from 1 to about 100 sprays or puffs of the intranasal immunogenic composition to the nostrils of the patient. In embodiments, the method comprises adding 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 In some embodiments, the spray or powder of the intranasal immunogenic composition is administered intranasally to one or more nostrils of the patient. In some embodiments, the spray or powder is delivered to each nostril of the patient.

[0330] In embodiments, the methods described herein allow the immunogenic composition to reach the lungs of a patient. In embodiments, the methods described herein allow the immunogenic composition to reach the upper respiratory tract of a patient. In embodiments, the methods described herein allow the immunogenic composition to reach the lower respiratory tract of a patient.

[0331] In embodiments, the method comprises intranasal administration of 0.05 mL to about 1 mL of an intranasal immunogenic composition as described herein. In embodiments, the method comprises intranasal administration of about 0.05 mL, about 0.1 mL, about 0.2 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 or about 1 mL of an intranasal immunogenic composition as described herein, including all values ​​and ranges therebetween. In embodiments, the method comprises intranasal administration of 0.2 mL of an intranasal immunogenic composition as described herein. In embodiments, the method comprises intranasal administration of 0.5 mL of an intranasal immunogenic composition as described herein. In embodiments, the method comprises intranasal administration of 0.25 mL of an intranasal immunogenic composition as described herein to each nostril of the patient.

[0332] In embodiments, provided herein are methods for producing IgA and / or IgG antibodies against SARS-CoV-2, influenza, and / or RSV in a subject. In embodiments, the antibodies are neutralizing antibodies. In embodiments, provided herein are methods for inducing mucosal immunity against SARS-CoV-2, influenza, RSV, or a combination thereof in a patient, the methods comprising intranasally administering an immunogenic composition as described herein. In embodiments, the antibodies are located in the bronchoalveolar lavage fluid of the subject.

[0333] In some embodiments, the present disclosure provides a co-formulation (i.e., prefilled syringe or premix) strategy for an immunogenic composition comprising RSV F glycoprotein and an adjuvant (e.g., a saponin adjuvant). The typical vaccine administration strategy currently in use is a bedside mixed formulation. That is, the vaccine composition and adjuvant are stored separately and mixed before administration. Due to concerns about the stability of the antigen (e.g., RSV F glycoprotein) and its subsequent immunogenicity, a premix, co-formulation, or prefilled syringe strategy for vaccines is less common. The present disclosure provides an immunogenic composition that can be pre-mixed and stored in advance. The disclosed vaccination strategy and formulation can improve the efficiency of vaccination and can reduce the risk of bedside mixing errors while maintaining overall safety and immunogenicity.

[0334] Various containers can be used to store and transport premixed formulations, including syringes and plastic ampoules for single administration. In some cases, plastic ampoules can be manufactured using blow-fill-seal manufacturing technology or methods. Generally speaking, the blow-fill-seal (BFS) manufacturing method includes extruding a plastic material (e.g., a resin) to form a parison, which is then placed in a mold and cut to the appropriate size. The plastic is then inflated using a filling needle or mandrel to form a hollow ampoule that substantially conforms to the mold shape. Once inflated, the desired amount of liquid can be injected into the ampoule, the filling needle or mandrel can be removed, and the ampoule can be sealed. Therefore, BFS can be an automated process that can be performed in a sterile environment without direct human intervention.

[0335] In some cases, the ability to aseptically manufacture sterile ampoules containing the desired liquid makes BFS-manufactured ampoules particularly well-suited for the pharmaceutical industry. However, BFS technology is not compatible with all pharmaceutical liquids, products, and the like. For example, some known BFS manufacturing methods involve delivering the liquid or product into the ampoule while the plastic is still relatively hot, which can adversely affect temperature-sensitive liquids and / or products (such as vaccines and biologics). However, advances in cold BFS technology have expanded the range of applicable products, liquids, and the like, enabling some vaccines, biologics, and / or other temperature-sensitive medications to be contained in BFS ampoules.

[0336] In some cases, a BFS ampoule may have a size, shape, and / or configuration based at least in part on its intended use and / or the desired medicinal liquid or dosage that the ampoule is configured to contain. For example, some known BFS ampoules may include a pierced top, a twist-off top, a top comprising a male or female Luer, etc. Some known BFS ampoules may have a size and / or shape based on the volume of the liquid or dosage configured to be placed therein. Furthermore, some known BFS ampoules may be manufactured as a strip of multiple temporarily connected ampoules, which may improve manufacturing, packaging, and / or storage efficiencies, among other things.

[0337] In embodiments, the immunogenic compositions described herein are provided in the form of prefilled syringes. When the immunogenic composition is prepared in a prefilled syringe, the antigen and adjuvant are combined before administration. In embodiments, the prefilled syringe contains hemagglutinin, SARS-CoV-2 S glycoprotein, RSV F glycoprotein, or a combination thereof. In embodiments, the immunogenic composition is administered intranasally to a subject from a prefilled syringe.

[0338] In embodiments, the prefilled syringe contains RSV F glycoprotein, HA glycoprotein, CoV S glycoprotein, or a combination thereof. In embodiments, the adjuvant is a saponin adjuvant. In embodiments, the saponin adjuvant comprises at least two iscom particles, wherein a first iscom particle comprises Quillaja Fraction A but not Quillaja Fraction C; and a second iscom particle comprises Quillaja Fraction C but not Quillaja Fraction A; wherein Quillaja Fraction A and Quillaja Fraction C comprise approximately 85% and approximately 15% by weight, respectively, of the combined weight of Quillaja Fraction A and Quillaja Fraction C in the adjuvant. In embodiments, the prefilled syringe contains RSV F glycoprotein and a saponin adjuvant, wherein the adjuvant comprises at least two iscom particles, wherein a first iscom particle comprises Quillaja Fraction A but does not comprise Quillaja Fraction C; and a second iscom particle comprises Quillaja Fraction C but does not comprise Quillaja Fraction A; wherein Quillaja Fraction A and Quillaja Fraction C comprise approximately 92% and approximately 8% by weight, respectively, of the total weight of Quillaja Fraction A and Quillaja Fraction C in the adjuvant. In embodiments, the prefilled syringe contains RSV F glycoprotein and a saponin adjuvant, wherein the adjuvant comprises at least two iscom particles, wherein a first iscom particle comprises Quillaja Fraction A but does not comprise Quillaja Fraction C; and a second iscom particle comprises Quillaja Fraction C but does not comprise Quillaja Fraction A; wherein Quillaja Fraction A comprises at least about 75% by weight of the sum of the weight of Quillaja Fraction A and Quillaja Fraction C in the adjuvant, and Quillaja Fraction C comprises the remainder of the sum of the weight of Quillaja Fraction A and Quillaja Fraction C in the adjuvant. Example

[0339] Example 1

[0340] Expression and purification of RSV F polypeptide nanoparticles

[0341] Native RSV F glycoprotein (SEQ ID NO: 329 and SEQ ID NO: 330) and RSV F glycoprotein with the amino acid sequence corresponding to SEQ ID NO: 329-336 were expressed in a baculovirus expression system, and recombinant plaques expressing RSV F glycoprotein were selected and confirmed. In each case, the signal peptide was SEQ ID NO: 360.

[0342] Table A: Selected RSV F glycoproteins

[0343]

[0344] Protein and nanoparticle production

[0345] Recombinant virus is amplified by infection of Sf9 insect cells. Insect cell culture is infected with baculovirus at about 0.6MOI (multiplicity of infection=virus ffu or pfu / cell). Culture and supernatant are harvested 48-72 hours after infection. Crude cell harvest (about 30mL) is clarified by centrifugation at about 800x g for 15 minutes. The crude cell harvest containing RSV F glycoprotein is purified into nanoparticles as described below.

[0346] To produce nanoparticles, use nonionic surfactants in membrane protein extraction protocols Nonylphenol ethoxylate NP-9. The crude extract was further purified by passing it through anion exchange chromatography, lentil lectin affinity / HIC, and cation exchange chromatography. The washed cells were lysed by detergent treatment and then subjected to a low pH treatment to precipitate BV and Sf9 host cell DNA and proteins. The neutralized low pH treated lysate was clarified and further purified by anion exchange and affinity chromatography before a second low pH treatment.

[0347] Affinity chromatography is used to remove Sf9 / BV protein, DNA and NP-9, and to concentrate RSV F glycoprotein. In short, Lentil Lectin is a metalloprotein containing calcium and manganese that reversibly binds to polysaccharides and glycosylated proteins containing glucose or mannose. The anion exchange flow-through fraction containing RSV F glycoprotein is loaded onto Lentil Lectin affinity chromatography resin (Capto Lentil Lectin, GE Healthcare). Glycosylated RSV F protein selectively binds to the resin, while non-glycosylated proteins and DNA are removed during column flow. Weakly bound glycoproteins are removed by a buffer containing high salt and a low molar concentration of methyl α-D-mannopyranoside (MMP).

[0348] Column cleaning solution is also used to exchange NP-9 detergent with surfactant polysorbate 80 (PS80). RSV F glycoprotein is eluted from a lentil lectin column with a high concentration of MMP as a nanoparticle structure. After elution, RSV F glycoprotein is assembled into nanoparticles consisting of RSV F glycoprotein trimer and PS80 contained in the detergent core.

[0349] Example 2

[0350] Immunogenicity of RSV F glycoprotein nanoparticle vaccine in cotton rats

[0351] Methods: RSV F glycoprotein nanoparticles were produced as described in Example 1. Cotton rats were immunized intramuscularly (IM) with the compositions of Table B. Each composition contained 0.01-0.0001 μg RSV F glycoprotein and a saponin adjuvant. The saponin adjuvant contained 85% w / w Fraction A ISCOM matrix and 15% w / w Fraction C ISCOM matrix. Each composition contained 20 μg of saponin adjuvant.

[0352] Table B. Composition

[0353] Composition RSV F glycoprotein adjuvant BV1184 BV1184 (SEQ ID NO: 332) Saponin adjuvant BV2279 BV2279 (SEQ ID NO: 333) 0.01-0.0001 μg

[0354] Animals were immunized on days 0 and 21. Blood was collected for immunogenicity evaluation on days 0, 21, and 42. Animals were challenged intranasally with 10<5> pfu RSV A / Long on day 42, and tissues were collected four days later.

[0355] Immune responses to RSV F compositions were measured by anti-RSV F IgG ELISA and RSV A / RSV B neutralization assay. Protective efficacy was evaluated by measuring lung and nasal tissue virus titers using the RSV plaque assay in HEp2 cells.

[0356] Neutralization assays and pre-fusion F specificity of antibodies were determined by Octet QK384 competition binning assays using antigenic site-specific antibodies against pre- and post-fusion epitopes. F protein bound to an amine-reactive biosensor tip was presented in successive association and competition steps with polyclonal antibodies in serum and competing mAbs against RSV F sites II, IV, Φ, V, and p27. Competitive antibody equivalents (CAEs) were calculated based on percentage competition, competing antibody concentration, and serum dilution.

[0357] Protective efficacy was evaluated by measuring lung and nasal tissue virus titers using the RSV plaque assay in HEp2 cells.

[0358] Results: Octet kinetic analysis of antigenic site-specific monoclonal antibodies confirmed that all prefusion and postfusion antigenic sites and neutralizing epitopes on the RSV F glycoprotein were intact in the BV2279 glycoprotein. Immunization with BV1184 or BV2279 elicited similar anti-F IgG responses and RSV A / B neutralization responses. Immunization with BV2279 generated stronger antibody responses against sites Φ, IV, and VIII compared with immunization with BV1184. The BV2279 nanoparticle vaccine provided bactericidal immunity in the lungs at a dose of 0.01 μg and reduced viral load in the upper respiratory tract of cotton rats after live RSV challenge. In contrast, the BV1184 vaccine did not reduce vaccine load in the upper respiratory tract. Together, these data demonstrate that the BV2279 vaccine is a potent immunogen that elicits strong RSV F-specific immune responses against prefusion and postfusion F epitopes.

[0359] The table below shows that the BV2279 protein retains the epitopes before and after fusion.

[0360]

[0361] Figure 1 Binding of the BV2279 glycoprotein to antibodies recognizing site II (palivizumab), site IV (RSV.42, referred to as "RSV.42.2"), site Φ (D25), and site VIII (hRSV90) epitopes is shown. Figure 2 Shown are anti-RSV F IgG titers in cotton rat sera 42 days after immunization with the BV2279 (labeled "prefusion RSV F Cav1") or BV1184 (labeled "prefusogenic RSV F") vaccine compositions of Example 2. Figure 3 Shown are RSV neutralization titers against RSV A (Long) and RSV B (18537) strains following immunization with the BV2279 (labeled "prefusion RSV F Cav1") or BV1184 (labeled "prefusion RSV F") vaccine compositions of Example 2. Figure 4 Shown are the competing antibody equivalent titers induced by immunization with the BV2279 (labeled "prefusion RSV F Cav1") or BV1184 (labeled "prefusion RSV F") vaccine compositions of Example 2. Figure 5 RSV virus titers in cotton rat tissues four days after viral challenge are shown. The rats were immunized with the vaccine compositions of Example 2, BV2279 (labeled "prefusion RSV F Cav1") or BV1184 (labeled "prefusion RSV F").

[0362] Incorporated by reference

[0363] All references, articles, publications, patents, patent publications, and patent applications cited herein are incorporated herein by reference in their entirety for all purposes. However, the reference to any reference, article, publication, patent, patent publication, and patent application cited herein is not and should not be taken as an admission or any form of suggestion that they constitute valid prior art or form part of the common general knowledge in any country in the world. The following patent documents are incorporated herein by reference in their entirety for all purposes: International Publication No. 2004 / 004762 and International Publication No. 2017 / 041100.

Claims

1. A respiratory syncytial virus (RSV) fusion (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) deletion of one or more of amino acids 137-146; (b) inactivated major furin cleavage site; (c) inactivated minor furin cleavage site; (d) S155C; (e) S290C; (f) S190F; (g) V207L; and (h)N116Q; The modifications are numbered according to the RSV F glycoprotein of SEQ ID NO:

330.

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 major furin cleavage site.

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

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

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

342.

6. The RSV F glycoprotein of any one of claims 1-5, wherein the RSV F glycoprotein comprises S155C and S290C mutations.

7. The RSV F glycoprotein of any one of claims 1-5, wherein the RSV F glycoprotein comprises S190F and V207L ​​mutations.

8. The RSV F glycoprotein of any one of claims 1-5, wherein the RSV F glycoprotein comprises S155C, S290C, S190F, and V207L ​​mutations.

9. The RSV F glycoprotein of any one of claims 1-8, wherein the RSV F glycoprotein comprises an N116Q mutation.

10. The RSV F glycoprotein of any one of claims 1-9, wherein the RSV F glycoprotein comprises an inactivated major 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.

11. The RSV F glycoprotein of any one of claims 1-9, wherein the RSV F glycoprotein comprises an inactivated major 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 is 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.

12. The RSV F glycoprotein of any one of claims 1-11, wherein the RSV F glycoprotein comprises: (i) an F1 domain having an amino acid sequence that is 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 that is 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. The RSV F glycoprotein of any one of claims 1-12, wherein the Fl 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 Fl domain of any one of SEQ ID NOs: 350-355.

14. The RSV F glycoprotein of any one of claims 1-13, 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. 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. The RSV F glycoprotein of any one of claims 1-10, wherein 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: 364-366.

17. The RSV F glycoprotein of any one of claims 1-10, wherein 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:

364.

18. The RSV F glycoprotein of any one of claims 1-10, wherein 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.

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

20. The RSV F glycoprotein of any one of claims 1-19, wherein the RSV F glycoprotein comprises a signal peptide.

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

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

24. A nanoparticle comprising the RSV F glycoprotein of any one of claims 1-21 and a nonionic detergent core.

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

26. The nanoparticle of claim 24, wherein the nonionic detergent is PS80.

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

28. The cell of claim 27, wherein the cell is an insect cell.

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

30. The immunogenic composition of claim 29, comprising one, two, three, four, five, six, seven, eight, nine, or ten different RSV F glycoproteins.

31. The immunogenic composition of any one of claims 29-30, comprising (i) a first RSV F glycoprotein that 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: 337-340; and (ii) a second RSV F glycoprotein that 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.

32. The immunogenic composition of any one of claims 29-30, comprising (i) a first RSV F glycoprotein that 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; and (ii) a second RSV F glycoprotein that 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 NO:

364.

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

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

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

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

37. The immunogenic composition of any one of claims 29-36, comprising 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 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 13, about 4 to 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 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 10, 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 13, about 9 to about 12, about 9 to about 11, about 9 to 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,from about 11 to about 20, from about 11 to about 19, from about 11 to about 18, from about 11 to about 17, from about 11 to about 16, from about 11 to about 15, from about 11 to about 14, from about 11 to about 13, from about 11 to about 12, from about 12 to about 20, from about 12 to about 19, from about 12 to about 18, from about 12 to about 17, from about 12 to about 16, from about 12 to about 15, from about 12 to about 14, from about 12 to about 13, from about 13 to about 20, from about 13 to about 19, from about 13 to about 18, from about 13 to about 17, from about 13 to about 16, from about 13 to about 15, 13 to about 14, about 14 to about 20, about 14 to about 19, about 14 to about 18, 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. The immunogenic composition of claim 36, wherein the at least one viral glycoprotein that is not RSV F glycoprotein is influenza hemagglutinin (HA) glycoprotein or SARS-CoV-2 spike (CoV S) glycoprotein.

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

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

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

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

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

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

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

46. ​​The immunogenic composition of claim 43, comprising: (i) a detergent core nanoparticle comprising the first HA glycoprotein and a non-ionic detergent; (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.

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

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

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

50. The immunogenic composition of any one of claims 44-48, wherein the HA glycoprotein of the detergent core nanoparticle is from an influenza B strain.

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

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

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

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

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

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, wherein the SARS-CoV-2 glycoprotein is numbered according to the polypeptide of SEQ ID NO:

2.

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

59. The immunogenic composition of any one of claims 55-58, wherein the CoV S 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 SARS-CoV-2 S glycoprotein of SEQ ID NO:

87.

60. The immunogenic composition of any one of claims 55-58, wherein the CoV S 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 SARS-CoV-2 S glycoprotein of SEQ ID NO:

274.

61. The immunogenic composition of any one of claims 55-58, wherein the CoV S glycoprotein is identical to the CoV S glycoprotein of SEQ ID NO: 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, The polypeptide of any of 278, 280, 284, 288, 292, 296, 300, 304, 308, 312, 316, 320, 324, and 328 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 at least 100% identity.

62. The immunogenic composition of any one of claims 29 to 61, comprising: (i) a first CoV S glycoprotein comprising (a) an inactive furin cleavage site, and (b) prolines at amino acid positions 973 and 974, wherein the SARS-CoV-2 glycoprotein is numbered according to the polypeptide of SEQ ID NO: 2; and (ii) a second CoV S glycoprotein comprising (a) an inactive furin cleavage site, and (b) prolines at amino acid positions 973 and 974, wherein the SARS-CoV-2 glycoprotein is numbered according to 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 the first CoV S glycoprotein.

63. The immunogenic composition of any one of claims 29 to 61, comprising: (i) a first CoV S glycoprotein comprising (a) an inactive furin cleavage site, and (b) prolines at amino acid positions 973 and 974, wherein the SARS-CoV-2 glycoprotein is numbered according to 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, wherein the SARS-CoV-2 glycoprotein is numbered according to 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 the first CoV 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, wherein the SARS-CoV-2 glycoprotein is numbered according to 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 the first CoV S glycoprotein.

64. The immunogenic composition of any one of claims 29 to 61, comprising: (i) a first CoV S glycoprotein comprising (a) an inactive furin cleavage site, and (b) prolines at amino acid positions 973 and 974, wherein the SARS-CoV-2 glycoprotein is numbered according to 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, wherein the SARS-CoV-2 glycoprotein is numbered according to 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 the first CoV 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, wherein the SARS-CoV-2 glycoprotein is numbered according to 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 the first CoV 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, wherein the SARS-CoV-2 glycoprotein is numbered according to 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 the first CoV S glycoprotein.

65. The immunogenic composition of any one of claims 29 to 61, comprising: (i) a first CoV S glycoprotein comprising (a) an inactive furin cleavage site, and (b) prolines at amino acid positions 973 and 974, wherein the SARS-CoV-2 glycoprotein is numbered according to 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, wherein the SARS-CoV-2 glycoprotein is numbered according to 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 the first CoV 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, wherein the SARS-CoV-2 glycoprotein is numbered according to 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 the first CoV 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, wherein the SARS-CoV-2 glycoprotein is numbered according to 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 the first CoV S glycoprotein; and (v) a fifth CoV S glycoprotein comprising (a) an inactive furin cleavage site, and (b) prolines at amino acid positions 973 and 974, wherein the SARS-CoV-2 glycoprotein is numbered according to 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 the first CoV S glycoprotein.

66. The immunogenic composition of any one of claims 29-65, wherein the first CoV S glycoprotein is identical to the first CoV S glycoprotein of SEQ ID NO: 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, The polypeptide of any of 278, 280, 284, 288, 292, 296, 300, 304, 308, 312, 316, 320, 324, and 328 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 at least 100% identity.

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, third, fourth, and fifth CoV S glycoproteins.

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

69. The immunogenic composition of any one of claims 65-67, wherein one or more of the modifications in the second, third, fourth, and fifth CoV S glycoproteins occur in the N-terminal domain (RBD).

70. The immunogenic composition of any one of claims 65-67, wherein one or more of the modifications in the second, third, fourth, and fifth CoV S glycoproteins occur in the S1 subunit.

71. The immunogenic composition of any one of claims 65-67, wherein one or more of the modifications in the second, third, fourth, and fifth CoV S glycoproteins occur in the S2 subunit.

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

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

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

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

76. The immunogenic composition of claim 75, wherein the adjuvant comprises at least two iscom particles, wherein: the first iscom particle comprises Quillaja Fraction A and does not comprise Quillaja Fraction C; and The second iscom particles comprise fraction C of Quillaja saponaria and do not comprise fraction A of Quillaja saponaria.

77. The immunogenic composition of claim 76, wherein: Quillaja Fraction A and Quillaja Fraction C comprise about 85% and about 15% by weight, respectively, of the total weight of Quillaja Fraction A and Quillaja Fraction C in the adjuvant.

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

79. The immunogenic composition of claim 76, wherein Quillaja Fraction A comprises at least about 85% by weight of the sum of the weight of Quillaja Fraction A and Quillaja Fraction C in the adjuvant, and Quillaja Fraction C comprises the remainder of the sum of the weight of Quillaja Fraction A and Quillaja Fraction C in the adjuvant.

80. The immunogenic composition of claim 76, wherein Quillaja Fraction A comprises 50-96 weight % of the total weight of Quillaja Fraction A and Quillaja Fraction C in the adjuvant, and Quillaja Fraction C comprises the remainder of the total weight of Quillaja Fraction A and Quillaja Fraction C in the adjuvant.

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

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

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

84. The immunogenic composition of any one of claims 29-83, wherein the immunogenic composition is an intramuscular immunogenic composition.

85. The immunogenic composition of any one of claims 29-83, wherein the immunogenic composition is an intranasal immunogenic composition.

86. A prefilled syringe comprising the immunogenic composition of any one of claims 29-85.

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

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. 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 twenty-one days after the first dose.

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

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

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

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

94. The method of any one of claims 87-93, wherein the method is performed 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 22 months, At least 23 months, or at least 24 months at 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 8 The present invention provides an efficacy of about 5%, about 60% to about 80%, 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% to prevent one or more of RSV, influenza, and SARS-CoV-2.

95. The method of any one of claims 87-93, wherein the method is performed 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 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 at 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 8 The present invention provides an efficacy of about 5%, about 60% to about 80%, 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% to prevent one or more of RSV, influenza, and SARS-CoV-2.

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

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

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

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

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

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

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

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