EP4658308A2 - Rsv f vaccine formulations - Google Patents

Abstract

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

Description

RSV F VACCINE FORMULATIONS

CROSS-REFERENCE TO RELATED APPLICATIONS

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

DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY

[0002] The contents of the electronic sequence listing (NOW_104_01WO_SeqList_ST26.xml; Size: 852,414 bytes; and Date of Creation: February 3, 2024) are herein incorporated by reference in its entirety.

FIELD

[0003] The present disclosure is generally related to RSV F glycoproteins and nanoparticles useful for stimulating immune responses. The nanoparticles provide antigens, for example, glycoprotein antigens, associated with a detergent core and are typically produced using recombinant approaches. The nanoparticles have improved stability and enhanced epitope presentation. The disclosure also provides compositions containing the nanoparticles, methods for producing them, and methods of stimulating immune responses.

BACKGROUND OF THE INVENTION

[0004] Infectious diseases remain a problem throughout the world. While progress has been made on developing vaccines against some pathogens, many remain a threat to human health. Each year respiratory syncytial virus (RSV) causes 6000- 10,000 deaths among adults 65 and older and 100-300 deaths in children younger than 5 years old. The development of vaccines to prevent or reduce the severity of life-threatening infectious diseases like RSV is desirable.

SUMMARY OF THE INVENTION

[0005] The present disclosure provides non-naturally occurring respiratory syncytial virus (RSV) fusion (F) glycoproteins suitable for inducing immune responses against respiratory syncytial virus (RSV). The disclosure also provides nanoparticles containing the glycoproteins as well as methods of stimulating immune responses against RSV.

[0006] 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) 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 glycoproteins comprise S155C and S290C mutations. In embodiments, the RSV F glycoprotein comprise S190F and V207L mutations. In embodiments, the RSV F glycoproteins comprise S155C, S290C, S190F, and V207L mutations. In embodiments, the RSV F glycoproteins comprise a N116Q mutation. In embodiments, the RSV F glycoproteins comprise an inactivated primary furin cleavage site having the amino acid sequence of KKQKQQ (SEQ ID NO: 342), an S190F mutation, a V207 mutation, and deletion of amino acids 137-146. In embodiments, the RSV F glycoproteins comprise an inactivated primary furin cleavage site having the amino acid sequence of KKQKQQ (SEQ ID NO: 342), an S190F mutation, a V207 mutation, and deletion of amino acids 137-146, wherein the RSV F glycoprotein has at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 %, or 100 % identity to an RSV F glycoprotein of SEQ ID NO: 337. In embodiments, the RSV F glycoproteins comprise: (i) an F1 domain having an amino acid sequence with at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 %, or 100 % identity to the F1 domain of SEQ ID NO: 352; and (ii) an F2 domain having an amino acid sequence with at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 %, or 100 % identity to the F2 domain of SEQ ID NO: 357. In 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

[0007] Fig. 1 shows that binding of the BV2279 glycoprotein to antibodies recognizing the site II (palivizumab), site IV (RSV.42, referred to as “RSV.42.2”), site <|) (D25), and site VIII (hRSV90) epitopes.

[0008] Fig. 2 shows anti-RSV F IgG titers in cotton rat sera 42 days after immunization with either the BV2279 (labeled “prefusion RSV F Cav1”) or BV1184 (labeled “prefusogenic RSV F”) vaccine compositions of Example 2.

[0009] Fig. 3 shows the RSV neutralization titers against the RSV A (Long) and RSV B (18537) strains after immunization with either the BV2279 (labeled “prefusion RSV F Cav1”) or BV1184 (labeled “prefusogenic RSV F”) vaccine compositions of Example 2.

[0010] Fig. 4 shows competing antibody equivalent titers induced by immunization with either the BV2279 (labeled “prefusion RSV F Cav1 ”) or BV1184 (labeled “prefusogenic RSV F”) vaccine compositions of Example 2.

[0011] Fig. 5 shows the RSV viral titers in cotton rat tissue four days post viral challenge. The rats were immunized with either the BV2279 (labeled “prefusion RSV F Cav1”) or BV1184 (labeled “prefusogenic RSV F”) vaccine compositions of Example 2.

[0012] Fig. 6 shows transmission electron microscopy (TEM) images of influenza HA detergent core nanoparticles alone (left), saponin adjuvant (i.e., a first iscom particle containing Fraction A of Quillaja Saponaria Molina and not Fraction C of Quillaja Saponaria Molina, and a second iscom particle containing Fraction C of Quillaja Saponaria Molina and not Fraction A of Quillaja Saponaria Molina, wherein Fraction A accounts for 85 % of the sum of the weights of fraction A of Quillaja Saponaria Molina and fraction C of Quillaja Saponaria Molina in the adjuvant, and fraction C of Quillaja Saponaria Molina accounts for the remainder) (middle), and Hemagglutinin Saponin Matrix Nanoparticles (HaSMaNs) (right).

[0013] Fig. 7A shows the primary structure of a wild-type SARS-CoV-2 S polypeptide, containing a signal peptide, numbered with respect to SEQ ID NO: 1. Fig. 7B shows the primary structure of a wild-type SARS-CoV-2 S polypeptide, without a signal peptide, numbered with respect to SEQ ID NO: 2. DETAILED DESCRIPTION OF THE INVENTION

Definitions

[0014]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” can referto one protein or to mixtures of such protein, and reference to “the method” includes reference to equivalent steps and/or methods known to those skilled in the art, and so forth.

[0015] As used herein, the term “adjuvant” refers to a compound that, when used in combination with an immunogen, augments or otherwise alters or modifies the immune response induced against the immunogen. Modification of the immune response may include intensification or broadening the specificity of either or both antibody and cellular immune responses.

[0016] As used herein, the term “about” or “approximately” when preceding a numerical value indicates the value plus or minus a range of 10%. For example, “about 100” encompasses 90 and 110.

[0017] As used herein, the terms “immunogen,” “antigen,” and “epitope” refer to substances such as proteins, including glycoproteins, and peptides that are capable of eliciting an immune response.

[0018] As used herein, an “immunogenic composition” is a composition that comprises an antigen where administration of the composition to a subject results in the development in the subject of a humoral and/or a cellular immune response to the antigen.

[0019] As used herein, “substantially” refers to isolation of a substance (e.g. a compound, polynucleotide, or polypeptide) such that the substance forms the majority percent 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 substantially replaced the amount remaining in a sample is less than or equal to about 0.5% to about 10%, preferably less than about 0.5% to about 1 .0%.

[0020] The terms “treat,” “treatment,” and “treating,” as used herein, refer to an approach for obtaining beneficial or desired results, for example, clinical results. For the purposes of this disclosure, beneficial or desired results may include inhibiting or suppressing the initiation or progression of an infection or a disease; ameliorating, or reducing the development of, symptoms of an infection or disease; or a combination thereof.

[0021] “Prevention,” as used herein, is used interchangeably with “prophylaxis” and can mean complete prevention of an infection or disease, or prevention of the development of symptoms of that infection or disease; a delay in the onset of an infection or disease or its symptoms; or a decrease in the severity of a subsequently developed infection or disease or its symptoms.

[0022] As used herein an “effective dose” or “effective amount” refers to an amount of an immunogen sufficient to induce an immune response that reduces at least one symptom of pathogen infection. An effective dose or effective amount may be determined e.g., by measuring amounts of neutralizing secretory and/or serum antibodies, e.g., by plaque neutralization, complement fixation, enzyme-linked immunosorbent (ELISA), or microneutralization assay.

[0023] As used herein, the term “vaccine” refers to an immunogenic composition, such as an immunogen derived from a pathogen, which is used to induce an immune response against the pathogen that provides protective immunity (e.g., immunity that protects a subject against infection with the pathogen and/or reduces the severity of the disease or condition caused by infection with the pathogen). The protective immune response may include formation of antibodies and/or a cell-mediated response. Depending on context, the term “vaccine” may also refer to a suspension or solution of an immunogen that is administered to a subject to produce protective immunity.

[0024]As used herein, the term “subject” includes humans and other animals. Typically, the subject is a human. For example, the subject may be an adult, a teenager, a child (2 years to 14 years of age), an infant (birth to 2 year), or a neonate (up to 2 months). In particular aspects, the subject is up to 4 months old, or up to 6 months old. In aspects, the adults are seniors about 65 years or older, or about 60 years 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 intending to become pregnant. In other aspects, subject is not a human; for example a non-human primate; for example, a baboon, a chimpanzee, a gorilla, or a macaque. In certain aspects, the subject may be a pet, such as a dog or cat. [0025] The term “percent identity” in the context of two or more nucleic acid or polypeptide sequences, refers to two or more sequences or subsequences that have a specified percentage of nucleotides or amino acid residues that are the same, when compared. Unless otherwise indicated, percent identity is calculated over the entire length of the compared sequences using the tool EMBOSS Needle, which is available online at ebi.ac.uk/jdispatcher/psa/emboss_needle. When the percent identity of two polypeptides is compared, the following default parameters are used: Output Format = pair; Matrix = BLOSUM62; Gap Open = 10; Gap Extend 0.5, End Gap = false, End Gap Open = 10, End Gap Extend =0.5. When the percent identity of two nucleic acids is compared, the following default parameters are used: Output Format = pair; Matrix = DNAfull; Gap Open = 10; Gap Extend 0.5, End Gap = false, End Gap Open = 10, End Gap Extend =0.5.

[0026] As used herein, the term “vaccine” refers to an immunogenic composition, such as an immunogen derived from a pathogen, which is used to induce an immune response against the pathogen that provides protective immunity (e.g., immunity that protects a subject against infection with the pathogen and/or reduces the severity of the disease or condition caused by infection with the pathogen). The protective immune response may include formation of antibodies and/or a cell-mediated response. Depending on context, the term “vaccine” may also refer to a suspension or solution of an immunogen that is administered to a subject to produce protective immunity.

[0027] In aspects, the subject is immunocompromised. In embodiments, the immunocompromised subject is administered a medication that causes immunosuppression. Non-limiting examples of medications that cause immunosuppression include corticosteroids (e.g., prednisone), alkylating agents (e.g., cyclophosphamide), antimetabolites (e.g., azathioprine or 6-mercaptopurine), transplant-related immunosuppressive drugs (e.g., cyclosporine, tacrolimus, sirolimus, or mycophenolate mofetil), mitoxantrone, chemotherapeutic agents, methotrexate, tumor necrosis factor (TNF)-blocking agents (e.g., etanercept, adalimumab, infliximab). In embodiments, the immunocompromised subject 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, parainfluenza, adenovirus, or a picornavirus. In embodiments, the immunocompromised subject has acquired immunodeficiency syndrome (AIDS). In embodiments, the immunocompromised subject is a person living with human immunodeficiency virus (HIV). In embodiments, the immunocompromised subject is immunocompromised due to a treatment regimen designed to prevent inflammation or prevent rejection of a transplant. In embodiments, the immunocompromised subject is a subject who has received a transplant. In embodiments, the immunocompromised subject has undergone radiation therapy or a splenectomy. In embodiments, the immunocompromised subject has been diagnosed with cancer, an autoimmune disease, tuberculosis, a substance use disorder (e.g., an alcohol, opioid, or cocaine use disorder), stroke or cerebrovascular disease, a solid organ or blood stem cell transplant, sickle cell disease, thalassemia, autoimmune lymphoproliferative syndrome (ALPS), autoimmune polyglandular syndrome type 1 (APS-1 ), B-cell expansion with NF-KB and T-cell anergy (BENTA) disease, caspase eight deficiency state (CEDS), chronic granulomatous disease (CGD), common variable immunodeficiency (CVID), congenital neutropenia syndromes, a deficiency in the cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4), a DOCK8 deficiency, a GATA2 deficiency, a glycosylation disorder with immunodeficiency, a hyperimmunoglobulin E syndrome (HIES), hyper-immunoglobulin M syndrome, diabetes, type 1 diabetes, type 2 diabetes, interferon gamma deficiency, interleukin 12 deficiency, interleukin 23 deficiency, leukocyte adhesion deficiency, lipopolysaccharide-responsive beige-like anchor (LRBA) deficiency, PI3 kinase disease, PLCG2-associated antibody deficiency and immune dysregulation (PLAID), severe combined immunodeficiency (SCID), STAT3 dominant-negative disease, STAT3 gain-of-function disease, warts, hypogammaglobulinemia, infections, and myelokathexis (WHIM) syndrome, Wisckott-Aldrich syndrome (WAS), X-linked agammaglobulinemia (XLA), X-linked lymphoproliferative disease (XLP), uremia, malnutrition, orXMEN disease. In embodiments, the immunocompromised subject is a current or former cigarette smoker. In embodiments, the immunocompromised subject has a B-cell defect, T-cell defect, macrophage defect, cytokine defect, phagocyte deficiency, phagocyte dysfunction, complement deficiency or a combination thereof.

[0028] In embodiments, the subject is overweight or obese. In embodiments, an overweight subject has a body mass index (BMI) > 25 kg/m² and < 30 kg/m². In embodiments, an obese subject has a BMI that is > 30 kg/m². In embodiments, the subject has a mental health condition. In embodiments, the mental health condition is depression, schizophrenia, or anxiety.

[0029]As used herein, the term "pharmaceutically acceptable" means being approved by a regulatory agency of a U.S. Federal or a state government or listed in the U.S. Pharmacopeia, European Pharmacopeia or other generally recognized pharmacopeia for use in mammals, and more particularly in humans. These compositions can be useful as a vaccine and/or antigenic compositions for inducing a protective immune response in a vertebrate.

[0030]As used herein, the term “modification” as it refers to an RSV F glycoprotein refers to mutation, deletion, or addition of one or more amino acids of the RSV F glycoprotein. The location of a modification within an RSV F glycoprotein can be determined based by aligning the sequence of the RSV F glycoprotein to a native RSV F glycoprotein (e.g., SEQ ID NO: 330 (an RSV F glycoprotein containing a signal peptide) or SEQ ID NO: 329 (a mature RSV F glycoprotein lacking a signal peptide). The location of a modification within a SARS-CoV-2 Spike (S) glycoprotein (alternatively referred to herein as a “CoV S glycoprotein”) can be determined by aligning the sequence of the polypeptide to SEQ ID NO: 1 (CoV S glycoprotein containing signal peptide) or SEQ ID NO: 2 (mature CoV S glycoprotein lacking a signal peptide). The sequences of the native RSV F glycoproteins having the amino acid sequences of SEQ ID NO: 329 and SEQ ID NO: 330 and the native CoV S glycoproteins having the amino acid sequences of SEQ ID NO: 1 and SEQ ID NO: 2 are found in the table below.

[0031]The term “efficacy” of an immunogenic composition or vaccine composition described herein refers to the percentage reduction of infection (e.g., with an RSV virus) in a group administered an immunogenic composition as compared to a group that is not administered the immunogenic composition. In embodiments, efficacy (E) is calculated using the following equation: E (%) = (1 - RR) x 100, where RR = relative risk of incidence rates between the group administered the immunogenic composition and the group that is not administered the immunogenic composition. In embodiments, immunogenic compositions described herein have an efficacy against an RSV virus that is 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 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 98 %, or between about 90 % and about 99 %.

[0032] The term SARS-CoV-2 “variant”, used interchangeably herein with a “heterogeneous SARS-CoV-2 strain,” refers to a SARS-CoV-2 virus comprising a CoV S polypeptide having one or more modifications as compared to a SARS-CoV S polypeptide having the amino acid sequence of SEQ ID NO: 2. For example, a SARS- CoV-2 variant may 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, as compared to a CoV S polypeptide having the amino acid sequence of SEQ ID NO: 2. For example, a SARS- CoV-2 variant may have at least one and up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9, up to 10, up to 11 , up to 12, up to 13, up to 14, up to 15, up to 16, up to 17, up to 18, up to 19, up to 20, up to 21 , up to 22, up to 23, up to 24, up to 25, up to 26, up to 27, up to 28, up to 29, up to 30, up to 31 , up to 32, up to 33, up to

34, up to 35 modifications, up to 40 modifications, up to 45 modifications, up to 50 modifications, up to 55 modifications, up to 60 modifications, up to 65 modifications, up to 70 modifications, up to 75 modifications, up to 80 modifications, up to 85 modifications, up to 90 modifications, up to 95 modifications, or up to 100 modifications as compared to a CoV S polypeptide having the amino acid sequence of SEQ ID NO: 2. In aspects, a SARS-CoV-2 variant may 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, as compared to a CoV S polypeptide having the amino acid sequence of SEQ ID NO: 2. [0033]As used herein, the terms "intranasal administration" and "nasal administration" refer to administration of an immunogenic composition described herein to the nasal cavity of a subject. In embodiments, intranasal administration is achieved using a liquid preparation (e.g., an aqueous preparation), an aerosolized preparation, or a dry powder preparation. In embodiments, the liquid preparation, aerosolized preparation, or dry powder preparation is administered via an externally propelled nasal delivery device. In embodiments, the liquid preparation, aerosolized preparation, or dry powder preparation is administered via a self-propelled (i.e., via inhalation) nasal delivery device. In embodiments, the liquid preparation, aerosolized preparation, or dry powder preparation is administered via nasal insufflation (when an immunogenic composition is blown into the nose) or nasal instillation (when an immunogenic composition is dropped into the nose). In embodiments, the liquid preparation, aerosolized preparation, or dry powder preparation is administered via a gel, cream, ointment, lotion, or paste applied to one or more nasal epithelium (e.g., olfactory epithelium or nasal respiratory epithelium). In embodiments, the immunogenic composition is applied to mucus in the nasal cavity of a subject.

[0034]As used herein, the term "non-invasive nasal delivery device" refers an instrument that is capable of delivering an immunogenic composition to the nasal cavity without piercing the epithelium of the subject. Non-limiting examples of non- invasive nasal delivery devices include propellant (e.g., a pressurized inhaler) and non-propellant (e.g., a pump-type inhaler) types of aerosol or atomizer devices, particle dispersion devices, nebulizers, and pressurized olfactory delivery devices for delivery of liquid or powder formulations.

[0035]As used herein, the term "dry powder composition" refers to a lyophilized or spray dried form of an immunogenic composition described herein. In embodiments, a dry powder composition contains less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1 %, or less residual water content.

[0036]As used herein, the term "permeability enhancer" refers to a component of an immunogenic composition formulated for intranasal administration which promotes the passage of a viral glycoprotein through the nasal epithelium.

[0037]As used herein, the terms “bedside mix,” “bedside formulations,” “bedside vaccine compositions,” “bedside vials,” “bedside vial formulations” refer to vaccine formulations that are prepared immediately prior to administration. Such vaccine formulations contain viral antigens and adjuvants that are separately stored in different containers and are administered to a subject (e.g., either administering two consecutive injections, or combining the antigens and the adjuvants into one injection prior to administration).

[0038]As used herein, the terms “co-formulation mix,” “co-formulation,” “coformulation immunogenic compositions,” “prefilled syringes,” “pre-mix,” refer to vaccine formulations that are prepared for short to long-term storage prior to the time of administration to a subject. Such vaccine formulations contain a combination of antigens and adjuvant in the same container and prepared in advance of administration. In embodiments, formulations contain hemagglutinin and adjuvant (e.g., saponin adjuvant) and form HaSMaNs (Hemagglutinin Saponin Matrix Nanoparticles).

[0039]As used herein, the term “split-virion” refers to a virus (e.g., an influenza virus or a SARS-CoV-2 virus), which has a viral membrane that has been disrupted with a surfactant. Examples of surfactants are described throughout this disclosure. Splitvirions do not undergo further purification, so they typically contain multiple viral proteins.

[0040]As used herein, the term “recombinant” as it refers to a protein (e.g. hemagglutinin) that is produced in a cell by transcription and translation of a nucleic acid that is introduced into a cell. The nucleic acid may be introduced via a vector or a virus encoding the nucleic acid.

[0041] As used herein, the term “whole influenza virus” refers to a virus that comprises all of its envelope, viral membrane, nucleocapsid, and genetic material. In embodiments, the whole influenza virus is inactivated.

[0042]As used herein, the term “inactivated virus” refers to a virus that has undergone treatment to substantially reduce or eliminate its virulence compared to the wild-type virus.

[0043] The term “bronchoalveolar lavage,” also referred to as “BAL” refers to a fluid sample retrieved from a patient’s lungs. In embodiments, the 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 subsequently collected from the lungs.

[0044]The term “mucosal immunity” refers to the cellular and humoral immune response that occurs in mucosal membranes. In embodiments, the methods of the disclosure provided herein result in mucosal immunity in the respiratory system. [0045]The term “atomization” refers to the generation of fine, inhalable droplets of a liquid. The typical dimensions of atomized droplets are in the range of several microns.

[0046] The term “aerosol” refers to a dispersion of solid or liquid particles in a gas phase. In embodiments, the gas phase is air.

Immunogenic Compositions and Vaccine Compositions Containing RSV F glycoproteins

[0047] The disclosure provides non-naturally occurring RSV F glycoproteins, nanoparticles containing RSV F glycoproteins, and immunogenic compositions and vaccine compositions containing either non-naturally occurring RSV F glycoproteins or nanoparticles containing RSV F glycoproteins. In embodiments, provided herein are methods of using RSV F glycoproteins, nanoparticles comprising the same, immunogenic compositions comprising RSV F glycoproteins and/or nanoparticles, and vaccine compositions to stimulate an immune response against an RSV virus.

[0048]Also provided herein are methods of manufacturing 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 baculovirus/Sf9 systems.

Non-naturally occurring RSV F Glycoprotein Antigens

[0049] In embodiments, provided herein are RSV F glycoproteins that have one or more modifications compared to a native (also referred to as “wild-type”) RSV F glycoprotein. The 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 the mature, fusion competent protein. The mature RSV F protein contains an F1 subunit and an F2 subunit connected by a disulfide bond. Additional sources that describe the RSV F protein structure are 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.

[0050] In embodiments, an RSV F glycoprotein from the wild type RSV F A2 strain is the native RSV F glycoprotein. The RSV F glycoprotein from the wild type RSV F A2 strain comprises the amino acid sequence of SEQ ID NO: 330. The wild type RSV F A2 strain has sequencing errors (A102P, V379I, V447M). These sequencing errors are corrected in the non-naturally occurring RSV F glycoproteins described herein, i.e. , amino acid 102 is alanine, amino acid 379 is valine, and amino acid 447 is valine. [0051] In embodiments, the RSV F glycoproteins have one or more modifications selected from the group consisting of:

(a) deletion of from 1 to 10 amino acids of the fusion peptide;

(b) inactivation of the primary furin cleavage site (also referred to as Site II) through mutation of one or more of amino acids 131-136;

(c) inactivation of the secondary furin cleavage site (also referred to as Site I) through mutation of one or more of amino acids 106-109;

(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.

[0052] In embodiments, the RSV F glycoproteins have one or more modifications selected from the group consisting of:

(a) deletion of from 1 to 10 amino acids of the fusion peptide;

(b) inactivation of the primary furin cleavage site (also referred to as Site II) through mutation of one or more of amino acids 131-136;

(c) inactivation of the secondary furin cleavage site (also referred to as Site I) through mutation of one or more of amino acids 106-109;

(d) S155C;

(e) S290C; (f) S190F; and

(g) V207L; wherein the modifications are numbered according to the RSV F glycoprotein of SEQ ID NO: 330.

[0053] In embodiments, the RSV F glycoproteins described herein exhibit one or more of the antigenic sites 0, II, IV, and V. In embodiments, the RSV F proteins described herein exhibit mutations, which result in the presence of one or more of the RSV F antigenic sites 0, II, IV, and V. The RSV F antigenic site 0, which is located at the membrane-distal apex of the prefusion RSV F trimer, is targeted by potent RSV F neutralizing antibodies, including 5C4, AM22, and D25. The RSV F antigenic site II, also referred to herein as the palivizumab site, is the target of the antibodies pavilizumab and motavizumab. Antigenic site II is composed of a helix-turn-helix motif that spans residues 253-278 of the RSV F glycoprotein of SEQ ID NO: 330. The 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. The RSV F antigenic site V is composed of a2-a3 and 3- 4.

[0054] In embodiments, a particular antigenic site contains epitopes associated with pre-fusion or post-fusion structure. For example, the RSV F antigenic sites 0 and V are exhibited in pre-fusion structures of the RSV F glycoprotein. In contrast, the RSV F antigenic sites II and IV are exhibited in both pre-fusion and post-fusion structures of the RSV F glycoprotein. In embodiments, RSV F proteins contain antigenic sites that induce the formation of neutralizing antibodies against the pre-fusion conformation of the RSV F glycoprotein. In embodiments, the RSV F glycoproteins contain antigenic sites that induce the formation of neutralizing antibodies against the post-fusion conformation of the RSV F glycoprotein. In embodiments, the RSV F glycoproteins described herein contain antigenic sites that induce the production of neutralizing antibodies against both pre-fusion and post-fusion conformations of the RSV F glycoprotein.

[0055] In embodiments, the RSV F glycoproteins comprise a deletion of amino acids 137-146; an inactivated primary furin cleavage site; a S155C mutation; and a S290C mutation, wherein the modifications are numbered according to the RSV F glycoprotein of SEQ ID NO: 330. In embodiments, the RSV F glycoproteins comprise a deletion of amino acids 137-146; an inactivated primary furin cleavage site; a S155C mutation; a S290C mutation; a 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 glycoproteins comprise a deletion of amino acids 1 S - S; an inactivated primary furin cleavage site; a S190F mutation; and a V207L mutation, wherein the modifications are numbered according to the RSV F glycoprotein of SEQ ID NO: 330.

[0056] In embodiments, the RSV F glycoproteins comprise a deletion of amino acids 137-146; an inactivated primary furin cleavage site; and a N116Q mutation, wherein the modifications are numbered according to the RSV F glycoprotein of SEQ ID NO: 330. In embodiments, the RSV F glycoproteins comprise a deletion of amino acids 137-146; an inactivated primary furin cleavage site; a S155C mutation; a S290C mutation; and a N116Q mutation, wherein the modifications are numbered according to the RSV F glycoprotein of SEQ ID NO: 330. In embodiments, the RSV F glycoproteins comprise a deletion of amino acids 137-146; an inactivated primary furin cleavage site; a S190F mutation; a V207L mutation; and a N116Q mutation, wherein the modifications are numbered according to the RSV F glycoprotein of SEQ ID NO: 330. In embodiments, the RSV F glycoproteins comprise a deletion of amino acids 137- 146; an inactivated primary furin cleavage site; a S155C mutation; a S290C mutation; a S190F mutation; a V207L mutation; and a N116Q mutation, wherein the modifications are numbered according to the RSV F glycoprotein of SEQ ID NO: 330. Non-naturally occurring RSV F Glycoprotein Antigens — Mutations in Furin Cleavage Sites

[0057] In embodiments, particular mutations cause the mature RSV F proteins of the disclosure to retain p27 at the N-terminus of the F1 subunit. RSV F proteins which retain p27 contain a p27-F1 subunit and an F2 subunit that are connected by a disulfide bond. In embodiments, a mutation in the primary furin cleavage site leads to retention of p27.

[0058] The primary 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 primary furin cleavage site has the amino acid sequence of KKRKRR (SEQ ID NO: 341). Inactivation of the primary fusion cleavage site may be achieved by mutating residues in the site, with the result that furin can no longer recognize the consensus site. For example, inactivation of the primary furin cleavage site may be accomplished by introducing at least one, at least two, or three amino acid substitutions at positions corresponding to arginine 133, arginine 135, and arginine 136 of the wild-type RSV F protein (SEQ ID NO: 330). In particular aspects, one, two, or all three of the arginines are mutated to glutamine. In other aspects, inactivation is accomplished by mutating the wild-type primary furin cleavage site to one of the following sequences: KKQKQQ (SEQ ID NO: 342), QKQKQQ (SEQ ID NO: 343), KKQKRQ (SEQ ID NO: 344), and GRRQQR (SEQ ID NO: 345).

[0059] In embodiments, RSV F glycoproteins described herein comprise a wild-type secondary furin cleavage site. The secondary furin cleavage site, also referred to as Site I herein, is located at residues 106 to 109 of SEQ ID NO: 330. The wild-type secondary furin cleavage site has the amino acid sequence RARR (SEQ ID NO: 6). In embodiments, RSV F glycoproteins described herein comprise an inactive secondary furin cleavage site. In embodiments, inactivation of the secondary furin cleavage site is accomplished by mutating the wild-type site to one of the following sequences: QQAQ (SEQ ID NO: 7), RAQQ (SEQ ID NO: 348), or RANN (SEQ ID NO: 349). In embodiments, the inactivated secondary furin cleavage site comprises the amino acid sequence of any one of SEQ ID NOS: 7-34, 97, 111 , 348, and 349. In embodiments the inactivated secondary furin cleavage site comprises the amino acid sequence of QQAQ (SEQ ID NO: 7).

Non-naturally occurring RSV F Glycoprotein Antigens — Modifications to the F1 Domain

[0060] In embodiments, the non-naturally occurring RSV F glycoproteins described herein may have a deletion, an insertion, or mutation of up to about 1 , up to about 2, up to about 3, up to about 4, up to about 5, up to about 10, up to about 15, up to about 20, up to about 25, or up to about 30 amino acids compared to the amino acid sequence of the F1 domain of SEQ ID NO: 330. The wild-type F1 domain has the amino acid sequence of SEQ ID NO: 350. In embodiments, the RSV F glycoproteins described herein comprise an F1 domain with 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 %, identity, or 100 % identity to the F1 domain of SEQ ID NO: 350. The F1 domain of an RSV F glycoprotein herein may have a deletion, an insertion, or mutation of from 1 to about 30 amino acids, from 1 to about 25 amino acids, from 1 to about 20 amino acids, from 1 to about 15 amino acids, from 1 to about 10 amino acids, from about 1 to about 5 amino acids, from about 3 to about 10 amino acids, from about 5 to 10 amino acids, from about 8 to 12 amino acids, from about 10 to 15 amino acids, from about 12 to 17 amino acids, from about 15 to 20 amino acids, from about 18 to 23 amino acids, from about 20 to 25 amino acids, from about 22 to about 27 amino acids, or from about 25 to 30 amino acids as compared to the F1 domain of SEQ ID NO: 350.

[0061] In embodiments, the F1 domain of the RSV F glycoproteins comprise 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 glycoproteins comprise S190F and V207 L 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 glycoproteins comprise 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 glycoproteins comprise S190F, V207L, S155C, and S290C mutations, wherein the mutations are numbered according to the RSV F glycoprotein of SEQ ID NO: 330.

[0062] In embodiments, the F1 domain of the RSV F glycoproteins comprises an amino acid sequence with 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 % identity, or 100 % identity any one of SEQ ID NOS: 350-355.

Non-naturally occurring RSV F Glycoprotein Antigens — Deletions in Fusion Peptide of the F1 Domain

[0063] In embodiments, the RSV F glycoproteins of the disclosure lack one or more amino acids of the RSV F fusion peptide (amino acids 137-146 of SEQ ID NO: 330). The fusion peptide of the RSV F glycoprotein of SEQ ID NO: 330 has the amino acid sequence of FLGFLLGVGS (SEQ ID NO: 363). In embodiments, provided herein are RSV F glycoproteins that lack the fusion peptide. In embodiments, provided herein are RSV F glycoproteins lacking from 1 to 10, from 1 to 9, from 1 to 8, from 1 to 7, from 1 to 6 from 1 to 5, from 1 to 4, from 1 to 3, or from 1 to 2 contiguous amino acids of the fusion peptide. In embodiments, provided herein are RSV F glycoproteins lacking from 1 to 10, from 1 to 9, from 1 to 8, from 1 to 7, from 1 to 6 from 1 to 5, from 1 to 4, from 1 to 3, or from 1 to 2 non-contiguous amino acids of the fusion peptide. In embdiments, 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 no more than 10 amino acids of the fusion peptide are deleted.

Non-naturally occurring RSV F Glycoprotein Antigens — Modifications to the F2 Domain

[0064] In embodiments, the non-naturally occurring RSV F glycoproteins described herein may have a deletion, an insertion, or mutation of up to about 1 , up to about 2, up to about 3, up to about 4, up to about 5, up to about 10, up to about 15, up to about 20, up to about 25, or up to about 30 amino acids compared to the amino acid sequence of the F2 domain of SEQ ID NO: 330. The wild-type F2 domain has the amino acid sequence of SEQ ID NO: 356. In embodiments, the RSV F glycoproteins described herein comprise an F1 domain with 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 %, identity, or 100 % identity to the F2 domain of SEQ ID NO: 356. The F2 domain of an RSV F glycoprotein herein may have a deletion, an 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 as compared to the F2 domain of SEQ ID NO: 356.

[0065] In embodiments, the F2 domain of the RSV F glycoproteins may comprise a mutation of P102A, wherein the mutation is numbered according to the RSV F glycoprotein of SEQ ID NO: 330.

[0066] In embodiments, the F2 domain of the RSV F glycoproteins comprises an amino acid sequence with 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 % identity, or 100 % identity any one of SEQ ID NOS: 356-357.

Non-naturally occurring RSV F Glycoprotein Antigens — Modifications to p27 [0067] In embodiments, the non-naturally occurring RSV F glycoproteins described herein may have a deletion, an insertion, or mutation of up to about 1 , up to about 2, up to about 3, up to about 4, up to about 5, up to about 10, up to about 15, up to about 20, up to about 25, or up to about 30 amino acids compared to the amino acid sequence of p27 of SEQ ID NO: 330. The wild-type p27 has the amino acid sequence of SEQ ID NO: 359. In embodiments, the RSV F glycoproteins described herein comprise a p27 with 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 %, identity, or 100 % identity to the p27 of SEQ ID NO: 330. The p27 of an RSV F glycoprotein herein may have a deletion, an insertion, or mutation of from about 1 to about 5 amino acids, from about 3 to about 10 amino acids, from about 5 to 10 amino acids, from about 8 to 12 amino acids, from about 10 to 15 amino acids, from about 12 to 17 amino acids, from about 15 to 20 amino acids, from about 18 to 23 amino acids, from about 20 to 25 amino acids, from about 22 to about 27 amino acids, or from about 25 and 30 amino acids as compared to the p27 of SEQ ID NO: 330.

[0068] In embodiments, the p27 domain of the RSV F glycoproteins may comprise a mutation of N116Q, wherein the mutation is numbered according to the RSV F glycoprotein of SEQ ID NO: 330.

[0069] In embodiments, p27 of the RSV F glycoproteins comprises an amino acid sequence with 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 % identity, or 100 % identity to any one of SEQ ID NOS: 358-359.

Non-naturally occurring RSV F Glycoprotein Antigens — Transmembrane domain

[0070] In embodiments, the RSV F glycoproteins described herein comprise a transmembrane domain. In embodiments, the transmembrane domain has the sequence of ITTIIIVIIVILLSLIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN (SEQ ID NO: 362). In embodiments, the RSV F glycoproteins described herein may have a deletion, an 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.

Non-naturally occurring RSV F Glycoprotein Antigens — Signal Peptide

[0071] In embodiments, the RSV F glycoproteins described herein are expressed with an N-terminal signal peptide. In embodiments, the N-terminal signal peptide has an amino acid sequence of SEQ ID NO: 360 (MELLILKANAITTILTAVTFCFASG). In embodiments, the signal peptide may 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 may be deleted or mutated. An initiating 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.

[0072] Following expression of the RSV F glycoprotein in a host cell, the N-terminal signal peptide is cleaved to provide the mature RSV F glycoprotein sequence (SEQ ID NOS: 331 and 337-340). In embodiments, the signal peptide is cleaved by host cell proteases. In aspects, the full-length protein may be isolated from the host cell and the signal peptide cleaved subsequently.

[0073] Following cleavage of the signal peptide from the RSV F glycoprotein with an amino acid sequence corresponding to SEQ ID NOS: 332-336 during expression and purification, a mature polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOS: 331 and 337-340 is obtained and used to produce an RSV F nanoparticle vaccine or RSV F nanoparticles.

[0074] Advantageously, the disclosed RSV F glycoproteins may have enhanced protein expression, stability, and immunogenicity relative to the native RSV F glycoproteins.

[0075] In embodiments, the RSV F glycoproteins described herein contain further modifications from the native RSV F glycoprotein (SEQ ID NO: 330). In embodiments, the RSV F glycoproteins described herein exhibit at least 80 %, at least 81 %, at least

82 %, at least 83 %, at least 84 %, at least 85 %, at least 86 %, at least 87 %, at least

88 %, at least 89 %, at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least

94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 %, or 100

% identity to the native RSV F glycoprotein. [0076] In embodiments, the RSV F glycoproteins described herein are at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95%, at least 96 %, at least 97%, at least 98 %, at least 99%, at least 99.5 % identical to an RSV F glycoprotein having an amino acid selected from the group consisting of SEQ ID NOS: 329-340. In embodiments, provided herein is an RSV F glycoprotein with at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95%, at least 96 %, at least 97%, at least 98 %, at least 99%, at least 99.5 %, or 100 % identity to an RSF glycoprotein of SEQ ID NO: 337. A RSV F glycoprotein may have a deletion, an insertion, or mutation of up to about 1 , up to about 2, up to about 3, up to about 4, up to about 5, up to about 10, up to about 15, up to about 20, up to about 25, up to about 30, up to about 35, up to about 40, up to about 45, or up to about 50 amino acids compared to the amino acid sequence of the RSV F glycoprotein having an amino acid sequence of any one of SEQ ID NO: 329-340. A RSV F glycoprotein may have may have a deletion, an insertion, or mutation of from about 1 to about 5 amino acids, from about 3 to about 10 amino acids, from about 5 to 10 amino acids, from about 8 to 12 amino acids, from about 10 to 15 amino acids, from about 12 to 17 amino acids, from about 15 to 20 amino acids, from about 18 to 23 amino acids, from about 20 to 25 amino acids, from about 22 to about 27 amino acids, from about 25 to 30 amino acids, from about 30 to 35 amino acids, from about 35 to 40 amino acids, from about 40 to 45 amino acids, or from about 45 to 50 amino acids, as compared from the RSV F glycoprotein having an amino acid sequence of any one of SEQ ID NOS: 329-340. In embodiments, RSV F glycoproteins described herein comprise 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 an amino acid sequence of any one of SEQ ID NOS: 329- 340.

[0077] In embodiments, the RSV F glycoprotein is extended at the N-terminus, the C- terminus, or both the N-terminus and the C-terminus. In aspects, the extension is a tag useful for a function, such as purification or detection. In aspects the tag contains an epitope. For example, the tag may be a polyglutamate tag, a FLAG-tag, a HA-tag, a polyHis-tag (having about 5-10 histidines) (SEQ ID NO: 34), a hexahistidine tag (SEQ ID NO: 35), an 8X-His-tag (having eight histidines) (SEQ ID NO: 36), a Myc-tag, a Glutathione-S-transferase-tag, a Green fluorescent protein-tag, Maltose binding protein-tag, a Thioredoxin-tag, or an Fc-tag. In other aspects, the extension may be an N-terminal signal peptide fused to the protein to enhance expression. While such signal peptides are often cleaved during expression in the cell, some nanoparticles may contain the antigen with an intact signal peptide. Thus, when a nanoparticle comprises an antigen, the antigen may contain an extension and thus may be a fusion protein when incorporated into nanoparticles. For the purposes of calculating identity to the sequence, extensions are not included. In embodiments, the tag is a protease cleavage site. Non-limiting examples of protease cleavage sites include the 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 embodiments, the protease cleavage site is an HRV3C protease cleavage site.

[0078] In embodiments, the RSV F glycoprotein comprises a fusion protein. In embodiments, the RSV F glycoprotein comprises an N-terminal fusion protein. In embodiments, the RSV F glycoprotein comprises a C-terminal fusion protein. In embodiments, the fusion protein encompasses a tag useful for protein expression, purification, or detection. In embodiments, the tag is a polyHis-tag (having about 5-10 histidines) (SEQ ID NO: 34), a Myc-tag, a Glutathione-S-transferase-tag, a Green fluorescent protein-tag, 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, e.g. a polyHis-tag which contains six histidines (SEQ ID NO: 35).

Immunogenic Compositions and/or Vaccine Compositions containing RSV F glycoproteins and Additional Viral Glycoproteins

[0079] 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 the RSV F glycoproteins. In embodiments, the immunogenic compositions comprise from about 1 to about 50, from about 1 to about 45, from about 1 to about 40, from 1 to about 35, from about 1 to about 30, from 1 to about 25, from 1 to about 20, from 1 to about 15, from 1 to about 10, or from 1 to about 5 different RSV F glycoproteins. [0080] In another embodiment, the disclosures provide for a pharmaceutical pack or kit comprising one or more containers filled with one or more of the components of the vaccine compositions.

[0081] Compositions disclosed herein may be used either prophylactically or therapeutically, but will typically be prophylactic. Accordingly, the disclosure includes methods for treating or preventing infection. The methods involve administering to the subject a therapeutic or prophylactic amount of the immunogenic compositions of the disclosure. Preferably, the pharmaceutical composition is a vaccine composition that provides a protective effect. In other aspects, the protective effect may include amelioration of a symptom associated with infection in a percentage of the exposed population. For example, the composition may prevent or reduce one or more virus disease symptoms selected from: fever fatigue, muscle pain, headache, sore throat, vomiting, diarrhea, rash, symptoms of impaired kidney and liver function, internal bleeding and external bleeding, compared to an untreated subject.

[0082] The nanoparticles may be formulated for administration as vaccines in the presence of various excipients, buffers, and the like. For example, the vaccine compositions may contain sodium phosphate, sodium chloride, and/or histidine. Sodium phosphate may be present at about 10 mM to about 50 mM, about 15 mM to about 25 mM, or about 25 mM; in particular cases, about 22 mM sodium phosphate is present. Histidine may be present about 0.1 % (w/v), about 0.5% (w/v), about 0.7% (w/v), about 1 % (w/v), about 1 .5% (w/v), about 2% (w/v), or about 2.5% (w/v). Sodium chloride, when present, may be about 150 mM. In certain compositions, the sodium chloride may be present in higher concentrations, for example from about 200 mM to about 500 mM. In embodiments, the sodium chloride is present in 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.

[0083] In embodiments, the nanoparticles described herein have improved stability at certain pH levels. In embodiments, the nanoparticles are stable at slightly acidic pH levels. For example, the nanoparticles that are stable at a slightly acidic pH, for example from pH 5.8 to pH 7.0. In embodiments, the nanoparticles and compositions containing nanoparticles may be stable at pHs ranging 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, the nanoparticles and compositions described herein are stabile at neutral pHs, including from about pH 7.0 to about pH 7.4. In embodiments, the nanoparticles and compositions described herein are stable at slightly alkaline pHs, for example from about pH 7.0 to about pH 8.5, from about pH 7.0 to about pH 8.0, or from about pH 7.0 to about pH 7.5, including all values and ranges in between.

[0084] In embodiments, in addition to RSV F glycoproteins, the compositions described herein comprise one or more additional viral glycoproteins. In embodiments, the additional viral glycoproteins are selected from the group consisting of: a SARS- CoV-2 S glycoprotein, an influenza hemagglutinin glycoprotein, and an influenza neuraminidase. In embodiments, the immunogenic compositions comprise an RSV F glycoprotein described herein, an influenza glycoprotein, and a SARS-CoV-2 S glycoprotein. In embodiments, the immunogenic compositions comprise an RSV F glycoprotein described herein and an influenza glycoprotein. In embodiments, the immunogenic compositions comprise an RSV F glycoprotein described herein, and a SARS-CoV-2 S glycoprotein.

[0085] In embodiments, the immunogenic compositions comprise from about 1 to about 50, from about 1 to about 45, from about 1 to about 40, from 1 to about 35, from about 1 to about 30, from 1 to about 25, from 1 to about 20, from 1 to about 15, from 1 to about 10, or from 1 to about 5 different viral glycoproteins. Viral glycoproteins that are “different” have different amino acid sequences. In embodiments, different viral glycoproteins may be from different viruses, different strains, or sub-types of the same virus, or may differ from one another by mutation.

[0086] In embodiments, in addition to an RSV F glycoprotein, the immunogenic composition comprises from 1 to about 50 different influenza glycoproteins (e.g., hemagglutinin and/or neuraminidase). In embodiments, the influenza glycoprotein (e.g., hemagglutinin or neuraminidase) may be from any influenza virus strain.

[0087] In embodiments, the immunogenic compositions comprise 1 , 2, 3, 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11 , about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21 , about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31 , about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41 , about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, or about 50 influenza glycoproteins, including all values and ranges therebetween. [0088] 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. Phylogenetically, the influenza is split into groups. In embodiments, the HA glycoprotein is a Group 1 HA glycoprotein (i.e., a glycoprotein from the H1 , H2, H5, H6, H8, H9, H11 , H12, H13, H16, H17, or H18 subtypes). In embodiments, the HA glycoprotein is a Group 2 HA glycoprotein (i.e, a glycoprotein from the H3, H4, H7, H10, H14, or H15 subtypes).

[0089] Multiple examples of influenza HA glycoproteins are described in the following patent documents, which are each incorporated by reference herein in their entirety for all purposes: International Publication No. 2017/041100 and International Publication No. 2019/022930.

[0090] In embodiments, an immunogenic composition of the disclosure comprises at least three different HA glycoproteins or at least four different HA glycoproteins. In embodiments, an immunogenic composition of the disclosure comprises three or four different HA glycoproteins. In embodiments, each of the HA glycoproteins are from a different influenza strain. In embodiments, three HA glycoproteins are from a Type A influenza strain, and one HA glycoprotein is from a Type B influenza strain. In embodiments, two HA glycoproteins are from a Type A influenza strain, and two HA glycoproteins are from a Type B influenza strain. In embodiments, two HA glycoproteins are from a Type A influenza strain, and one HA glycoprotein is from a Type B influenza strain.

[0091] In embodiments, each of the at least three different HA glycoproteins is isolated separately using an egg-based manufacturing process. In embodiments, an eggbased manufacturing process comprises (a) propagating an influenza virus in an egg and (b) harvesting the influenza virus.

[0092] 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 an egg. In embodiments, the optimized influenza virus lacks the polybasic cleavage site of hemagglutinin. The following article describes the development of an optimized influenza virus (referred to as a “candidate vaccine virus”) in detail and is incorporated by reference herein in its entirety: Belser et al. Virology. 2017 Nov; 511 : 135-141. [0093] In embodiments, the egg is a chicken egg. In embodiments, the chicken egg is an embryonated chicken egg. In embodiments, the egg is a pathogen-free egg. In embodiments, an influenza virus is propagated by inoculating the virus in the allantoic cavity of an embryonated chicken egg. The following article describes an exemplary inoculation method and is incorporated by reference herein in its entirety: Brauer et al. J Vis Exp. 2015; (97): 52421.

[0094] In embodiments, the egg-based manufacturing process comprises purifying the influenza virus. In embodiments, an 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.

[0095] In embodiments, the egg-based manufacturing process comprises inactivating (also referred to herein as “killing”) an influenza virus. In embodiments, an influenza virus is inactivated using low pH (e.g., a pH from about 3.5-5.5), heat, ethanol, ultraviolet light, exposure to a detergent (e.g, octylphenol), or exposure to a chemical (e.g., 2-propanol, ethanol, iodopovidone). In embodiments, the purified virus is inactivated with ultraviolet light, betapropiolactone, sodium deoxycholate, formaldehyde, or any combination thereof.

[0096] In embodiments, the egg-based manufacturing process comprises exposing the influenza virus to a surfactant. The surfactant may be sodium taurodeoxycholate, octylphenol ethoxylate (Triton®-X 100), or cetyl trimethyl ammonium bromide. Exposing an influenza virus to a surfactant results in the formation of an influenza splitvirion. In embodiments, the at least three HA glycoproteins are in the form of an influenza split-virion.

[0097] In embodiments, the egg-based manufacturing process comprises purifying an influenza antigen (e.g., hemagglutinin) from the virus.

[0098]The following Food and Drug Administration (FDA) approved influenza vaccines are produced using an egg-based manufacturing process: AFLURIA ® QUADRIVALENT, FLUARIX® QUADRIVALENT, FLULAVAL QUADRIVALENT, FLUZONE® QUADRIVALENT, FLUZONE® HIGH-DOSE QUADRIVALENT, FLUMIST® QUADRIVALENT, FLUAD®, and FLUAD® QUADRIVALENT.

[0099] In embodiments, each of the at least three HA glycoproteins is isolated using a cell-culture based process. In embodiments, a cell-culture based process comprises (i) growing an influenza virus in a cell and (ii) harvesting the virus from the cell. In embodiments, a cell-culture based process comprises (i) transfecting a cell with a vector comprising a hemagglutinin and (ii) harvesting the hemagglutinin from the cell. In embodiments, a cell-culture based process comprises (i) transducing a cell with a virus encoding a hemagglutinin and (ii) harvesting the hemagglutinin from the cell. In embodiments, the harvested hemagglutinin is recombinant hemagglutinin.

[00100] 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., a chicken), a dog, a reptile, a goat, a pig, a mouse, a rabbit, or a rat.

[00101] In embodiments, the virus encoding a hemagglutinin is a baculovirus, a lentivirus, or an adeno-associated virus.

[00102] In embodiments, an influenza virus produced using a cell-culture based process is purified. Any of the purification techniques described to purify influenza virus produced using an egg-based manufacturing process can be used to purify influenza virus produced using a cell-cultured based process.

[00103] In embodiments, a hemagglutinin produced using a cell-culture based process is purified. Purification techniques include chromatography, centrifugation, precipitation, and nanofiltration.

[00104] The Food and Drug Administration (FDA) approved influenza vaccine FLUCELVAX® QUADRIVALENT is produced using a cell-culture based process.

[00105] In embodiments, each of the at least three HA glycoproteins is a recombinant hemagglutinin. The Food and Drug Administration (FDA) approved influenza vaccine FLUBLOK® QUADRIVALENT is a recombinant hemagglutinin.

[00106] In embodiments, the at least three hemagglutinins are in the form of recombinant hemagglutinin. Recombinant hemagglutinin is isolated from a cell that produces hemagglutinin. In embodiments, a cell that produces hemagglutinin has been transfected with a vector encoding hemagglutinin. In embodiments, a cell that produces hemagglutinin has been transduced with a virus encoding hemagglutinin.

[00107] In embodiments, the compositions disclosed herein comprise detergentcore nanoparticles comprising hemagglutinin from an influenza virus. The aforementioned detergent-core nanoparticles are described in detail in U.S. Patent No. 10,426,829, which is incorporated herein by reference in its entirety for all purposes. [00108] Detergent-core nanoparticles comprise a hemagglutinin from an influenza virus, which is associated with a detergent core. In embodiments, the hemagglutinin is a trimer. Each nanoparticle may contain 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, or 15 trimers. In embodiments, the nanoparticle contains between about 2 to about 9, about 2 to about 6, or about 5 hemagglutinin trimers. The hemagglutinin is associated with the non-ionic detergent containing core of the nanoparticle. In embodiments, the detergent is selected from polysorbate-20 (PS20), polysorbate-40 (PS40), polysorbate-60 (PS60), polysorbate-65 (PS65) and polysorbate-80 (PS80). The presence of the detergent facilitates formation of the nanoparticles by forming a core that organizes and presents the antigens. In embodiments, the nanoparticles may contain the antigens assembled into multi-oligomeric glycoprotein-PS80 proteindetergent nanoparticles with the head regions projecting outward and hydrophobic regions and PS80 detergent forming a central core surrounded by the antigens.

[00109] In embodiments, the detergent-core nanoparticles or HaSMaNs are trypsin-resistant nanoparticles produced using neutral pH purification. Trypsin resistance is achieved by neutral pH range of above 6.9 to 8.5 during purification and formulation of the HA nanoparticles. Trypsin resistant influenza glycoproteins and trypsin resistant influenza nanoparticles; and methods of making thereof are described in detail in U.S. Patent No. 10,426,829.

[00110] In embodiments, the hemagglutinin of the detergent-core nanoparticles or HaSMaNs described herein comprises the 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 the wild-type hemagglutinin protein.

[00111] HA is a homotrimer with each monomer consisting of -550 amino acid residues. Each monomer of HA has been conceptually divided into three domains: the ectodomain of -515 residues constitutes the extraviral part of the molecule; a single stretch of 27 residues defines the transmembrane (TM) domain; and -10 residues constitute the cytoplasmic tail (CT). While some changes may be made to hemagglutinin, formation of both detergent core nanoparticles and HaSMaNs requires an intact transmembrane domain (TM). Thus, in particular examples, a modified HA protein sequence comprises 100% identity to the wild-type TM and CT domains with some flexibility in the remaining ectodomain portion, where identity may be at least 90% or at least 95%. The domains may be identified by homology to the amino acid sequences of the TM domains and CT of Japan/305/57 HA shown in Figure 1 of Melikyan et al. (Mol Biol Cell. 1999 Jun; 10(6): 1821-1836) though it should be noted that the boundaries between ectodomain, TM, and CT domains may vary from HA protein to HA protein by up to three amino acids.

[00112] In embodiments, the immunogenic compositions and vaccine compositions described herein comprise a HaSMaN (Hemagglutinin Saponin Matrix Nanoparticle.) Fig. 6 (right panel) illustrates HaSMaN structures as observed under an electron microscope. The HA glycoproteins decorate the Matrix cage-like structures. The HaSMaN structures are formed by preparing detergent-core nanoparticles comprising hemagglutinin from an influenza virus and then incubating them with ISCOM matrix adjuvant particles for a period of time. ISCOM matrix particles are shown in the center panel of Fig. 6. Notably, the HaSMaNs form readily with Type A influenza HA proteins, but not Type B influenza HA proteins.

[00113] HaSMaNs disclosed herein are produced by incubating the detergentcore nanoparticles with an ISCOM Matrix adjuvant comprising a saponin fraction, cholesterol and a phospholipid. In embodiments, a HaSMaN is formed by incubating a detergent-core nanoparticle with an ISCOM matrix adjuvant for between about 24 hours and about 48 hours. For example, the detergent-core nanoparticle may be incubated with an 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, a HaSMaN is formed by incubating a detergent-core nanoparticle 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, a HaSMaN is formed by incubating a detergent-core nanoparticle with an ISCOM matrix adjuvant at a temperature from about 4oC to about 25oC. For example, the HaSMaN may be formed by incubating a detergent-core nanoparticle with an ISCOM matrix adjuvant at a temperature of about 40, 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 ^cC, about 23 C, about 24 C, about 25^cC, or higher. In embodiments, a HaSMaN is formed by incubating a detergent-core nanoparticle 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 ⁷C, 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, about 24 to 48 hours at 4oC or 25oC incubation is required for formation. Formation of HaSMaNs is promoted by higher temperatures. In embodiments, formation of HaSMaN occurs by incubation of detergent-core nanoparticles with an ISCOM Matrix adjuvant for at least 24 hours at about 25oC. Mixing detergent core nanoparticles with ISCOM Matrix adjuvant shortly prior to administering to a subject — i.e. bedside mix, does not produce HaSMaNs. Longer incubation periods do not negatively impact HaSMaNs formation.

[00114] In embodiments, in addition to an RSV F glycoprotein, the immunogenic composition comprises from 1 to about 50 different SARS-CoV-2 S glycoproteins (“CoV S glycoproteins). In embodiments, in addition to an RSV F glycoprotein, the immunogenic composition comprises about 5 different SARS-CoV-2 CoV S glycoproteins. The CoV S glycoprotein is synthesized as an inactive precursor (SO) that is proteolytically cleaved at the furin cleavage site into S1 and S2 subunits which remain non-covalently linked to form prefusion trimers. The S2 domain of the CoV S glycoprotein comprises a fusion peptide (FP), two heptad repeats (HR1 and HR2), a transmembrane (TM) domain, and a cytoplasmic tail (CT). The S1 domain of the SARS-CoV-2 S protein folds into four distinct domains: the N-terminal domain (NTD) and the C-terminal domain, which contains the receptor binding domain (RBD) and two subdomains SD1 and SD2. The prefusion SARS-CoV-2 S protein trimers undergo a structural rearrangement from a prefusion to a postfusion conformation upon S- protein receptor binding and cleavage. In embodiments, the CoV S polypeptides are glycoproteins, due to post-translational glycosylation. The glycoproteins comprise one or more domains, including a signal peptide, an S1 subunit, an S2 subunit, a NTD, a, RBD, two subdomains (SD1 and SD2, labeled SD1/2 in Figs. 1A-B and referred to as “SD1/2” herein), an intact or modified fusion peptide, an HR1 domain, an HR2 domain, a TM, and a CD. In embodiments, the amino acids for each domain are given in Fig. 7A (shown according to SEQ ID NO: 1), Fig. 7B (shown according to SEQ ID NO: 2). SARS-CoV-2 S glycoproteins also contain a furin cleavage site.

[00115] In embodiments, 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.

[00116] In embodiments, the CoV S glycoproteins described herein are expressed with an N-terminal signal peptide. In embodiments, the N-terminal signal peptide has an amino acid sequence of SEQ ID NO: 5 (MFVFLVLLPLVSS). In embodiments, the N-terminal signal peptide has an amino acid sequence of SEQ ID NO: 117 (MFVFLVLLPLVSI). In embodiments, the N-terminal signal peptide has an amino acid sequence of SEQ ID NO: 154 (MFVFFVLLPLVSS). In embodiments, the N-terminal signal peptide has an amino acid sequence of SEQ ID NO: 193 (MFGFLVLLPLVSS). In embodiments, the signal peptide may be replaced with any signal peptide that enables expression of the CoV S glycoprotein. In embodiments, one or more of the CoV S glycoprotein signal peptide amino acids may be deleted or mutated. An initiating methionine residue is maintained to initiate expression. In embodiments, the CoV S glycoproteins are 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:N 208; SEQ ID NO: 210; SEQ ID NO: 212; SEQ ID NO: 214; and SEQ ID NO: 216. In embodiments, the N- terminal signal peptide of the CoV S glycoprotein contains a mutation at Ser-13 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.

[00117] Following expression of the CoV S glycoprotein in a host cell, the N- terminal signal peptide is cleaved to provide the mature CoV glycoprotein sequence (SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 106, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 132, SEQ ID NO: 144, SEQ ID NO: 151 , SEQ ID NO: 153, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 181 , SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 186, SEQ ID NO: 188, SEQ ID NO: 190, SEQ ID NO: 192, SEQ ID NO: 195, SEQ ID NO: 217, SEQ ID NO: 218, SEQ ID NO: 219, SEQ ID NO: 220, SEQ ID NO: 221 , SEQ ID NO: 222, SEQ ID NO: 223, SEQ ID NO: 224, SEQ ID NO: 225, SEQ ID NO: 226, SEQ ID NO: 227, SEQ ID NO: 228, SEQ ID NO: 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 host cell proteases. In aspects, the full-length protein may be isolated from the host cell and the signal peptide cleaved subsequently.

[00118] Following cleavage of the signal peptide from a CoV Spike (S) polypeptide with an amino acid sequence corresponding to any one of SEQ ID NO: 88, SEQ ID NO: 105, SEQ ID NO: 130, SEQ ID NO: 136, SEQ ID NO: 143, SEQ ID NO: 149, SEQ ID NO: 152, SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 167, SEQ ID NO: 170, SEQ ID NO: 173, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 185, SEQ ID NO: 187, SEQ ID NO: 189, SEQ ID NO: 191 , SEQ ID NO: 194, SEQ ID NO: 200, SEQ ID NO: 203, SEQ ID NO: 205, SEQ ID NO: 207, SEQ ID NO: 209, SEQ ID NO: 211 , SEQ ID NO: 213, SEQ ID NO: 215, SEQ ID NO: 229, SEQ ID NO: 230, SEQ ID NO: 231 , SEQ ID NO: 232, SEQ ID NO: 242, SEQ ID NO: 243, SEQ ID NO: 244, SEQ ID NO: 250, SEQ ID NO: 251 , SEQ ID NO: 252, SEQ ID NO: 253, SEQ ID NO: 254, SEQ ID NO: 266, SEQ ID NO: 268, SEQ ID NO: 270, SEQ ID NO: 272, SEQ ID NO: 282, SEQ ID NO: 286, SEQ ID NO: 290, SEQ ID NO: 295, SEQ ID NO: 299, SEQ ID NO: 303, SEQ ID NO: 307, SEQ ID NO: 311 , SEQ ID NO: 315, SEQ ID NO: 319, SEQ ID NO: 323, and SEQ ID NO: 327 during expression and purification, a mature polypeptide having an amino acid sequence selected from the group consisting of SEQ SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 106, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 132, SEQ ID NO: 144, SEQ ID NO: 151 , SEQ ID NO: 153, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 181 , SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 186, SEQ ID NO: 188, SEQ ID NO: 190, SEQ ID NO: 192, SEQ ID NO: 195, SEQ ID NO: 217, SEQ ID NO: 218, SEQ ID NO: 219, SEQ ID NO: 220, SEQ ID NO: 221 , SEQ ID NO: 222, SEQ ID NO: 223, SEQ ID NO: 224, SEQ ID NO: 225, SEQ ID NO: 226, SEQ ID NO: 227, SEQ ID NO: 228, SEQ ID NO: 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 is obtained and used to produce a CoV S nanoparticle vaccine orCoV S nanoparticles. [00119] Advantageously, the disclosed CoV S polypeptides may have enhanced protein expression and stability relative to a native CoV Spike (S) protein.

[00120] In embodiments, the CoV S polypeptides described herein contain further modifications from a native coronavirus S protein (e.g., SEQ ID NO: 2). In embodiments, the coronavirus S proteins described herein exhibit at least 80 %, at least 85 %, at least 86 %, at least 87 %, at least 88 %, at least 89 %, at least 90 % , at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 % identity to a native CoV S glycoprotein. A person of skill in the art would use known techniques to calculate the percent identity of the recombinant coronavirus S protein to the native protein or to any of the CoV S glycoproteins described herein.

[00121] In embodiments, the CoV S polypeptides described herein are at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95%, at least 96 %, at least 97%, at least 98 %, at least 99%, or at least 99.5 % identical to the CoV S polypeptide having an 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. A CoV S polypeptide may have a deletion, an insertion, or mutation of up to about 1 , up to about 2, up to about 3, up to about 4, up to about 5, up to about 10, up to about 15, up to about 20, up to about 25, up to about 30, up to about 35, up to about 40, up to about 45, or up to about 50 amino acids compared to the amino acid sequence of the CoV S polypeptide having an 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, SEQ ID NO: 243, and SEQ ID NOS: 255-328. A CoV S polypeptide may have may have a deletion, an insertion, or mutation of from 1 to about 70 amino acids, from 1 to about 5 amino acids, from about 3 to about 10 amino acids, from about 5 to about 10 amino acids, from about 8 to about 12 amino acids, from about 10 to about 15 amino acids, from about 12 to about 17 amino acids, from about 15 to about 20 amino acids, from about 18 to about 23 amino acids, from about 20 to about 25 amino acids, from about 22 to about 27 amino acids, from about 25 to about 30 amino acids, from about 30 to about 35 amino acids, from about 35 to about 40 amino acids, from about 40 to about 45 amino acids, or from about 45 to about 50 amino acids, as compared to the CoV S polypeptide having an 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, SEQ ID NO: 243, and SEQ ID NOS: 255-328. In embodiments, the CoV S polypeptides described herein comprise about 1 , about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11 , about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21 , about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31 , about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41 , about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 51 , about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, about 60, about 61 , about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71 , about 72, about 73, about 74, or about 75 deletions, insertions, or mutations compared to the coronavirus S protein having an 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, SEQ ID NO: 243, and SEQ ID NOS: 255-328.

[00122] 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, the extension is a tag useful for a function, such as purification or detection. In aspects the tag contains an epitope. For example, the tag may be a polyglutamate tag, a FLAG-tag, a 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 proteintag, Maltose binding protein-tag, a Thioredoxin-tag, or an Fc-tag. In other aspects, the extension may be an N-terminal signal peptide fused to the protein to enhance expression. While such signal peptides are often cleaved during expression in the cell, some nanoparticles may contain the antigen with an intact signal peptide. Thus, when a nanoparticle comprises an antigen, the antigen may contain an extension and thus may be a fusion protein when incorporated into nanoparticles. For the purposes of calculating identity to the sequence, extensions are not included. In embodiments, the tag is a protease cleavage site. Non-limiting examples of protease cleavage sites include the 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 embodiments, the protease cleavage site is an HRV3C protease cleavage site. In embodiments, the protease cleavage site comprises an amino acid sequence of SEQ ID NO: 98.

[00123] In embodiments, the CoV S glycoprotein comprises a fusion protein. In embodiments, the CoV S glycoprotein comprises an N-terminal fusion protein. In embodiments, the Cov S glycoprotein comprises a C-terminal fusion protein. In embodiments, the fusion protein encompasses a tag useful 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 proteintag, 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, e.g. a polyHis-tag which contains six histidines (SEQ ID NO: 100). In embodiments, the tag is a Twin-Strep-tag with an amino acid sequence of SEQ ID NO: 99. [00124] In embodiments, the CoV S polypeptide is a fusion protein comprising another coronavirus protein. In embodiments, the other coronavirus protein is from the same coronavirus. In embodiments, the other coronavirus protein is from a different coronavirus.

[00125] In aspects, the CoV S glycoprotein may be truncated. For example, the N-terminus may 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 may be truncated instead of or in addition to the N-terminus. For example, the C-terminus may 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. For purposes of calculating identity to the protein having truncations, identity is measured over the remaining portion of the protein.

[00126] In embodiments, the CoV S glycoproteins contain one or more modifications to the S1 subunit having an amino acid sequence of SEQ ID NO: 121 .

[00127] The amino acid sequence of the S1 subunit (SEQ ID NO: 121) is shown below.

[00128] QCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFS NVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSL LIVNNATNWIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQ PFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPI GINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDA VDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATR FASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVI RGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRK SNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRWVLS FELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIA DTTDAVRDPQTLEI LDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAI HAD QLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPRR AR

[00129] In embodiments, the CoV S polypeptides described herein comprise an

51 subunit with at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95%, at least 96 %, at least 97%, at least 98 %, at least 99%, at least 99.5 %, or 100 % identity to the S1 subunit of SEQ ID NO: 1 or SEQ ID NO: 2. The S1 subunit may have a deletion, an 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 may have a deletion, an 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 as compared to the S1 subunit of SEQ ID NO: 1 or SEQ ID NO: 2.

[00130] In embodiments, the S1 subunit may contain any combination of modifications shown in Table 1A.

Table 1A

[00131] In embodiments, the CoV S polypeptides contain 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.

[00132] The amino acid sequence of an NTD (SEQ ID NO: 118) is shown below.

[00133] QCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFS NVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSL LIVNNATNWIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQ PFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPI GINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDA VDCALDPLSETKCTLKS

[00134] In embodiments, the NTD has an 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 an NTD (SEQ ID NO: 45) is shown below.

[00135] QCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFS NVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSL LIVNNATNWIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQ PFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPI GINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDA VDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPN

[00136] In embodiments, the NTD and RBD overlap by up to about 1 amino acid, up to about 5 amino acids, up to about 10 amino acids, or up to about 20 amino acids. [00137] In embodiments, an NTD as provided herein may be extended at the C- terminus by up to 5, up to 10, up to 15, up to 20, up to 25, or up to 30 amino acids.

[00138] In embodiments, the CoV S polypeptides described herein comprise a NTD with at least 95%, at least 96 %, at least 97%, at least 98 %, at least 99%, at least 99.5 %, or 100% identity to the NTD of SEQ ID NO: 1 or SEQ ID NO: 2. The NTD may have a deletion, an 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 NTD of SEQ ID NO: 1 or SEQ ID NO: 2. The NTD may have a deletion, an 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 as compared to the NTD of SEQ ID NO: 1 or SEQ ID NO: 2.

[00139] In embodiments, the CoV S polypeptides contain 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 polypeptides contain 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.

[00140] In embodiments, the CoV S polypeptides contain 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 polypeptides contain a deletion of amino acids 1-318 of the NTD of SEQ ID NO: 2. In embodiments, deletion of the NTD enhances protein expression of the CoV Spike (S) polypeptide. In embodiments, the CoV S polypeptides which have an NTD deletion have amino acid sequences represented by SEQ ID NOS: 46, 48, 49, 51 , 52, and 54. In embodiments, the CoV S polypeptides which have an NTD deletion are 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.

[00141] 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, the mature S polypeptide sequence for reference.

Table 1B

[00142] In embodiments, the CoV S polypeptides contain one or more modifications to the RBD. [00143] In embodiments, 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.

[00144] The amino acid sequence of the RBD (SEQ ID NO: 126) is shown below: [00145] NITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTF KCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVI AWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYF PLQSYGFQPTNGVGYQPYRVWLSFELLHAPATVCGP

[00146] In embodiments, 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.

[00147] The amino acid sequence of the RBD (SEQ ID NO: 116) is shown below. [00148] LCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYG VSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNS NNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQS YGFQPTNGVGYQPYRWVLSFELLHAPATVCGPKKS

[00149] In embodiments, an RBD as provided herein may 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.

[00150] In embodiments, the CoV S polypeptides described herein comprise a RBD with at least 95%, at least 96 %, at least 97%, at least 98 %, at least 99%, at least 99.5 %, or 100% identity to the RBD of SEQ ID NO: 1 or SEQ ID NO: 2. The RBD may have a deletion, an 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 may have a deletion, an insertion, or mutation of from about 1 to about 50 amino acids, from about 1 to about 5 amino acids, from about 3 to about 10 amino acids, from about 5 to about 10 amino acids, from about 8 to about 12 amino acids, from about 10 to about 15 amino acids, from about 12 to about 17 amino acids, from about 15 to about 20 amino acids, from about 18 to about 23 amino acids, from about 20 to about 25 amino acids, from about 22 to about 27 amino acids, or from about 25 to about 30 amino acids as compared to the RBD of SEQ ID NO: 1 or SEQ ID NO: 2. [00151] In embodiments, the CoV S polypeptide has at least one, at least two, at least three, at least four, at least four, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11 , at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 modifications in the RBD. In 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 the RBD. In embodiments, the RBD may contain any combination of modifications as shown in Table 1C.

Table 1C

[00152] In embodiments, the CoV S polypeptides contain one or more modifications to the SD1/2 having an amino acid sequence of SEQ ID NO: 122, which corresponds to amino acids 542-681 of SEQ ID NO: 1 or amino acids 529-668 of SEQ ID NO: 2.

[00153] The amino acid sequence of the SD1/2 (SEQ ID NO: 122) is shown below. [00154] NFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITP CSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQT RAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSP

[00155] In embodiments, the CoV S polypeptides described herein comprise a SD1/2 with at least 95%, at least 96 %, at least 97%, at least 98 %, at least 99%, at least 99.5%, or 100 % identity to the SD1/2 of SEQ ID NO: 1 or SEQ ID NO: 2. The SD1/2 may have a deletion, an 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 SD1/2 of SEQ ID NO: 1 or SEQ ID NO: 2. The SD1/2 may have a deletion, an 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 as compared to the SD1/2 of SEQ ID NO: 1 or SEQ ID NO: 2.

[00156] In embodiments, the CoV S polypeptide has at least one, at least two, at least three, at least four, at least four, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11 , at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 modifications in the 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 the SD1/2. In embodiments, the SD1/2 may contain any combination of modifications as shown in Table 1 D.

Table 1D

[00157] In embodiments, the CoV S polypeptides contain a furin site (RRAR), which corresponds to amino acids 682-685 of SEQ ID NO: 1 or amino acids 669-672 of SEQ ID NO: 2, that is inactivated by one or more mutations. Inactivation of the furin cleavage site prevents furin from cleaving the CoV S polypeptide. In embodiments, the CoV S polypeptides described herein which contain an inactivated furin cleavage site are expressed as a single chain.

[00158] In embodiments, one or more of the amino acids comprising the native furin cleavage site is mutated to any natural amino acid. In embodiments, the amino acids are L-amino acids. Non-limiting examples of amino acids include alanine, arginine, glycine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, serine, threonine, histidine, lysine, methionine, proline, valine, isoleucine, leucine, tyrosine, tryptophan, and phenylalanine.

[00159] In embodiments, one or more of the amino acids comprising the native furin cleavage site is mutated to glutamine. In embodiments, 1 , 2, 3, or 4 amino acids may be mutated to glutamine. In embodiments, one of the arginines comprising the native furin cleavage site is mutated to glutamine. In embodiments, two of the arginines comprising the native furin cleavage site are mutated to glutamine. In embodiments, three of the arginines comprising the native furin cleavage site are mutated to glutamine.

[00160] In embodiments, one or more of the amino acids comprising the native furin cleavage site, is mutated to alanine. In embodiments, 1 , 2, 3, or 4 amino acids may be mutated to alanine, embodiments, one of the arginines comprising the native furin cleavage site is mutated to alanine. In embodiments, two of the arginines comprising the native furin cleavage site are mutated to alanine. In embodiments, three of the arginines comprising the native furin cleavage site are mutated to alanine. [00161] In embodiments, one or more of the amino acids comprising the native furin cleavage site is mutated to glycine. In embodiments, 1 , 2, 3, or 4 amino acids may be mutated to glycine. In embodiments, one of the arginines of the native furin cleavage site is mutated to glycine. In embodiments, two of the arginines comprising the native furin cleavage site are mutated to glycine. In embodiments, three of the arginines comprising the native furin cleavage site are mutated to glycine.

[00162] In embodiments, one or more of the amino acids comprising the native furin cleavage site, is mutated to asparagine. For example 1 , 2, 3, or 4 amino acids may be mutated to asparagine. In embodiments, one of the arginines comprising the native furin cleavage site is mutated to asparagine. In embodiments, two of the arginines comprising the native furin cleavage site are mutated to asparagine. In embodiments, three of the arginines comprising the native furin cleavage site are mutated to asparagine.

[00163] In embodiments, in lieu of an active furin cleavage site (SEQ ID NO: 6) the CoV S polypeptides described herein contain an inactivated furin cleavage site. In embodiments, the amino acid sequence of the inactivated furin cleavage site is represented by any one of SEQ ID 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).

[00164] Non-limiting examples of the amino acid sequences of the inactivated furin sites contained within the CoV S polypeptides are found in Table 1 E.

Table 1 E

[00165] In embodiments, the CoV S polypeptides contain one or more modifications to the S2 subunit having an 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.

[00166] The amino acid sequence of the S2 subunit (SEQ ID NO: 120) is shown below.

[00167] SVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTS VDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKT PPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLI CAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFN GIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVK QLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASAN LAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTA PAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDWIGIVNN TVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASWNIQKEIDRLNEVAKNL NESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSC GSCCKFDEDDSEPVLKGVKLHYT

[00168] In embodiments, the CoV S polypeptides described herein comprise an S2 subunit with at least 95%, at least 96 %, at least 97%, at least 98 %, at least 99%, at least 99.5 %, at least 99.7 %, at least 99.9 %, or 100% identity to the S2 subunit of SEQ ID NO: 1 or SEQ ID NO: 2. The S2 subunit may have a deletion, an 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, an insertion, or mutation of from about 1 to about 50 amino acids, from about 1 to about 5 amino acids, from about 3 to about 10 amino acids, from about 5 to 10 amino acids, from about 8 to 12 amino acids, from about 10 to 15 amino acids, from about 12 to 17 amino acids, from about 15 to 20 amino acids, from about 18 to 23 amino acids, from about 20 to 25 amino acids, from about 22 to about 27 amino acids, or from about 25 to 30 amino acids as compared to the S2 subunit of SEQ ID NO: 1 or SEQ ID NO: 2.

[00169] In embodiments, the CoV S polypeptides contain 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 polypeptides containing a mutation at amino acid 973 are selected from the group consisting of SEQ ID NO: 84-89, 105-106, and 109-110.

[00170] In embodiments, the CoV S polypeptides contain 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 polypeptides containing a mutation at amino acid 974 are selected from the group consisting of SEQ ID NO: 84-89, 105-106, and 109-110.

[00171] In embodiments, the CoV S polypeptides contain a mutation at Lys-973 and Val-974 of the native CoV Spike (S) polypeptide (SEQ ID NO: 2). In embodiments, Lys-973 and Val-974 are mutated to any natural amino acid. In embodiments, Lys-973 and Val-974 are mutated to proline. In embodiments, the CoV S polypeptides containing a mutation at amino acids 973 and 974 are selected from SEQ ID NOS: 84- 89, 105-106, 109-110, 175, 220, and 217-228.

[00172] In embodiments, the S2 subunit may contain any combination of modifications as shown in Table 1 F.

Table 1F

[00173] In embodiments, the immunogenic compositions comprise 1 , 2, 3, 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11 , about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21 , about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31 , about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41 , about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, or about 50 SARS-CoV-2 S glycoproteins, including all values and ranges therebetween.

[00174] Multiple examples of SARS-CoV-2 S glycoproteins are described in the following patent documents, which are each incorporated by reference herein in their entirety for all purposes: International Publication No. 2021/015220, International Publication No. 2022/020974, International Publication No. 2022/235662, International Publication No. 2023/102448.

Nanoparticles comprising Viral Glycoproteins

[00175] In embodiments, the immunogenic compositions and vaccine compositions described herein comprise nanoparticles. The nanoparticles of the present disclosure are non-naturally occurring products, the components of which do not occur together in nature. In embodiments, the nanoparticles are detergent-core nanoparticles comprising a viral glycoprotein and a non-ionic detergent. In embodiments, provided herein are compositions comprising detergent-core nanoparticles comprising an RSV F glycoprotein and a non-ionic detergent. In embodiments, provided herein are compositions comprising detergent-core nanoparticles comprising a HA glycoprotein and a non-ionic detergent. In embodiments, provided herein are compositions comprising detergent-core nanoparticles comprising a CoV S glycoprotein and a non-ionic detergent.

[00176] In embodiments, a single nanoparticle comprises one or more different viral glycoproteins. In embodiments, provided herein are detergent-core nanoparticles comprising an RSV F glycoprotein and a HA glycoprotein. In embodiments, provided herein are detergent-core nanoparticles comprising an RSV F glycoprotein and a CoV S glycoprotein. In embodiments, provided herein are detergent-core nanoparticles comprising an RSV F glycoprotein, a HA glycoprotein, and a CoV S glycoprotein.

[00177] In embodiments, the detergent-core nanoparticle comprises a viral glycoprotein with 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 glycoproteins form trimers.

[00178] In embodiments, to produce detergent-core nanoparticles, a detergent exchange approach is utilized, wherein a first detergent is used to isolate a viral glycoprotein and then that first detergent is exchanged for a second detergent to form the nanoparticles.

[00179] The viral glycoproteins contained in the nanoparticles are typically produced by recombinant expression in host cells. Standard recombinant techniques may be used. In embodiments, the viral glycoproteins are expressed in insect host cells using a baculovirus system. In embodiments, from 1-50 viral glycoproteins are co-expressed in a host cell. In embodiments, the baculovirus is a cathepsin-L knockout baculovirus, a chitinase knock-out baculovirus. Optionally, the baculovirus is a double knock-out for both cathepsin-L and chitinase. High level expression may be obtained in insect cell expression systems. Non limiting examples of insect cells are, Spodoptera frugiperda (Sf) cells, e.g. Sf9, Sf21 , Trichoplusiani cells, e.g. High Five cells, and Drosophila S2 cells. In embodiments, the viral glycoproteins described herein are produced in any suitable host cell. In embodiments, the host cell is an insect cell. In embodiments, the insect cell is an Sf9 cell.

[00180] Typical transfection and cell growth methods can be used to culture the cells. Vectors, e.g., vectors comprising polynucleotides that encode fusion proteins, can be transfected into host cells according to methods well known in the art. For example, introducing nucleic acids into eukaryotic cells can be achieved by calcium phosphate co-precipitation, electroporation, microinjection, lipofection, and transfection employing polyamine transfection reagents. In one embodiment, the vector is a recombinant baculovirus.

[00181] Methods to grow host cells include, but are not limited to, batch, batch- fed, continuous and perfusion cell culture techniques. Cell culture means the growth and propagation of cells in a bioreactor (a fermentation chamber) where cells propagate and express protein (e.g. recombinant proteins) for purification and isolation. Typically, cell culture is performed under sterile, controlled temperature and atmospheric conditions in a bioreactor. A bioreactor is a chamber used to culture 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. Cellbag®, Wave Biotech, Bridgewater, N.J.). In other embodiment, the pre-sterilized plastic bags are about 50 L to 3500 L bags.

[00182] After growth of the host cells, the protein may be harvested from the host cells using detergents and purification protocols. In embodiments, multiple viral glycoproteins are purified simultaneously. In embodiments, host cells expressing multiple viral glycoproteins are pooled together. Once the host cells have grown for 48 to 96 hours, the cells are isolated from the media and a detergent-containing solution is added to solubilize the cell membrane, releasing the protein in a detergent extract. Triton X-100 and TERGITOL® nonylphenol ethoxylate, also known as NP-9, are each preferred detergents for extraction. The detergent may be added to a final concentration of about 0.1 % to about 1.0%. For example, the concentration may 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 may be about 0.1 % to about 0.3%. In aspects, the concentration is about 0.5%.

[00183] In other aspects, different first detergents may be used to isolate the protein from the host cell. For example, the first detergent may be Bis(polyethylene glycol bis[imidazoylcarbonyl]), nonoxynol-9, Bis(polyethylene glycol bis[imidazoyl carbonyl]), BRU® Polyethylene glycol dodecyl ether 35, BRU® Polyethylene glycol (3) cetyl ether 56, BRU® alcohol ethoxylate 72, BRU® Polyoxyl 2 stearyl ether 76, BRU® polyethylene glycol monoolelyl ether 92V, BRU® Polyoxyethylene (10) oleyl ether 97, BRU® Polyethylene glycol hexadecyl ether 58P, CREMOPHOR® EL Macrogolglycerol ricinoleate, Decaethyleneglycol monododecyl ether, N-Decanoyl-N- methylglucamine, n-Decyl alpha-Dglucopyranoside, Decyl beta-D-maltopyranoside, n- Dodecanoyl-N-methylglucamide, nDodecyl alpha-D-maltoside, n-Dodecyl beta-D- maltoside, n-Dodecyl beta-D-maltoside, Heptaethylene glycol monodecyl ether, Heptaethylene glycol monododecyl ether, Heptaethylene glycol monotetradecyl ether, n-Hexadecyl beta-D-maltoside, Hexaethylene glycol monododecyl ether, Hexaethylene glycol monohexadecyl ether, Hexaethylene glycol monooctadecyl ether, Hexaethylene glycol monotetradecyl ether, Igepal CA-630,lgepal CA -630, Methyl-6-0-(N -heptylcarbamoyl)-alpha-D-glucopyranoside, Nonaethylene glycol monododecyl ether, N-Nonanoyl-N-methylglucamine, N-NonanoylN- methylglucamine, Octaethylene glycol monodecyl ether, Octaethylene glycolmonododecyl ether, Octaethylene glycol monohexadecyl ether, Octaethylene glycol monooctadecyl ether, Octaethylene glycol monotetradecyl ether, Octyl-beta-D glucopyranoside, Pentaethylene glycol monodecyl ether, Pentaethylene glycol monododecyl ether, Pentaethylene glycol monohexadecyl ether, Pentaethylene glycol monohexyl ether, Pentaethylene glycol monooctadecyl ether, Pentaethylene glycol monooctyl ether, Polyethylene glycol diglycidyl ether, Polyethylene glycol ether W-1 , Polyoxyethylene 10 tridecyl ether, Polyoxyethylene 100 stearate, Polyoxyethylene 20 isohexadecyl ether, Polyoxyethylene 20 oleyl ether, Polyoxyethylene 40 stearate, Polyoxyethylene 50 stearate, Polyoxyethylene 8 stearate, Polyoxyethylene bis(imidazolyl carbonyl), Polyoxyethylene 25 propylene glycol stearate, Saponin from Quillaja bark, SPAN® 20 sorbitan laurate, SPAN® 40 sorbitan monopalmitate, SPAN® 60 sorbitan stearate, SPAN® 65 sorbitan tristearate, SPAN® 80 sorbitane monooleate, SPAN® 85 sorbitane trioleate, TERGITOL® secondary alcohol ethoxylate Type 15-S-12, TERGITOL® secondary alcohol ethoxylate Type 15-S-30, TERGITOL® secondary alcohol ethoxylate Type 15-S-5, TERGITOL® secondary alcohol ethoxylate Type 15-S-7, TERGITOL® secondary alcohol ethoxylate Type 15- S-9, TERGITOL® nonylphenol ethoxylate Type NP-10, TERGITOL® nonylphenol ethoxylate Type NP-4, TERGITOL® nonylphenol ethoxylate Type NP-40, TERGITOL® nonylphenol ethoxylate Type NP-7, TERGITOL® nonylphenol ethoxylate Type NP-9, TERGITOL® branched secondary alcohol ethoxylate Type TMN-10, TERGITOL® branched secondary alcohol ethoxylate Type TMN-6, TRITONTM X-100 Polyethylene glycol tert-octyl phenyl ether or combinations thereof. [00184] The nanoparticles may then be isolated from cellular debris using centrifugation. In embodiments, gradient centrifugation, such as using cesium chloride, sucrose and iodixanol, may be used. Other techniques may be used as alternatives or in addition, such as standard purification techniques including, e.g., ion exchange, affinity, and gel filtration chromatography.

[00185] For example, the first column may be an ion exchange chromatography resin, such as FRACTOGEL® EMD methacrylate based polymeric beads TMAE (EMD Millipore), the second column may be a lentil (Lens culinaris) lectin affinity resin, and the third column may be a cation exchange column such as a FRACTOGEL® EMD methacrylate based polymeric beads SO3 (EMD Millipore) resin. In other aspects, the cation exchange column may be an MMC column or a Nuvia C Prime column (BioRad Laboratories, Inc). Preferably, the methods disclosed herein do not use a detergent extraction column; for example a hydrophobic interaction column. Such a column is often used to remove detergents during purification but may negatively impact the methods disclosed here.

[00186] To form detergent-core nanoparticles, the first detergent, used to extract the protein from the host cell is substantially replaced with a second detergent to arrive at the nanoparticle structure. In embodiments, the first detergent is NP-9. Typically, the nanoparticles do not contain detectable NP-9 when measured by HPLC. The second detergent is typically selected from the group consisting of PS20, PS40, PS60, PS65, and PS80. In embodiments, the second detergent is PS80.

[00187] In particular aspects, detergent exchange is performed using affinity chromatography to bind glycoproteins via their carbohydrate moiety. For example, the affinity chromatography may use a legume lectin column. Legume lectins are proteins originally identified in plants and found to interact specifically and reversibly with carbohydrate residues. See, for example, Sharon and Lis, “Legume lectins-a large family of homologous proteins,” FASEB J. 1990 Nov;4(14):3198-208; Liener, “The Lectins: Properties, Functions, and Applications in Biology and Medicine,” Elsevier, 2012. Suitable lectins include concanavalin A (con A), pea lectin, sainfoin lect, 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 Lentil Lectin, is available from GE Healthcare. In certain aspects, the lentil lectin column may use a recombinant lectin. At the molecular level, it is thought that the carbohydrate moieties bind to the lentil lectin, freeing the amino acids of the protein to coalesce around the detergent resulting in the formation of a detergent core providing nanoparticles having multiple copies of the antigen, e.g., glycoprotein oligomers which can be dimers, trimers, or tetramers anchored in the detergent. In embodiments, the viral glycoproteins form trimers. In embodiments, the viral glycoprotein trimers are anchored in detergent. In embodiments, each viral glycoprotein nanoparticle contains at least one trimer associated with a non-ionic core.

[00188] The detergent, when incubated with the protein to form the nanoparticles during detergent exchange, may be present at up to about 0.1 % (w/v) during early purifications steps and this amount is lowered to achieve the final nanoparticles having optimum stability. For example, the non-ionic detergent (e.g., PS80) may be about 0.005% (v/v) to about 0.1 % (v/v), for example, 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 0.04 % (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 embodiments, the nanoparticle contains about 0.03% to about 0.05% PS80. In embodiments, the nanoparticle contains about 0.01 % (v/v) PS80.

[00189] In embodiments, purified viral glycoproteins are dialyzed. In embodiments, dialysis occurs after purification. In embodiments, the viral glycoproteins are dialyzed in a solution comprising sodium phosphate, NaCI, and PS80. In embodiments, the dialysis solution comprising sodium phosphate contains from about 5 mM to about 100 mM of sodium phosphate, for example, about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, about 80 mM, about 85 mM, about 90 mM, about 95 mM, or about 100 mM sodium phosphate. In 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 NaCI to about 750 mM NaCI, 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 510 mM, about 520 mM, about 530 mM, about 540 mM, about 550 mM, about 560 mM, about 570 mM, about 580 mM, about 590 mM, about 600 mM, about 610 mM, about 620 mM, about 630 mM, about 640 mM, about 650 mM, about 660 mM, about 670 mM, about 680 mM, about 690, about 700 mM, about 710 mM, about 720 mM, about

730 mM, about 740 mM, or about 750 mM NaCI. 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 0.04 % (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 embodiments, the dialysis solution comprises about 25 mM sodium phosphate (pH 7.2), about 300 mM NaCI, and about 0.01 % (v/v) PS80.

[00190] In embodiments, the pharmaceutically acceptable buffer comprises 10 mM sodium phosphate, 150 mM NaCI, 100 mM arginine, 5 % trehalose, and 0.03 % PS80 at a pH of 7.5. In embodiments, the pharmaceutically acceptable buffer comprises 25 mM sodium phosphate, 300 mM NaCI, and 0.03 % PS80 at a pH of 7.2. In embodiments, the pharmaceutically acceptable buffer comprises 25 mM sodium phosphate, 600 mM NaCI, and 0.01 % PS80 at a pH of 6.8.

[00191] Detergent exchange may be performed with proteins purified as discussed above and purified, frozen for storage, and then thawed for detergent exchange.

[00192] Stability of compositions disclosed herein may be measured in a variety of ways. In one approach, a peptide map may be prepared to determine the integrity of the antigen protein after various treatments designed to stress the nanoparticles by mimicking harsh storage conditions. Thus, a measure of stability is the relative abundance of antigen peptides in a stressed sample compared to a control sample. For example, the stability of nanoparticles containing the viral glycoproteins may be evaluated by exposing the nanoparticles to various pHs, proteases, salt, oxidizing agents, including but not limited to hydrogen peroxide, various temperatures, freeze/thaw cycles, and agitation. It is thought that the position of the glycoprotein anchored into the detergent core provides enhanced stability by reducing undesirable interactions. For example, the improved protection against protease-based degradation may be achieved through a shielding effect whereby anchoring the glycoproteins into the core at the molar ratios disclosed herein results in steric hindrance blocking protease access. Stability may also be measured by monitoring intact proteins.

[00193] In embodiments, provided herein are immunogenic compositions and vaccine compositions containing nanoparticles comprising RSV F glycoproteins, HA glycoproteins, SARS-CoV-2 S glycoproteins, or a combination thereof.

[00194] In embodiments, the mature RSV F glycoproteins are used to produce a vaccine comprising RSV F glycoprotein nanoparticles. In embodiments, nanoparticles of the present disclosure comprise the RSV F glycoproteins described herein. In embodiments, immunogenic compositions and vaccine compositions provided herein comprise nanoparticles comprising CoV S glycoproteins, influenza HA glycoproteins, RSV F glycoproteins, or any combination thereof.

[00195] In embodiments, the nanoparticles of the present disclosure comprise viral glycoproteins (e.g., RSV F, CoV S, influenza HA glycoproteins, or a combination thereof) associated with a detergent core. The presence of the detergent facilitates formation of the nanoparticles by forming a core that organizes and presents the antigens. In embodiments, the nanoparticles may contain the viral glycoprotein assembled into multi-oligomeric glycoprotein-detergent (e.g. PS80) nanoparticles with the head regions projecting outward and hydrophobic regions and detergent forming a central core surrounded by the glycoprotein. In embodiments, the viral glycoprotein inherently contains or is adapted to contain a transmembrane domain to promote association of the protein into a detergent core. In embodiments, the viral glycoprotein contains a head domain. Primarily the transmembrane domains of a viral glycoprotein trimer associate with detergent; however, other portions 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 due to organization of multiple copies of the protein around the detergent.

[00196] In embodiments, the detergent core is a non-ionic detergent core. In embodiments, RSV F glycoprotein is associated with the non-ionic 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).

[00197] In embodiments, the detergent is PS80.

[00198] In embodiments, the RSV F glycoprotein forms a trimer. In embodiments, the RSV F glycoprotein nanoparticles are composed of multiple polypeptide trimers surrounding a non-ionic detergent core. In embodiments, the nanoparticles contain at least about 1 trimer or more. In embodiments, the nanoparticles contain at least about 5 trimers to about 30 trimers of the Spike protein. In embodiments, each nanoparticle may contain 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, or 15, 20, 25, or 30 trimers, including all values and ranges in between. Compositions disclosed herein may contain nanoparticles having different numbers of trimers. For example, a composition may contain nanoparticles where the number of trimers ranges from 2-9; in embodiments, the nanoparticles in a composition may contain from 2-6 trimers. In embodiments, the compositions contain a heterogeneous population of nanoparticles having 2 to 6 trimers per nanoparticle, or 2 to 9 trimers per nanoparticle. In embodiments, the compositions may contain a substantially homogenous population of nanoparticles. For example, the population may contain about 95% nanoparticles having 5 trimers.

[00199] The nanoparticles disclosed herein range in particle size. In embodiments, the nanoparticles disclosed herein range in particle size from a Z-ave size from about 20 nm to about 60 nm, about 20 nm to about 50 nm, about 20 nm to about 45 nm, about 20 nm to about 35 nm, about 20 nm to about 30 nm, about 25 nm to about 35 nm, about 25 nm to about 45 nm, about 30 nm to about 120 nm, about 30 nm to about 80 nm, about 30 nm to about 60 nm, about 30 nm to about 65 nm, or from about 30 nm to about 50 nm. Particle size (Z-ave) is measured by dynamic light scattering (DLS) using a Zetasizer NanoZS (Malvern, UK), unless otherwise specified. [00200] Several nanoparticle types may be included in vaccine compositions disclosed herein. In aspects, the nanoparticle type is in the form of an anisotropic rod, which may be a dimer or a monomer. In other aspects, the nanoparticle type is a spherical oligomer. In yet other aspects, the nanoparticle may be described as an intermediate nanoparticle, having sedimentation properties intermediate between the first two types. Formation of nanoparticle types may be regulated by controlling detergent and protein concentration during the production process. Nanoparticle type may be determined by measuring sedimentation co-efficient. Amounts of Viral Glycoproteins In Immunogenic Compositions and Vaccine Compositions

[00201] In embodiments, the compositions described herein comprise from about 1 pg to about 400 pg, from 1 pg to about 350 pg, from 1 pg to about 300 pg, from 1 pg to about 250 pg, from 1 pg to about 200 pg, from 1 pg to about 150 pg, from 1 pg to about 100 pg, from 1 pg to about 50 pg, or from 1 pg to about 25 pg of each viral glycoprotein. In embodiments, the compositions comprise about 1 pg, about 2 pg, about 3 pg, about 4 pg, about 5 pg, about 6 pg, about 7 pg, about 8 pg, about 9 pg, about 10 pg, about 11 pg, about 12 pg, about 13 pg, about 14 pg, about 15 pg, about 16 pg, about 17 pg, about 18 pg, about 19 pg, about 20 pg, about 21 pg, about 22 pg, about 23 pg, about 24 pg, about 25 pg, about 26 pg, about 27 pg, about 28 pg, about 29 pg, about 30 pg, about 31 pg, about 32 pg, about 33 pg, about 34 pg, about 35 pg, about 36 pg, about 37 pg, about 38 pg, about 39 pg, about 40 pg, about 41 pg, about 42 pg, about 43 pg, about 44 pg, about 45 pg, about 46 pg, about 47 pg, about 48 pg, about 49 pg, about 50 pg, about 51 pg, about 52 pg, about 53 pg, about 54 pg, about 55 pg, about 56 pg, about 57 pg, about 58 pg, about 59 pg, about 60 pg, about 61 pg, about 62 pg, about 63 pg, about 64 pg, about 65 pg, about 66 pg, about 67 pg, about 68 pg, about 69 pg, about 70 pg, about 71 pg, about 72 pg, about 73 pg, about 74 pg, about 75 pg, about 76 pg, about 77 pg, about 78 pg, about 79 pg, about 80 pg, about 81 pg, about 82 pg, about 83 pg, about 84 pg, about 85 pg, about 86 pg, about 87 pg, about 88 pg, about 89 pg, about 90 pg, about 91 pg, about 92 pg, about 93 pg, about 94 pg, about 95 pg, about 96 pg, about 97 pg, about 98 pg, about 99 pg, about 100 pg, about 101 pg, about 102 pg, about 103 pg, about 104 pg, about 105 pg, about 106 pg, about 107 pg, about 108 pg, about 109 pg, about 110 pg, about 111 pg, about 112 pg, about 113 pg, about 114 pg, about 115 pg, about 116 pg, about 117 pg, about 118 pg, about 119 pg, about 120 pg, about 121 pg, about 122 pg, about 123 pg, about 124 pg, about 125 pg, about 126 pg, about 127 pg, about 128 pg, about 129 pg, about 130 pg, about 131 pg, about 132 pg, about 133 pg, about 134 pg, about 135 pg, about 136 pg, about 137 pg, about 138 pg, about 139 pg, about 140 pg, about 141 pg, about 142 pg, about 143 pg, about 144 pg, about 145 pg, about 146 pg, about 147 pg, about 148 pg, about 149 pg, about 150 pg, about 151 pg, about 152 pg, about 153 pg, about 154 pg, about 155 pg, about 156 pg, about 157 pg, about 158 pg, about 159 pg, about 160 pg, about 161 pg, about 162 pg, about 163 pg, about 164 pg, about 165 pg, about 166 pg, about 167 pg, about 168 pg, about 169 pg, about 170 pg, about 171 pg, about 172 pg, about 173 pg, about 174 pg, about 175 pg, about 176 pg, about 177 pg, about 178 pg, about 179 pg, about 180 pg, about 181 pg, about 182 pg, about 183 pg, about 184 pg, about 185 pg, about 186 pg, about 187 pg, about 188 pg, about 189 pg, about 190 pg, about 191 pg, about 192 pg, about 193 pg, about 194 pg, about 195 pg, about 196 pg, about 197 pg, about 198 pg, about 199 pg, about 200 pg, about 201 pg, about 202 pg, about 203 pg, about 204 pg, about 205 pg, about 206 pg, about 207 pg, about 208 pg, about 209 pg, about 210 pg, about 211 pg, about 212 pg, about 213 pg, about 214 pg, about 215 pg, about 216 pg, about 217 pg, about 218 pg, about 219 pg, about 220 pg, about 221 pg, about 222 pg, about 223 pg, about 224 pg, about 225 pg, about 226 pg, about 227 pg, about 228 pg, about 229 pg, about 230 pg, about 231 pg, about 232 pg, about 233 pg, about 234 pg, about 235 pg, about 236 pg, about 237 pg, about 238 pg, about 239 pg, about 240 pg, about 241 pg, about 242 pg, about 243 pg, about 244 pg, about 245 pg, about 246 pg, about 247 pg, about 248 pg, about 249 pg, about 250 pg, about 251 pg, about 252 pg, about 253 pg, about 254 pg, about 255 pg, about 256 pg, about 257 pg, about 258 pg, about 259 pg, about 260 pg, about 261 pg, about 262 pg, about 263 pg, about 264 pg, about 265 pg, about 266 pg, about 267 pg, about 268 pg, about 269 pg, about 270 pg, about 271 pg, about 272 pg, about 273 pg, about 274 pg, about 275 pg, about 276 pg, about 277 pg, about 278 pg, about 279 pg, about 280 pg, about 281 pg, about 282 pg, about 283 pg, about 284 pg, about 285 pg, about 286 pg, about 287 pg, about 288 pg, about 289 pg, about 290 pg, about 291 pg, about 292 pg, about 293 pg, about 294 pg, about 295 pg, about 296 pg, about 297 pg, about 298 pg, about 299 pg, about 300 pg, about 301 pg, about 302 pg, about 303 pg, about 304 pg, about 305 pg, about 306 pg, about 307 pg, about 308 pg, about 309 pg, about 310 pg, about 311 pg, about 312 pg, about 313 pg, about 314 pg, about 315 pg, about 316 pg, about 317 pg, about 318 pg, about 319 pg, about 320 pg, about 321 pg, about 322 pg, about 323 pg, about 324 pg, about 325 pg, about 326 pg, about 327 pg, about 328 pg, about 329 pg, about 330 pg, about 331 pg, about 332 pg, about 333 pg, about 334 pg, about 335 pg, about 336 pg, about 337 pg, about 338 pg, about 339 pg, about 340 pg, about 341 pg, about 342 pg, about 343 pg, about 344 pg, about 345 pg, about 346 pg, about 347 pg, about 348 pg, about 349 pg, about 350 pg, about 351 pg, about 352 pg, about 353 pg, about 354 pg, about 355 pg, about 356 pg, about 357 pg, about 358 pg, about 359 pg, about 360 pg, about 361 pg, about 362 pg, about 363 pg, about 364 pg, about 365 pg, about 366 pg, about 367 pg, about 368 pg, about 369 pg, about 370 pg, about 371 pg, about 372 pg, about 373 pg, about 374 pg, about 375 pg, about 376 pg, about 377 pg, about 378 pg, about 379 pg, about 380 pg, about 381 pg, about 382 pg, about 383 pg, about 384 pg, about 385 pg, about 386 pg, about 387 pg, about 388 pg, about 389 pg, about 390 pg, about 391 pg, about 392 pg, about 393 pg, about 394 pg, about 395 pg, about 396 pg, about 397 pg, about 398 pg, about 399 pg, or about

400 pg of each viral glycoprotein.

[00202] In embodiments, the immunogenic compositions comprise about 1 pg of each viral glycoprotein. In embodiments, the immunogenic compositions comprise about 5 pg of each viral glycoprotein. In embodiments, the immunogenic compositions comprise about 25 pg of each viral glycoprotein. In embodiments, the immunogenic compositions comprise about 60 pg of each viral glycoprotein. In embodiments, the immunogenic compositions comprise about 240 pg of each viral glycoprotein.

[00203] In embodiments, the immunogenic compositions described herein comprise from about 1 pg to about 300 pg of each RSV F glycoprotein. In embodiments, the immunogenic compositions described herein comprise from about 5 pg to about 60 pg of each RSV F glycoprotein. In embodiments, the immunogenic compositions described herein comprise from about 24 pg to about 40 pg of each RSV F glycoprotein. In embodiments, the immunogenic compositions described herein comprise from about 30 pg to about 40 pg of each RSV F glycoprotein. In embodiments, the immunogenic compositions described herein comprise from about 30 pg to about 60 pg of each RSV F glycoprotein. In embodiments, the immunogenic compositions described herein comprise from about 33 pg to about 39 pg of each RSV F glycoprotein. In embodiments, the immunogenic compositions described herein comprise from about 20 pg to about 240 pg of each RSV F glycoprotein. In embodiments, the immunogenic compositions described herein comprise from about 30 pg to about 60 pg of each RSV F glycoprotein. In embodiments, the immunogenic compositions described herein comprise from about 24 pg to about 40 pg of each RSV F glycoprotein. In embodiments, the immunogenic compositions comprise about 1 pg, about 2 pg, about 3 pg, about 4 pg, about 5 pg, about 6 pg, about 7 pg, about 8 pg, about 9 pg, about 10 pg, about 11 pg, about 12 pg, about 13 pg, about 14 pg, about 15 pg, about 16 pg, about 17 pg, about 18 pg, about 19 pg, about 20 pg, about 21 pg, about 22 pg, about 23 pg, about 24 pg, about 25 pg, about 26 pg, about 27 pg, about 28 pg, about 29 pg, about 30 pg, about 31 pg, about 32 pg, about 33 pg, about 34 pg, about 35 pg, about 36 pg, about 37 pg, about 38 pg, about 39 pg, about 40 pg, about 41 pg, about 42 pg, about 43 pg, about 44 pg, about 45 pg, about 46 pg, about 47 pg, about 48 pg, about 49 pg, about 50 pg, about 51 pg, about 52 pg, about 53 pg, about 54 pg, about 55 pg, about 56 pg, about 57 pg, about 58 pg, about 59 pg, about 60 pg, about 61 pg, about 62 pg, about 63 pg, about 64 pg, about 65 pg, about 66 pg, about 67 pg, about 68 pg, about 69 pg, about 70 pg, about 71 pg, about 72 pg, about 73 pg, about 74 pg, about 75 pg, about 76 pg, about 77 pg, about 78 pg, about 79 pg, about 80 pg, about 81 pg, about 82 pg, about 83 pg, about 84 pg, about 85 pg, about 86 pg, about 87 pg, about 88 pg, about 89 pg, about 90 pg, about 91 pg, about 92 pg, about 93 pg, about 94 pg, about 95 pg, about 96 pg, about 97 pg, about 98 pg, about 99 pg, about 100 pg, about 101 pg, about 102 pg, about 103 pg, about 104 pg, about 105 pg, about 106 pg, about 107 pg, about 108 pg, about 109 pg, about 110 pg, about 111 pg, about 112 pg, about 113 pg, about 114 pg, about 115 pg, about 116 pg, about 117 pg, about 118 pg, about 119 pg, about 120 pg, about 121 pg, about 122 pg, about 123 pg, about 124 pg, about 125 pg, about 126 pg, about 127 pg, about 128 pg, about 129 pg, about 130 pg, about 131 pg, about 132 pg, about 133 pg, about 134 pg, about 135 pg, about 136 pg, about 137 pg, about 138 pg, about 139 pg, about 140 pg, about 141 pg, about 142 pg, about 143 pg, about 144 pg, about 145 pg, about 146 pg, about 147 pg, about 148 pg, about 149 pg, about 150 pg, about 151 pg, about 152 pg, about 153 pg, about 154 pg, about 155 pg, about 156 pg, about 157 pg, about 158 pg, about 159 pg, about 160 pg, about 161 pg, about 162 pg, about 163 pg, about 164 pg, about 165 pg, about 166 pg, about 167 pg, about 168 pg, about 169 pg, about 170 pg, about 171 pg, about 172 pg, about 173 pg, about 174 pg, about 175 pg, about 176 pg, about 177 pg, about 178 pg, about 179 pg, about 180 pg, about 181 pg, about 182 pg, about 183 pg, about 184 pg, about 185 pg, about 186 pg, about 187 pg, about 188 pg, about 189 pg, about 190 pg, about 191 pg, about 192 pg, about 193 pg, about 194 pg, about 195 pg, about 196 pg, about 197 pg, about 198 pg, about 199 pg, about 200 pg, about 201 pg, about 202 pg, about 203 pg, about 204 pg, about 205 pg, about 206 pg, about 207 pg, about 208 pg, about 209 pg, about 210 pg, about 211 pg, about 212 pg, about 213 pg, about 214 pg, about 215 pg, about 216 pg, about 217 pg, about 218 pg, about 219 pg, about 220 pg, about 221 pg, about 222 pg, about 223 pg, about 224 pg, about 225 pg, about 226 pg, about 227 pg, about 228 pg, about 229 pg, about 230 pg, about 231 pg, about 232 pg, about 233 pg, about 234 pg, about 235 pg, about 236 pg, about 237 pg, about 238 pg, about 239 pg, about 240 pg, about 241 pg, about 242 pg, about 243 pg, about 244 pg, about 245 pg, about 246 pg, about 247 pg, about 248 pg, about 249 pg, about 250 pg, about 251 pg, about 252 pg, about 253 pg, about 254 pg, about 255 pg, about 256 pg, about 257 pg, about 258 pg, about 259 pg, about 260 pg, about 261 pg, about 262 pg, about 263 pg, about 264 pg, about 265 pg, about 266 pg, about 267 pg, about 268 pg, about 269 pg, about 270 pg, about 271 pg, about 272 pg, about 273 pg, about 274 pg, about 275 pg, about 276 pg, about 277 pg, about 278 pg, about 279 pg, about 280 pg, about 281 pg, about 282 pg, about 283 pg, about 284 pg, about 285 pg, about 286 pg, about 287 pg, about 288 pg, about 289 pg, about 290 pg, about 291 pg, about 292 pg, about 293 pg, about 294 pg, about 295 pg, about 296 pg, about 297 pg, about 298 pg, about 299 pg, or about 300 pg of each RSV F glycoprotein, including all values and ranges therebetween. In embodiments, the immunogenic compositions described herein comprise about 60 pg of each RSV F glycoprotein. In embodiments, the immunogenic compositions described herein comprise about 240 pg of each RSV F glycoprotein.

[00204] In embodiments, the immunogenic compositions described herein comprise from about 1 pg to about 1000 pg of total RSV F glycoproteins. In embodiments, the immunogenic compositions comprise about 1 pg, about 2 pg, about 3 pg, about 4 pg, about 5 pg, about 6 pg, about 7 pg, about 8 pg, about 9 pg, about 10 pg, about 11 pg, about 12 pg, about 13 pg, about 14 pg, about 15 pg, about 16 pg, about 17 pg, about 18 pg, about 19 pg, about 20 pg, about 21 pg, about 22 pg, about 23 pg, about 24 pg, about 25 pg, about 26 pg, about 27 pg, about 28 pg, about 29 pg, about 30 pg, about 31 pg, about 32 pg, about 33 pg, about 34 pg, about 35 pg, about 36 pg, about 37 pg, about 38 pg, about 39 pg, about 40 pg, about 41 pg, about 42 pg, about 43 pg, about 44 pg, about 45 pg, about 46 pg, about 47 pg, about 48 pg, about 49 pg, about 50 pg, about 51 pg, about 52 pg, about 53 pg, about 54 pg, about 55 pg, about 56 pg, about 57 pg, about 58 pg, about 59 pg, about 60 pg, about 61 pg, about 62 pg, about 63 pg, about 64 pg, about 65 pg, about 66 pg, about 67 pg, about 68 pg, about 69 pg, about 70 pg, about 71 pg, about 72 pg, about 73 pg, about 74 pg, about 75 pg, about 76 pg, about 77 pg, about 78 pg, about 79 pg, about 80 pg, about 81 pg, about 82 pg, about 83 pg, about 84 pg, about 85 pg, about 86 pg, about 87 pg, about 88 pg, about 89 pg, about 90 pg, about 91 pg, about 92 pg, about 93 pg, about 94 pg, about 95 pg, about 96 pg, about 97 pg, about 98 pg, about 99 pg, about 100 pg, about 101 pg, about 102 pg, about 103 pg, about 104 pg, about 105 pg, about 106 pg, about

107 pg, about 108 pg, about 109 pg, about 110 pg, about 111 pg, about 112 pg, about

113 pg, about 114 pg, about 115 pg, about 116 pg, about 117 pg, about 118 pg, about

119 pg, about 120 pg, about 121 pg, about 122 pg, about 123 pg, about 124 pg, about pg, about 126 pg, about 127 pg, about 128 pg, about 129 pg, about 130 pg, about pg, about 132 pg, about 133 pg, about 134 pg, about 135 pg, about 136 pg, about pg, about 138 pg, about 139 pg, about 140 pg, about 141 pg, about 142 pg, about pg, about 144 pg, about 145 pg, about 146 pg, about 147 pg, about 148 pg, about pg, about 150 pg, about 151 pg, about 152 pg, about 153 pg, about 154 pg, about pg, about 156 pg, about 157 pg, about 158 pg, about 159 pg, about 160 pg, about pg, about 162 pg, about 163 pg, about 164 pg, about 165 pg, about 166 pg, about pg, about 168 pg, about 169 pg, about 170 pg, about 171 pg, about 172 pg, about pg, about 174 pg, about 175 pg, about 176 pg, about 177 pg, about 178 pg, about pg, about 180 pg, about 181 pg, about 182 pg, about 183 pg, about 184 pg, about pg, about 186 pg, about 187 pg, about 188 pg, about 189 pg, about 190 pg, about pg, about 192 pg, about 193 pg, about 194 pg, about 195 pg, about 196 pg, about pg, about 198 pg, about 199 pg, about 200 pg, about 201 pg, about 202 pg, about pg, about 204 pg, about 205 pg, about 206 pg, about 207 pg, about 208 pg, about pg, about 210 pg, about 211 pg, about 212 pg, about 213 pg, about 214 pg, about pg, about 216 pg, about 217 pg, about 218 pg, about 219 pg, about 220 pg, about pg, about 222 pg, about 223 pg, about 224 pg, about 225 pg, about 226 pg, about pg, about 228 pg, about 229 pg, about 230 pg, about 231 pg, about 232 pg, about pg, about 234 pg, about 235 pg, about 236 pg, about 237 pg, about 238 pg, about pg, about 240 pg, about 241 pg, about 242 pg, about 243 pg, about 244 pg, about pg, about 246 pg, about 247 pg, about 248 pg, about 249 pg, about 250 pg, about pg, about 252 pg, about 253 pg, about 254 pg, about 255 pg, about 256 pg, about pg, about 258 pg, about 259 pg, about 260 pg, about 261 pg, about 262 pg, about pg, about 264 pg, about 265 pg, about 266 pg, about 267 pg, about 268 pg, about pg, about 270 pg, about 271 pg, about 272 pg, about 273 pg, about 274 pg, about pg, about 276 pg, about 277 pg, about 278 pg, about 279 pg, about 280 pg, about pg, about 282 pg, about 283 pg, about 284 pg, about 285 pg, about 286 pg, about pg, about 288 pg, about 289 pg, about 290 pg, about 291 pg, about 292 pg, about pg, about 294 pg, about 295 pg, about 296 pg, about 297 pg, about 298 pg, about pg, about 300 pg, about 301 pg, about 302 pg, about 303 pg, about 304 pg, about pg, about 306 pg, about 307 pg, about 308 pg, about 309 pg, about 310 pg, about pg, about 312 pg, about 313 pg, about 314 pg, about 315 pg, about 316 pg, about pg, about 318 pg, about 319 pg, about 320 pg, about 321 pg, about 322 pg, about pg, about 324 pg, about 325 pg, about 326 pg, about 327 pg, about 328 pg, about pg, about 330 pg, about 331 pg, about 332 pg, about 333 pg, about 334 pg, about pg, about 336 pg, about 337 pg, about 338 pg, about 339 pg, about 340 pg, about pg, about 342 pg, about 343 pg, about 344 pg, about 345 pg, about 346 pg, about pg, about 348 pg, about 349 pg, about 350 pg, about 351 pg, about 352 pg, about pg, about 354 pg, about 355 pg, about 356 pg, about 357 pg, about 358 pg, about pg, about 360 pg, about 361 pg, about 362 pg, about 363 pg, about 364 pg, about pg, about 366 pg, about 367 pg, about 368 pg, about 369 pg, about 370 pg, about pg, about 372 pg, about 373 pg, about 374 pg, about 375 pg, about 376 pg, about pg, about 378 pg, about 379 pg, about 380 pg, about 381 pg, about 382 pg, about pg, about 384 pg, about 385 pg, about 386 pg, about 387 pg, about 388 pg, about pg, about 390 pg, about 391 pg, about 392 pg, about 393 pg, about 394 pg, about pg, about 396 pg, about 397 pg, about 398 pg, about 399 pg, about 400 pg, about pg, about 402 pg, about 403 pg, about 404 pg, about 405 pg, about 406 pg, about pg, about 408 pg, about 409 pg, about 410 pg, about 411 pg, about 412 pg, about pg, about 414 pg, about 415 pg, about 416 pg, about 417 pg, about 418 pg, about pg, about 420 pg, about 421 pg, about 422 pg, about 423 pg, about 424 pg, about pg, about 426 pg, about 427 pg, about 428 pg, about 429 pg, about 430 pg, about pg, about 432 pg, about 433 pg, about 434 pg, about 435 pg, about 436 pg, about pg, about 438 pg, about 439 pg, about 440 pg, about 441 pg, about 442 pg, about pg, about 444 pg, about 445 pg, about 446 pg, about 447 pg, about 448 pg, about pg, about 450 pg, about 451 pg, about 452 pg, about 453 pg, about 454 pg, about pg, about 456 pg, about 457 pg, about 458 pg, about 459 pg, about 460 pg, about pg, about 462 pg, about 463 pg, about 464 pg, about 465 pg, about 466 pg, about pg, about 468 pg, about 469 pg, about 470 pg, about 471 pg, about 472 pg, about pg, about 474 pg, about 475 pg, about 476 pg, about 477 pg, about 478 pg, about pg, about 480 pg, about 481 pg, about 482 pg, about 483 pg, about 484 pg, about pg, about 486 pg, about 487 pg, about 488 pg, about 489 pg, about 490 pg, about pg, about 492 pg, about 493 pg, about 494 pg, about 495 pg, about 496 pg, about pg, about 498 pg, about 499 pg, about 500 pg, about 501 pg, about 502 pg, about pg, about 504 pg, about 505 pg, about 506 pg, about 507 pg, about 508 pg, about pg, about 510 pg, about 511 pg, about 512 pg, about 513 pg, about 514 pg, about pg, about 516 pg, about 517 pg, about 518 pg, about 519 pg, about 520 pg, about pg, about 522 pg, about 523 pg, about 524 pg, about 525 pg, about 526 pg, about pg, about 528 pg, about 529 pg, about 530 pg, about 531 pg, about 532 pg, about pg, about 534 pg, about 535 pg, about 536 pg, about 537 pg about 538 pg, about pg, about 540 pg, about 541 pg, about 542 pg, about 543 pg about 544 pg, about pg, about 546 pg, about 547 pg, about 548 pg, about 549 pg about 550 pg, about pg, about 552 pg, about 553 pg, about 554 pg, about 555 pg about 556 pg, about pg, about 558 pg, about 559 pg, about 560 pg, about 561 pg about 562 pg, about pg, about 564 pg, about 565 pg, about 566 pg, about 567 pg about 568 pg, about pg, about 570 pg, about 571 pg, about 572 pg, about 573 pg about 574 pg, about pg, about 576 pg, about 577 pg, about 578 pg, about 579 pg about 580 pg, about pg, about 582 pg, about 583 pg, about 584 pg, about 585 pg about 586 pg, about pg, about 588 pg, about 589 pg, about 590 pg, about 591 pg about 592 pg, about pg, about 594 pg, about 595 pg, about 596 pg, about 597 pg about 598 pg, about pg, about 600 pg, about 601 pg, about 602 pg, about 603 pg about 604 pg, about pg, about 606 pg, about 607 pg, about 608 pg, about 609 pg about 610 pg, about pg, about 612 pg, about 613 pg, about 614 pg, about 615 pg about 616 pg, about pg, about 618 pg, about 619 pg, about 620 pg, about 621 pg about 622 pg, about pg, about 624 pg, about 625 pg, about 626 pg, about 627 pg about 628 pg, about pg, about 630 pg, about 631 pg, about 632 pg, about 633 pg about 634 pg, about pg, about 636 pg, about 637 pg, about 638 pg, about 639 pg about 640 pg, about pg, about 642 pg, about 643 pg, about 644 pg, about 645 pg about 646 pg, about pg, about 648 pg, about 649 pg, about 650 pg, about 651 pg about 652 pg, about pg, about 654 pg, about 655 pg, about 656 pg, about 657 pg about 658 pg, about pg, about 660 pg, about 661 pg, about 662 pg, about 663 pg about 664 pg, about pg, about 666 pg, about 667 pg, about 668 pg, about 669 pg about 670 pg, about pg, about 672 pg, about 673 pg, about 674 pg, about 675 pg about 676 pg, about pg, about 678 pg, about 679 pg, about 680 pg, about 681 pg about 682 pg, about pg, about 684 pg, about 685 pg, about 686 pg, about 687 pg about 688 pg, about pg, about 690 pg, about 691 pg, about 692 pg, about 693 pg about 694 pg, about pg, about 696 pg, about 697 pg, about 698 pg, about 699 pg about 700 pg, about pg, about 702 pg, about 703 pg, about 704 pg, about 705 pg about 706 pg, about pg, about 708 pg, about 709 pg, about 710 pg, about 711 pg about 712 pg, about pg, about 714 pg, about 715 pg, about 716 pg, about 717 pg about 718 pg, about pg, about 720 pg, about 721 pg, about 722 pg, about 723 pg about 724 pg, about pg, about 726 pg, about 727 pg, about 728 pg, about 729 pg about 730 pg, about pg, about 732 pg, about 733 pg, about 734 pg, about 735 pg about 736 pg, about pg, about 738 pg, about 739 pg, about 740 pg, about 741 pg, about 742 pg, about pg, about 744 pg, about 745 pg, about 746 pg, about 747 pg, about 748 pg, about pg, about 750 pg, about 751 pg, about 752 pg, about 753 pg, about 754 pg, about pg, about 756 pg, about 757 pg, about 758 pg, about 759 pg, about 760 pg, about pg, about 762 pg, about 763 pg, about 764 pg, about 765 pg, about 766 pg, about pg, about 768 pg, about 769 pg, about 770 pg, about 771 pg, about 772 pg, about pg, about 774 pg, about 775 pg, about 776 pg, about 777 pg, about 778 pg, about pg, about 780 pg, about 781 pg, about 782 pg, about 783 pg, about 784 pg, about pg, about 786 pg, about 787 pg, about 788 pg, about 789 pg, about 790 pg, about pg, about 792 pg, about 793 pg, about 794 pg, about 795 pg, about 796 pg, about pg, about 798 pg, about 799 pg, about 800 pg, about 801 pg, about 802 pg, about pg, about 804 pg, about 805 pg, about 806 pg, about 807 pg, about 808 pg, about pg, about 810 pg, about 811 pg, about 812 pg, about 813 pg, about 814 pg, about pg, about 816 pg, about 817 pg, about 818 pg, about 819 pg, about 820 pg, about pg, about 822 pg, about 823 pg, about 824 pg, about 825 pg, about 826 pg, about pg, about 828 pg, about 829 pg, about 830 pg, about 831 pg, about 832 pg, about pg, about 834 pg, about 835 pg, about 836 pg, about 837 pg, about 838 pg, about pg, about 840 pg, about 841 pg, about 842 pg, about 843 pg, about 844 pg, about pg, about 846 pg, about 847 pg, about 848 pg, about 849 pg, about 850 pg, about pg, about 852 pg, about 853 pg, about 854 pg, about 855 pg, about 856 pg, about pg, about 858 pg, about 859 pg, about 860 pg, about 861 pg, about 862 pg, about pg, about 864 pg, about 865 pg, about 866 pg, about 867 pg, about 868 pg, about pg, about 870 pg, about 871 pg, about 872 pg, about 873 pg, about 874 pg, about pg, about 876 pg, about 877 pg, about 878 pg, about 879 pg, about 880 pg, about pg, about 882 pg, about 883 pg, about 884 pg, about 885 pg, about 886 pg, about pg, about 888 pg, about 889 pg, about 890 pg, about 891 pg, about 892 pg, about pg, about 894 pg, about 895 pg, about 896 pg, about 897 pg, about 898 pg, about pg, about 900 pg, about 901 pg, about 902 pg, about 903 pg, about 904 pg, about pg, about 906 pg, about 907 pg, about 908 pg, about 909 pg, about 910 pg, about pg, about 912 pg, about 913 pg, about 914 pg, about 915 pg, about 916 pg, about pg, about 918 pg, about 919 pg, about 920 pg, about 921 pg, about 922 pg, about pg, about 924 pg, about 925 pg, about 926 pg, about 927 pg, about 928 pg, about pg, about 930 pg, about 931 pg, about 932 pg, about 933 pg, about 934 pg, about pg, about 936 pg, about 937 pg, about 938 pg, about 939 pg, about 940 pg, about 941 pg, about 942 pg, about 943 pg, about 944 pg, about 945 pg, about 946 pg, about

947 pg, about 948 pg, about 949 pg, about 950 pg, about 951 pg, about 952 pg, about

953 pg, about 954 pg, about 955 pg, about 956 pg, about 957 pg, about 958 pg, about

959 pg, about 960 pg, about 961 pg, about 962 pg, about 963 pg, about 964 pg, about

965 pg, about 966 pg, about 967 pg, about 968 pg, about 969 pg, about 970 pg, about

971 pg, about 972 pg, about 973 pg, about 974 pg, about 975 pg, about 976 pg, about

977 pg, about 978 pg, about 979 pg, about 980 pg, about 981 pg, about 982 pg, about

983 pg, about 984 pg, about 985 pg, about 986 pg, about 987 pg, about 988 pg, about

989 pg, about 990 pg, about 991 pg, about 992 pg, about 993 pg, about 994 pg, about

995 pg, about 996 pg, about 997 pg, about 998 pg, about 999 pg, about 1000 pg, of total RSV F glycoproteins, including all values and ranges therebetween.

[00205] In embodiments, the immunogenic compositions described herein comprise from about 1 pg to about 300 pg of each HA glycoprotein. In embodiments, the immunogenic compositions described herein comprise from about 5 pg to about 60 pg of each HA glycoprotein. In embodiments, the immunogenic compositions described herein comprise from about 24 pg to about 40 pg of each HA glycoprotein. In embodiments, the immunogenic compositions described herein comprise from about 30 pg to about 40 pg of each HA glycoprotein. In embodiments, the immunogenic compositions described herein comprise from about 30 pg to about 60 pg of each HA glycoprotein. In embodiments, the immunogenic compositions described herein comprise from about 33 pg to about 39 pg of each HA glycoprotein. In embodiments, the immunogenic compositions described herein comprise from about 20 pg to about 240 pg of each HA glycoprotein. In embodiments, the immunogenic compositions described herein comprise from about 30 pg to about 60 pg of each HA glycoprotein. In embodiments, the immunogenic compositions described herein comprise from about 24 pg to about 40 pg of each HA glycoprotein. In embodiments, the immunogenic compositions comprise about 1 pg, about 2 pg, about 3 pg, about 4 pg, about 5 pg, about 6 pg, about 7 pg, about 8 pg, about 9 pg, about 10 pg, about 11 pg, about 12 pg, about 13 pg, about 14 pg, about 15 pg, about 16 pg, about 17 pg, about 18 pg, about 19 pg, about 20 pg, about 21 pg, about 22 pg, about 23 pg, about 24 pg, about 25 pg, about 26 pg, about 27 pg, about 28 pg, about 29 pg, about 30 pg, about 31 pg, about 32 pg, about 33 pg, about 34 pg, about 35 pg, about 36 pg, about 37 pg, about 38 pg, about 39 pg, about 40 pg, about 41 pg, about 42 pg, about 43 pg, about 44 pg, about 45 pg, about 46 pg, about 47 pg, about 48 pg, about 49 pg, about 50 pg, about 51 pg, about 52 pg, about 53 pg, about 54 pg, about 55 pg, about 56 pg, about 57 pg, about 58 pg, about 59 pg, about 60 pg, about 61 pg, about 62 pg, about 63 pg, about 64 pg, about 65 pg, about 66 pg, about 67 pg, about 68 pg, about 69 pg, about 70 pg, about 71 pg, about 72 pg, about 73 pg, about 74 pg, about 75 pg, about 76 pg, about 77 pg, about 78 pg, about 79 pg, about 80 pg, about 81 pg, about 82 pg, about 83 pg, about 84 pg, about 85 pg, about 86 pg, about 87 pg, about 88 pg, about 89 pg, about 90 pg, about 91 pg, about 92 pg, about 93 pg, about 94 pg, about 95 pg, about 96 pg, about 97 pg, about 98 pg, about 99 pg, about 100 pg, about 101 pg, about 102 pg, about 103 pg, about 104 pg, about 105 pg, about 106 pg, about 107 pg, about 108 pg, about 109 pg, about 110 pg, about 111 pg, about 112 pg, about 113 pg, about 114 pg, about 115 pg, about 116 pg, about 117 pg, about 118 pg, about 119 pg, about 120 pg, about 121 pg, about 122 pg, about 123 pg, about 124 pg, about 125 pg, about 126 pg, about 127 pg, about 128 pg, about 129 pg, about 130 pg, about 131 pg, about 132 pg, about 133 pg, about 134 pg, about 135 pg, about 136 pg, about 137 pg, about 138 pg, about 139 pg, about 140 pg, about 141 pg, about 142 pg, about 143 pg, about 144 pg, about 145 pg, about 146 pg, about 147 pg, about 148 pg, about 149 pg, about 150 pg, about 151 pg, about 152 pg, about 153 pg, about 154 pg, about 155 pg, about 156 pg, about 157 pg, about 158 pg, about 159 pg, about 160 pg, about 161 pg, about 162 pg, about 163 pg, about 164 pg, about 165 pg, about 166 pg, about 167 pg, about 168 pg, about 169 pg, about 170 pg, about 171 pg, about 172 pg, about 173 pg, about 174 pg, about 175 pg, about 176 pg, about 177 pg, about 178 pg, about 179 pg, about 180 pg, about 181 pg, about 182 pg, about 183 pg, about 184 pg, about 185 pg, about 186 pg, about 187 pg, about 188 pg, about 189 pg, about 190 pg, about 191 pg, about 192 pg, about 193 pg, about 194 pg, about 195 pg, about 196 pg, about 197 pg, about 198 pg, about 199 pg, about 200 pg, about 201 pg, about 202 pg, about 203 pg, about 204 pg, about 205 pg, about 206 pg, about 207 pg, about 208 pg, about 209 pg, about 210 pg, about 211 pg, about 212 pg, about 213 pg, about 214 pg, about 215 pg, about 216 pg, about 217 pg, about 218 pg, about 219 pg, about 220 pg, about 221 pg, about 222 pg, about 223 pg, about 224 pg, about 225 pg, about 226 pg, about 227 pg, about 228 pg, about 229 pg, about 230 pg, about 231 pg, about 232 pg, about 233 pg, about 234 pg, about 235 pg, about 236 pg, about 237 pg, about 238 pg, about 239 pg, about 240 pg, about 241 pg, about 242 pg, about 243 pg, about 244 pg, about 245 pg, about 246 pg, about 247 pg, about 248 pg, about 249 pg, about 250 pg, about 251 pg, about 252 pg, about 253 pg, about 254 pg, about 255 pg, about 256 pg, about 257 pg, about 258 pg, about 259 pg, about 260 pg, about 261 pg, about 262 pg, about 263 pg, about 264 pg, about 265 pg, about 266 pg, about 267 pg, about 268 pg, about 269 pg, about 270 pg, about 271 pg, about 272 pg, about 273 pg, about 274 pg, about 275 pg, about 276 pg, about 277 pg, about 278 pg, about 279 pg, about 280 pg, about 281 pg, about 282 pg, about 283 pg, about 284 pg, about 285 pg, about 286 pg, about 287 pg, about 288 pg, about 289 pg, about 290 pg, about 291 pg, about 292 pg, about 293 pg, about 294 pg, about 295 pg, about 296 pg, about 297 pg, about 298 pg, about 299 pg, or about 300 pg of each HA glycoprotein, including all values and ranges therebetween. In embodiments, the immunogenic compositions described herein comprise about 30 pg of each HA glycoprotein. In embodiments, the immunogenic compositions described herein comprise about 33 pg of each HA glycoprotein. In embodiments, the immunogenic compositions described herein comprise about 39 pg of each HA glycoprotein. In embodiments, the immunogenic compositions described herein comprise about 54 pg of each HA glycoprotein. In embodiments, the immunogenic compositions described herein comprise about 60 pg of each HA glycoprotein. In embodiments, the immunogenic compositions described herein comprise about 240 pg of each HA glycoprotein.

[00206] In embodiments, the immunogenic compositions described herein comprise from about 1 pg to about 1000 pg of total HA glycoproteins. In embodiments, the immunogenic compositions comprise about 1 pg, about 2 pg, about 3 pg, about 4 pg, about 5 pg, about 6 pg, about 7 pg, about 8 pg, about 9 pg, about 10 pg, about 11 pg, about 12 pg, about 13 pg, about 14 pg, about 15 pg, about 16 pg, about 17 pg, about 18 pg, about 19 pg, about 20 pg, about 21 pg, about 22 pg, about 23 pg, about 24 pg, about 25 pg, about 26 pg, about 27 pg, about 28 pg, about 29 pg, about 30 pg, about 31 pg, about 32 pg, about 33 pg, about 34 pg, about 35 pg, about 36 pg, about 37 pg, about 38 pg, about 39 pg, about 40 pg, about 41 pg, about 42 pg, about 43 pg, about 44 pg, about 45 pg, about 46 pg, about 47 pg, about 48 pg, about 49 pg, about 50 pg, about 51 pg, about 52 pg, about 53 pg, about 54 pg, about 55 pg, about 56 pg, about 57 pg, about 58 pg, about 59 pg, about 60 pg, about 61 pg, about 62 pg, about 63 pg, about 64 pg, about 65 pg, about 66 pg, about 67 pg, about 68 pg, about 69 pg, about 70 pg, about 71 pg, about 72 pg, about 73 pg, about 74 pg, about 75 pg, about 76 pg, about 77 pg, about 78 pg, about 79 pg, about 80 pg, about 81 pg, about 82 pg, about 83 pg, about 84 pg, about 85 pg, about 86 pg, about 87 pg, about 88 pg, about 89 pg, about 90 pg, about 91 pg, about 92 pg, about 93 pg, about 94 pg, about 95 pg, about 96 pg, about 97 pg, about 98 pg, about 99 pg, about 100 pg, about 101 pg, about 102 pg, about 103 pg, about 104 pg, about 105 pg, about 106 pg, about 107 pg, about 108 pg, about 109 pg, about 110 pg, about 111 pg, about 112 pg, about 113 pg, about 114 pg, about 115 pg, about 116 pg, about 117 pg, about 118 pg, about 119 pg, about 120 pg, about 121 pg, about 122 pg, about 123 pg, about 124 pg, about 125 pg, about 126 pg, about 127 pg, about 128 pg, about 129 pg, about 130 pg, about 131 pg, about 132 pg, about 133 pg, about 134 pg, about 135 pg, about 136 pg, about 137 pg, about 138 pg, about 139 pg, about 140 pg, about 141 pg, about 142 pg, about 143 pg, about 144 pg, about 145 pg, about 146 pg, about 147 pg, about 148 pg, about 149 pg, about 150 pg, about 151 pg, about 152 pg, about 153 pg, about 154 pg, about 155 pg, about 156 pg, about 157 pg, about 158 pg, about 159 pg, about 160 pg, about 161 pg, about 162 pg, about 163 pg, about 164 pg, about 165 pg, about 166 pg, about 167 pg, about 168 pg, about 169 pg, about 170 pg, about 171 pg, about 172 pg, about 173 pg, about 174 pg, about 175 pg, about 176 pg, about 177 pg, about 178 pg, about 179 pg, about 180 pg, about 181 pg, about 182 pg, about 183 pg, about 184 pg, about 185 pg, about 186 pg, about 187 pg, about 188 pg, about 189 pg, about 190 pg, about 191 pg, about 192 pg, about 193 pg, about 194 pg, about 195 pg, about 196 pg, about 197 pg, about 198 pg, about 199 pg, about 200 pg, about 201 pg, about 202 pg, about 203 pg, about 204 pg, about 205 pg, about 206 pg, about 207 pg, about 208 pg, about 209 pg, about 210 pg, about 211 pg, about 212 pg, about 213 pg, about 214 pg, about 215 pg, about 216 pg, about 217 pg, about 218 pg, about 219 pg, about 220 pg, about 221 pg, about 222 pg, about 223 pg, about 224 pg, about 225 pg, about 226 pg, about 227 pg, about 228 pg, about 229 pg, about 230 pg, about 231 pg, about 232 pg, about 233 pg, about 234 pg, about 235 pg, about 236 pg, about 237 pg, about 238 pg, about 239 pg, about 240 pg, about 241 pg, about 242 pg, about 243 pg, about 244 pg, about 245 pg, about 246 pg, about 247 pg, about 248 pg, about 249 pg, about 250 pg, about 251 pg, about 252 pg, about 253 pg, about 254 pg, about 255 pg, about 256 pg, about 257 pg, about 258 pg, about 259 pg, about 260 pg, about 261 pg, about 262 pg, about 263 pg, about 264 pg, about 265 pg, about 266 pg, about 267 pg, about 268 pg, about 269 pg, about 270 pg, about 271 pg, about 272 pg, about 273 pg, about 274 pg, about 275 pg, about 276 pg, about 277 pg, about 278 pg, about 279 pg, about 280 pg, about 281 pg, about 282 pg, about 283 pg, about 284 pg, about 285 pg, about 286 pg, about 287 pg, about 288 pg, about 289 pg, about 290 pg, about 291 pg, about 292 pg, about 293 pg, about 294 pg, about 295 pg, about 296 pg, about 297 pg, about 298 pg, about 299 pg, about 300 pg, about 301 pg, about 302 pg, about 303 pg, about 304 pg, about 305 pg, about 306 pg, about 307 pg, about 308 pg, about 309 pg, about 310 pg, about 311 pg, about 312 pg, about 313 pg, about 314 pg, about 315 pg, about 316 pg, about 317 pg, about 318 pg, about 319 pg, about 320 pg, about 321 pg, about 322 pg, about 323 pg, about 324 pg, about 325 pg, about 326 pg, about 327 pg, about 328 pg, about 329 pg, about 330 pg, about 331 pg, about 332 pg, about 333 pg, about 334 pg, about 335 pg, about 336 pg, about 337 pg, about 338 pg, about 339 pg, about 340 pg, about 341 pg, about 342 pg, about 343 pg, about 344 pg, about 345 pg, about 346 pg, about 347 pg, about 348 pg, about 349 pg, about 350 pg, about 351 pg, about 352 pg, about 353 pg, about 354 pg, about 355 pg, about 356 pg, about 357 pg, about 358 pg, about 359 pg, about 360 pg, about 361 pg, about 362 pg, about 363 pg, about 364 pg, about 365 pg, about 366 pg, about 367 pg, about 368 pg, about 369 pg, about 370 pg, about 371 pg, about 372 pg, about 373 pg, about 374 pg, about 375 pg, about 376 pg, about 377 pg, about 378 pg, about 379 pg, about 380 pg, about 381 pg, about 382 pg, about 383 pg, about 384 pg, about 385 pg, about 386 pg, about 387 pg, about 388 pg, about 389 pg, about 390 pg, about 391 pg, about 392 pg, about 393 pg, about 394 pg, about 395 pg, about 396 pg, about 397 pg, about 398 pg, about 399 pg, about 400 pg, about 401 pg, about 402 pg, about 403 pg, about 404 pg, about 405 pg, about 406 pg, about 407 pg, about 408 pg, about 409 pg, about 410 pg, about 411 pg, about 412 pg, about 413 pg, about 414 pg, about 415 pg, about 416 pg, about 417 pg, about 418 pg, about 419 pg, about 420 pg, about 421 pg, about 422 pg, about 423 pg, about 424 pg, about 425 pg, about 426 pg, about 427 pg, about 428 pg, about 429 pg, about 430 pg, about 431 pg, about 432 pg, about 433 pg, about 434 pg, about 435 pg, about 436 pg, about 437 pg, about 438 pg, about 439 pg, about 440 pg, about 441 pg, about 442 pg, about 443 pg, about 444 pg, about 445 pg, about 446 pg, about 447 pg, about 448 pg, about 449 pg, about 450 pg, about 451 pg, about 452 pg, about 453 pg, about 454 pg, about 455 pg, about 456 pg, about 457 pg, about 458 pg, about 459 pg, about 460 pg, about 461 pg, about 462 pg, about 463 pg, about 464 pg, about 465 pg, about 466 pg, about 467 pg, about 468 pg, about 469 pg, about 470 pg, about 471 pg, about 472 pg, about 473 pg, about 474 pg, about 475 pg, about 476 pg, about 477 pg, about 478 pg, about 479 pg, about 480 pg, about 481 pg, about 482 pg, about 483 pg, about 484 pg, about 485 pg, about 486 pg, about 487 pg, about 488 pg, about 489 pg, about 490 pg, about 491 pg, about 492 pg, about 493 pg, about 494 pg, about 495 pg, about 496 pg, about 497 pg, about 498 pg, about 499 pg, about 500 pg, about 501 pg, about 502 pg, about 503 pg, about 504 pg, about 505 pg, about 506 pg, about 507 pg, about 508 pg, about 509 pg, about 510 pg, about 511 pg, about 512 pg, about 513 pg, about 514 pg, about 515 pg, about 516 pg, about 517 pg, about 518 pg, about 519 pg, about 520 pg, about 521 pg, about 522 pg, about 523 pg, about 524 pg, about 525 pg, about 526 pg, about 527 pg, about 528 pg, about 529 pg, about 530 pg, about 531 pg, about 532 pg, about 533 pg, about 534 pg, about 535 pg, about 536 pg, about 537 pg, about 538 pg, about 539 pg, about 540 pg, about 541 pg, about 542 pg, about 543 pg, about 544 pg, about 545 pg, about 546 pg, about 547 pg, about 548 pg, about 549 pg, about 550 pg, about 551 pg, about 552 pg, about 553 pg, about 554 pg, about 555 pg, about 556 pg, about 557 pg, about 558 pg, about 559 pg, about 560 pg, about 561 pg, about 562 pg, about 563 pg, about 564 pg, about 565 pg, about 566 pg, about 567 pg, about 568 pg, about 569 pg, about 570 pg, about 571 pg, about 572 pg, about 573 pg, about 574 pg, about 575 pg, about 576 pg, about 577 pg, about 578 pg, about 579 pg, about 580 pg, about 581 pg, about 582 pg, about 583 pg, about 584 pg, about 585 pg, about 586 pg, about 587 pg, about 588 pg, about 589 pg, about 590 pg, about 591 pg, about 592 pg, about 593 pg, about 594 pg, about 595 pg, about 596 pg, about 597 pg, about 598 pg, about 599 pg, about 600 pg, about 601 pg, about 602 pg, about 603 pg, about 604 pg, about 605 pg, about 606 pg, about 607 pg, about 608 pg, about 609 pg, about 610 pg, about 611 pg, about 612 pg, about 613 pg, about 614 pg, about 615 pg, about 616 pg, about 617 pg, about 618 pg, about 619 pg, about 620 pg, about 621 pg, about 622 pg, about 623 pg, about 624 pg, about 625 pg, about 626 pg, about 627 pg, about 628 pg, about 629 pg, about 630 pg, about 631 pg, about 632 pg, about 633 pg, about 634 pg, about 635 pg, about 636 pg, about 637 pg, about 638 pg, about 639 pg, about 640 pg, about 641 pg, about 642 pg, about 643 pg, about 644 pg, about 645 pg, about 646 pg, about 647 pg, about 648 pg, about 649 pg, about 650 pg, about 651 pg, about 652 pg, about 653 pg, about 654 pg, about 655 pg, about 656 pg, about 657 pg, about 658 pg, about 659 pg, about 660 pg, about 661 pg, about 662 pg, about 663 pg, about 664 pg, about 665 pg, about 666 pg, about 667 pg, about 668 pg, about 669 pg, about 670 pg, about 671 pg, about 672 pg, about 673 pg, about 674 pg, about 675 pg, about 676 pg, about 677 pg, about 678 pg, about 679 pg, about 680 pg, about 681 pg, about 682 pg, about 683 pg, about 684 pg, about 685 pg, about 686 pg, about 687 pg, about 688 pg, about 689 pg, about 690 pg, about 691 pg, about 692 pg, about 693 pg, about 694 pg, about 695 pg, about 696 pg, about 697 pg, about 698 pg, about 699 pg, about 700 pg, about 701 pg, about 702 pg, about 703 pg, about 704 pg, about 705 pg, about 706 pg, about 707 pg, about 708 pg, about 709 pg, about 710 pg, about 711 pg, about 712 pg, about 713 pg, about 714 pg, about 715 pg, about 716 pg, about 717 pg, about 718 pg, about 719 pg, about 720 pg, about 721 pg, about 722 pg, about 723 pg, about 724 pg, about 725 pg, about 726 pg, about 727 pg, about 728 pg, about 729 pg, about 730 pg, about 731 pg, about 732 pg, about 733 pg, about 734 pg, about 735 pg, about 736 pg, about 737 pg, about 738 pg, about 739 pg, about 740 pg, about 741 pg, about 742 pg, about 743 pg, about 744 pg, about 745 pg, about 746 pg, about 747 pg, about 748 pg, about 749 pg, about 750 pg, about 751 pg, about 752 pg, about 753 pg, about 754 pg, about 755 pg, about 756 pg, about 757 pg, about 758 pg, about 759 pg, about 760 pg, about 761 pg, about 762 pg, about 763 pg, about 764 pg, about 765 pg, about 766 pg, about 767 pg, about 768 pg, about 769 pg, about 770 pg, about 771 pg, about 772 pg, about 773 pg, about 774 pg, about 775 pg, about 776 pg, about 777 pg, about 778 pg, about 779 pg, about 780 pg, about 781 pg, about 782 pg, about 783 pg, about 784 pg, about 785 pg, about 786 pg, about 787 pg, about 788 pg, about 789 pg, about 790 pg, about 791 pg, about 792 pg, about 793 pg, about 794 pg, about 795 pg, about 796 pg, about 797 pg, about 798 pg, about 799 pg, about 800 pg, about 801 pg, about 802 pg, about 803 pg, about 804 pg, about 805 pg, about 806 pg, about 807 pg, about 808 pg, about 809 pg, about 810 pg, about 811 pg, about 812 pg, about 813 pg, about 814 pg, about 815 pg, about 816 pg, about 817 pg, about 818 pg, about 819 pg, about 820 pg, about 821 pg, about 822 pg, about 823 pg, about 824 pg, about 825 pg, about 826 pg, about 827 pg, about 828 pg, about 829 pg, about 830 pg, about 831 pg, about 832 pg, about 833 pg, about 834 pg, about 835 pg, about 836 pg, about 837 pg, about 838 pg, about 839 pg, about 840 pg, about 841 pg, about 842 pg, about 843 pg, about 844 pg, about 845 pg, about 846 pg, about 847 pg, about 848 pg, about 849 pg, about 850 pg, about 851 pg, about 852 pg, about 853 pg, about 854 pg, about 855 pg, about 856 pg, about 857 pg, about 858 pg, about 859 pg, about 860 pg, about 861 pg, about 862 pg, about 863 pg, about 864 pg, about 865 pg, about 866 pg, about 867 pg, about 868 pg, about 869 pg, about 870 pg, about 871 pg, about 872 pg, about 873 pg, about 874 pg, about 875 pg, about 876 pg, about 877 pg, about 878 pg, about 879 pg, about 880 pg, about 881 pg, about 882 pg, about 883 pg, about 884 pg, about 885 pg, about 886 pg, about 887 pg, about 888 pg, about 889 pg, about 890 pg, about 891 pg, about 892 pg, about 893 pg, about 894 pg, about 895 pg, about 896 pg, about 897 pg, about 898 pg, about 899 pg, about 900 pg, about 901 pg, about 902 pg, about 903 pg, about 904 pg, about 905 pg, about 906 pg, about 907 pg, about 908 pg, about 909 pg, about 910 pg, about 911 pg, about 912 pg, about 913 pg, about 914 pg, about 915 pg, about 916 pg, about 917 pg, about 918 pg, about 919 pg, about 920 pg, about 921 pg, about 922 pg, about 923 pg, about 924 pg, about 925 pg, about 926 pg, about 927 pg, about 928 pg, about 929 pg, about 930 pg, about 931 pg, about 932 pg, about 933 pg, about 934 pg, about 935 pg, about 936 pg, about 937 pg, about 938 pg, about 939 pg, about 940 pg, about 941 pg, about 942 pg, about 943 pg, about 944 pg, about 945 pg, about 946 pg, about 947 pg, about 948 pg, about 949 pg, about 950 pg, about 951 pg, about 952 pg, about 953 pg, about 954 pg, about 955 pg, about 956 pg, about 957 pg, about 958 pg, about 959 pg, about 960 pg, about 961 pg, about 962 pg, about 963 pg, about 964 pg, about 965 pg, about 966 pg, about 967 pg, about 968 pg, about 969 pg, about 970 pg, about 971 pg, about 972 pg, about 973 pg, about 974 pg, about 975 pg, about 976 pg, about 977 pg, about 978 pg, about 979 pg, about 980 pg, about 981 pg, about 982 pg, about 983 pg, about 984 pg, about 985 pg, about 986 pg, about 987 pg, about 988 pg, about 989 pg, about 990 pg, about 991 pg, about 992 pg, about 993 pg, about 994 pg, about 995 pg, about 996 pg, about 997 pg, about 998 pg, about 999 pg, about 1000 pg, of total

HA glycoproteins, including all values and ranges therebetween.

[00207] In embodiments, the immunogenic compositions described herein comprise from about 1 pg to about 100 pg of each CoV S glycoprotein. In embodiments, the immunogenic compositions described herein comprise from about 2.5 pg to about 35 pg of each CoV S glycoprotein. In embodiments, the immunogenic compositions described herein comprise from about 5 pg to about 25 pg of each CoV S glycoprotein. In embodiments, the immunogenic compositions described herein comprise from about 14 pg to about 15 pg of each CoV S glycoprotein. In embodiments, the immunogenic compositions described herein comprise from about 15 pg to about 35 pg of each CoV S glycoprotein. In embodiments, the immunogenic compositions described herein comprise from about 1 pg to about 100 pg of total CoV S glycoproteins. In embodiments, the immunogenic compositions comprise about 1 pg, about 2 pg, about 3 pg, about 4 pg, about 5 pg, about 6 pg, about 7 pg, about 8 pg, about 9 pg, about 10 pg, about 11 pg, about 12 pg, about 13 pg, about 14 pg, about 15 pg, about 16 pg, about 17 pg, about 18 pg, about 19 pg, about 20 pg, about 21 pg, about 22 pg, about 23 pg, about 24 pg, about 25 pg, about 26 pg, about 27 pg, about 28 pg, about 29 pg, about 30 pg, about 31 pg, about 32 pg, about 33 pg, about 34 pg, about 35 pg, about 36 pg, about 37 pg, about 38 pg, about 39 pg, about 40 pg, about 41 pg, about 42 pg, about 43 pg, about 44 pg, about 45 pg, about 46 pg, about 47 pg, about 48 pg, about 49 pg, about 50 pg, about 51 pg, about 52 pg, about 53 pg, about 54 pg, about 55 pg, about 56 pg, about 57 pg, about 58 pg, about 59 pg, about 60 pg, about 61 pg, about 62 pg, about 63 pg, about 64 pg, about 65 pg, about 66 pg, about 67 pg, about 68 pg, about 69 pg, about 70 pg, about 71 pg, about 72 pg, about 73 pg, about 74 pg, about 75 pg, about 76 pg, about 77 pg, about 78 pg, about 79 pg, about 80 pg, about 81 pg, about 82 pg, about 83 pg, about 84 pg, about 85 pg, about 86 pg, about 87 pg, about 88 pg, about 89 pg, about 90 pg, about 91 pg, about 92 pg, about 93 pg, about 94 pg, about 95 pg, about 96 pg, about 97 pg, about 98 pg, about 99 pg, about 100 pg, including all values and ranges therebetween, of each CoV S glycoprotein. In embodiments, the immunogenic compositions comprise about 1 pg, about 2 pg, about 3 pg, about 4 pg, about 5 pg, about 6 pg, about 7 pg, about 8 pg, about 9 pg, about 10 pg, about 11 pg, about 12 pg, about 13 pg, about 14 pg, about 15 pg, about 16 pg, about 17 pg, about 18 pg, about 19 pg, about 20 pg, about 21 pg, about 22 pg, about 23 pg, about 24 pg, about 25 pg, about 26 pg, about 27 pg, about 28 pg, about 29 pg, about 30 pg, about 31 pg, about 32 pg, about 33 pg, about 34 pg, about 35 pg, about 36 pg, about 37 pg, about 38 pg, about 39 pg, about 40 pg, about 41 pg, about 42 pg, about 43 pg, about 44 pg, about 45 pg, about 46 pg, about 47 pg, about 48 pg, about 49 pg, about 50 pg, about 51 pg, about 52 pg, about 53 pg, about 54 pg, about 55 pg, about 56 pg, about 57 pg, about 58 pg, about 59 pg, about 60 pg, about 61 pg, about 62 pg, about 63 pg, about 64 pg, about 65 pg, about 66 pg, about 67 pg, about 68 pg, about 69 pg, about 70 pg, about 71 pg, about 72 pg, about 73 pg, about 74 pg, about 75 pg, about 76 pg, about 77 pg, about 78 pg, about 79 pg, about 80 pg, about 81 pg, about 82 pg, about 83 pg, about 84 pg, about 85 pg, about 86 pg, about 87 pg, about 88 pg, about 89 pg, about 90 pg, about 91 pg, about 92 pg, about 93 pg, about 94 pg, about 95 pg, about 96 pg, about 97 pg, about 98 pg, about 99 pg, about 100 pg of total CoV S glycoproteins, including all values and ranges therebetween. In embodiments, the immunogenic compositions comprise about 1 pg of a CoV S glycoprotein. In embodiments, the immunogenic compositions comprise about 13 pg of a CoV S glycoprotein. In embodiments, the immunogenic compositions comprise about 5 pg of a CoV S glycoprotein. In embodiments, the immunogenic compositions comprise about 25 pg of a CoV S glycoprotein.

Adjuvants In Immunogenic Compositions and Vaccine Compositions [00208] In certain embodiments, the compositions disclosed herein comprise one or more adjuvants. In embodiments, the one or more adjuvants enhance an immune response. In other embodiments, the compositions are prepared without adjuvants, and are thus available to be administered as adjuvant-free compositions. Advantageously, adjuvant-free compositions disclosed herein may provide protective immune responses when administered as a single dose.

Aluminum-based adjuvants

[00209] In embodiments, the adjuvant may be alum (e.g. AIPO4 or AI(OH)s). Typically, the nanoparticle is substantially bound to the alum. For example, the nanoparticle may be at least 80% bound, at least 85% bound, at least 90% bound or at least 95% bound to the alum. Often, the nanoparticle is 92% to 97% bound to the alum in a composition. The amount of alum is present per dose is typically in a range between about 400 pg to about 1250 pg. For example, the alum may be present in a per dose amount of about 300 pg to about 900 pg, about 400 pg to about 800 pg, about 500 pg to about 700 pg, about 400 pg to about 600 pg, or about 400 pg to about 500 pg. Typically, the alum is present at about 400 pg for a dose of 120 pg of the protein nanoparticle.

Saponin Adjuvants

[00210] Adjuvants containing saponin may also be combined with the immunogens disclosed herein. Saponins are glycosides derived from the bark of the Quillaja saponaria Molina tree. Typically, saponin is prepared using a multi-step purification process resulting in multiple fractions. As used, herein, the term “a saponin fraction from Quillaja saponaria Molina” is used generically to describe a semi-purified or defined saponin fraction of Quillaja saponaria or a substantially pure fraction thereof.

Saponin Fractions

[00211] Several approaches for producing saponin fractions are suitable. Fractions A, B, and C are described in U.S. Pat. No. 6,352,697 and may be prepared as follows. A lipophilic fraction from Quil A, a crude aqueous Quillaja saponaria Molina extract, is separated by chromatography and eluted with 70% acetonitrile in water to recover the lipophilic fraction. This lipophilic fraction is then separated by semipreparative HPLC with elution using a gradient of from 25% to 60% acetonitrile in acidic water. The fraction referred to herein as “Fraction A” or “QH-A” is, or corresponds to, the fraction, which is eluted at approximately 39% acetonitrile. The fraction referred to herein as “Fraction B” or “QH-B” is, or corresponds to, the fraction, which is eluted at approximately 47% acetonitrile. The fraction referred to herein as “Fraction C” or “QH-C” is, or corresponds to, the fraction, which is eluted at approximately 49% acetonitrile. Additional information regarding purification of Fractions is found in U.S Pat. No. 5,057,540. When prepared as described herein, Fractions A, B and C of Quillaja saponaria Molina each represent groups or families of chemically closely related molecules with definable properties. The chromatographic conditions under which they are obtained are such that the batch-to- batch reproducibility in terms of elution profile and biological activity is highly consistent.

[00212] Other saponin fractions have been described. Fractions B3, B4 and B4b are described in EP 0436620. Fractions QA1-QA22 are described EP03632279 B2, Q-VAC (Nor-Feed, AS Denmark), Quillaja saponaria Molina Spikoside (Isconova AB, Ultunaallen 2B, 756 51 Uppsala, Sweden). Fractions QA-1 , QA-2, QA-3, QA-4, QA-5, QA-6, QA-7, QA-8, QA-9, QA-10, QA-11 , QA-12, QA-13, QA-14, QA-15, QA-16, QA- 17, QA-18, QA-19, QA-20, QA-21 , and QA-22 of EP 0 3632 279 B2, especially QA-7, QA-17, QA-18, and QA-21 may be used. They are obtained as described in EP 03632 279 B2, especially at page 6 and in Example 1 on page 8 and 9.

[00213] The saponin fractions described herein and used for forming adjuvants are often substantially pure fractions; that is, the fractions are substantially free of the presence of contamination from other materials. In particular aspects, a substantially pure saponin fraction may contain up to 40% by weight, up to 30% by weight, up to 25% by weight, up to 20% by weight, up to 15% by weight, up to 10% by weight, up to 7% by weight, up to 5% by weight, up to 2% by weight, up to 1 % by weight, up to 0.5% by weight, or up to 0.1 % by weight of other compounds such as other saponins or other adjuvant materials.

ISCOM Structures

[00214] Saponin fractions may be administered in the form of a cage-like particle referred to as an ISCOM (Immune Stimulating COMplex). ISCOMs may be prepared as described in EP0109942B1 , EP0242380B1 and EP0180546 B1. In particular embodiments a transport and/or a passenger antigen may be used, as described in EP 9600647-3 (PCT/SE97/00289).

Matrix Adjuvants [00215] In embodiments, the ISCOM is an ISCOM matrix complex. An ISCOM matrix complex comprises at least one saponin fraction and a lipid. The lipid is at least a sterol, such as cholesterol. In particular aspects, the ISCOM matrix complex also contains a phospholipid. The ISCOM matrix complexes may also contain one or more other immunomodulatory (adjuvant-active) substances, not necessarily a glycoside, and may be produced as described in EP0436620B1 , which is incorporated by reference in its entirety herein.

[00216] In other aspects, the ISCOM is an ISCOM complex. An ISCOM complex contains at least one saponin, at least one lipid, and at least one kind of antigen or epitope. The ISCOM complex contains antigen associated by detergent treatment such that that a portion of the antigen integrates into the particle. In contrast, ISCOM matrix is formulated as an admixture with antigen and the association between ISCOM matrix particles and antigen is mediated by electrostatic and/or hydrophobic interactions.

[00217] According to one embodiment, the saponin fraction integrated into an ISCOM matrix complex or an ISCOM complex, or at least one additional adjuvant, which also is integrated into the ISCOM or ISCOM matrix complex or mixed therewith, is selected from fraction A, fraction B, or fraction C of Quillaja saponaria, a semipurified preparation of Quillaja saponaria, a purified preparation of Quillaja saponaria, or any purified sub-fraction e.g., QA 1-21.

[00218] In particular aspects, each ISCOM particle may contain at least two saponin fractions. Any combinations of weight % of different saponin fractions may be used. Any combination of weight % of any two fractions may be used. For example, the particle may contain any weight % of fraction A and any weight % of another saponin fraction, such as a crude saponin fraction or fraction C, respectively. Accordingly, in particular aspects, each ISCOM matrix particle or each ISCOM complex particle may contain from 0.1 to 99.9 by weight, 5 to 95% by weight, 10 to 90% by weight 15 to 85% by weight, 20 to 80% by weight, 25 to 75% by weight, 30 to 70% by weight, 35 to 65% by weight, 40 to 60% by weight, 45 to 55% by weight, 40 to 60% by weight, or 50% by weight of one saponin fraction, e.g. fraction A and the rest up to 100% in each case of another saponin e.g. any crude fraction or any other faction e.g. fraction C. The weight is calculated as the total weight of the saponin fractions. Examples of ISCOM matrix complex and ISCOM complex adjuvants are disclosed in U.S Published Application No. 2013/0129770, which is incorporated by reference in its entirety herein.

[00219] In particular embodiments, the ISCOM matrix or ISCOM complex comprises from 5-99% by weight of one fraction, e.g. fraction A and the rest up to 100% of weight of another fraction e.g. a crude saponin fraction or fraction C. The weight is calculated as the total weight of the saponin fractions.

[00220] In another embodiment, the ISCOM matrix or ISCOM complex comprises from 40% to 99% by weight of one fraction, e.g. fraction A and from 1 % to 60% by weight of another fraction, e.g. a crude saponin fraction or fraction C. The weight is calculated as the total weight of the saponin fractions.

[00221] In yet another embodiment, the ISCOM matrix or ISCOM complex comprises from 70% to 95% by weight of one fraction e.g., fraction A, and from 30% to 5% by weight of another fraction, e.g., a crude saponin fraction, or fraction C. The weight is calculated as the total weight of the saponin fractions. In other embodiments, the saponin fraction from Quillaja saponaria Molina is selected from any one of QA 1- 21.

[00222] In addition to particles containing mixtures of saponin fractions, ISCOM matrix particles and ISCOM complex particles may each be formed using only one saponin fraction. Compositions disclosed herein may contain multiple particles wherein each particle contains only one saponin fraction. That is, certain compositions may contain one or more different types of ISCOM-matrix complexes particles and/or one or more different types of ISCOM complexes particles, where each individual particle contains one saponin fraction from Quillaja saponaria Molina, wherein the saponin fraction in one complex is different from the saponin fraction in the other complex particles.

[00223] In particular aspects, one type of saponin fraction or a crude saponin fraction may be integrated into one ISCOM matrix complex or particle and another type of substantially pure saponin fraction, or a crude saponin fraction, may be integrated into another ISCOM matrix complex or particle. A composition or vaccine may comprise at least two types of complexes or particles each type having one type of saponins integrated into physically different particles.

[00224] In the compositions, mixtures of ISCOM matrix complex particles and/or ISCOM complex particles may be used in which one saponin fraction Quillaja saponaria Molina and another saponin fraction Quillaja saponaria Molina are separately incorporated into different ISCOM matrix complex particles and/or ISCOM complex particles.

[00225] The ISCOM matrix or ISCOM complex particles, which each have one saponin fraction, may be present in composition at any combination of weight %. In particular aspects, a composition may contain 0.1 % to 99.9% by weight, 5% to 95% by weight, 10% to 90% by weight, 15% to 85% by weight, 20% to 80% by weight, 25% to 75% by weight, 30% to 70% by weight, 35% to 65% by weight, 40% to 60% by weight, 45% to 55% by weight, 40 to 60% by weight, or 50% by weight, of an ISCOM matrix or complex containing a first saponin fraction with the remaining portion made up by an ISCOM matrix or complex containing a different saponin fraction. In aspects, the remaining portion is one or more ISCOM matrix or complexes where each matrix or complex particle contains only one saponin fraction. In other aspects, the ISCOM matrix or complex particles may contain more than one saponin fraction.

[00226] In particular 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.

[00227] In embodiments, the Fraction A of Quillaja Saponaria Molina accounts for at least about 80 %, 81 %, 82 %, 83 %, 84 %, 85 %, 86 %, 87 %, 88 %, 89 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, or 99 % by weight, and fraction C of Quillaja Saponaria Molina accounts for the remainder, respectively, of the sum of the weights of fraction A of Quillaja Saponaria Molina and fraction C of Quillaja Saponaria Molina in the adjuvant.

[00228] Preferred compositions comprise a first ISCOM matrix containing Fraction A and a second ISCOM matrix containing Fraction C, wherein the Fraction A ISCOM matrix constitutes about 70% per weight of the total saponin adjuvant, and the Fraction C ISCOM matrix constitutes about 30% per weight of the total saponin adjuvant. In another preferred composition, the Fraction A ISCOM matrix constitutes about 85% per weight of the total saponin adjuvant, and the Fraction C ISCOM matrix constitutes about 15% per weight of the total saponin adjuvant. In another preferred composition, the Fraction A ISCOM matrix constitutes about 92% per weight of the total saponin adjuvant, and the Fraction C ISCOM matrix constitutes about 8% per 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%, and Fraction C ISCOM matrix is present in a range of about 15% to about 30%, of the total weight amount of saponin adjuvant in the composition. In certain compositions, the Fraction A ISCOM matrix is present in a range of about 70% to about 92%, and Fraction C ISCOM matrix is present in a range of about 8% to about 30%, of the total weight amount of saponin adjuvant in the composition. In embodiments, the Fraction A ISCOM matrix accounts for 50-96 % by weight and Fraction C ISCOM matrix accounts for the remainder, respectively, of the sums of the weights of Fraction A ISCOM matrix and Fraction C ISCOM in the adjuvant. In a particularly preferred composition, referred to herein as MATRIX-M™, the Fraction A ISCOM matrix is present at about 85 % and Fraction C ISCOM matrix is present at about 15% of the total weight amount of saponin adjuvant in the composition. MATRIX-M™ may be referred to interchangeably as Matrix-M1.

[00229] Exemplary QS-7 and QS-21 fractions, their production and their use is described in U.S Pat. 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 by reference herein.

[00230] In embodiments, other adjuvants may be used in addition or as an alternative. The inclusion of any adjuvant described in Vogel et al., "A Compendium of Vaccine Adjuvants and Excipients (2nd Edition)," herein incorporated by reference in its entirety for all purposes, is envisioned within the scope of this disclosure. Other adjuvants include complete Freund's adjuvant (a non-specific stimulator of the immune response containing killed Mycobacterium tuberculosis), incomplete Freund's adjuvants and aluminum hydroxide adjuvant. Other adjuvants comprise 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 cell wall skeleton (CWS) in a 2% squalene/TWEEN® polysorbate 80 emulsion. In embodiments, the adjuvant may be a paucilamellar lipid vesicle; for example, NOVASOMES®. NOVASOMES® are paucilamellar nonphospholipid vesicles ranging from about 100 nm to about 500 nm. They comprise BRU® alcohol ethoxylate 72, cholesterol, oleic acid and squalene. NOVASOMES® have been shown to be an effective adjuvant (see, U.S. Pat. Nos. 5,629,021 , 6,387,373, and 4,911 ,928.

Amount of Adjuvant in Compositions

[00231] In embodiments, the immunogenic compositions described herein comprise from about 1 pg to about 400 pg, from 1 pg to about 350 pg, from 1 pg to about 300 pg, from 1 pg to about 250 pg, from 1 pg to about 200 pg, from 1 pg to about 150 pg, from 1 pg to about 100 pg, from 1 pg to about 50 pg, or from 1 pg to about 25 pg of adjuvant. In embodiments, the immunogenic compositions comprise about 1 pg, about 2 pg, about 3 pg, about 4 pg, about 5 pg, about 6 pg, about 7 pg, about 8 pg, about 9 pg, about 10 pg, about 11 pg, about 12 pg, about 13 pg, about 14 pg, about 15 pg, about 16 pg, about 17 pg, about 18 pg, about 19 pg, about 20 pg, about 21 pg, about 22 pg, about 23 pg, about 24 pg, about 25 pg, about 26 pg, about 27 pg, about 28 pg, about 29 pg, about 30 pg, about 31 pg, about 32 pg, about 33 pg, about 34 pg, about 35 pg, about 36 pg, about 37 pg, about 38 pg, about 39 pg, about 40 pg, about 41 pg, about 42 pg, about 43 pg, about 44 pg, about 45 pg, about 46 pg, about 47 pg, about 48 pg, about 49 pg, about 50 pg, about 51 pg, about 52 pg, about 53 pg, about 54 pg, about 55 pg, about 56 pg, about 57 pg, about 58 pg, about 59 pg, about 60 pg, about 61 pg, about 62 pg, about 63 pg, about 64 pg, about 65 pg, about 66 pg, about 67 pg, about 68 pg, about 69 pg, about 70 pg, about 71 pg, about 72 pg, about 73 pg, about 74 pg, about 75 pg, about 76 pg, about 77 pg, about 78 pg, about 79 pg, about 80 pg, about 81 pg, about 82 pg, about 83 pg, about 84 pg, about 85 pg, about 86 pg, about 87 pg, about 88 pg, about 89 pg, about 90 pg, about 91 pg, about 92 pg, about 93 pg, about 94 pg, about 95 pg, about 96 pg, about 97 pg, about 98 pg, about 99 pg, about 100 pg, about 101 pg, about 102 pg, about 103 pg, about 104 pg, about 105 pg, about 106 pg, about 107 pg, about 108 pg, about 109 pg, about 110 pg, about 111 pg, about 112 pg, about 113 pg, about 114 pg, about 115 pg, about 116 pg, about 117 pg, about 118 pg, about 119 pg, about 120 pg, about 121 pg, about 122 pg, about 123 pg, about 124 pg, about 125 pg, about 126 pg, about 127 pg, about 128 pg, about 129 pg, about 130 pg, about 131 pg, about 132 pg, about 133 pg, about 134 pg, about 135 pg, about 136 pg, about 137 pg, about 138 pg, about 139 pg, about 140 pg, about 141 pg, about 142 pg, about 143 pg, about 144 pg, about 145 pg, about 146 pg, about 147 pg, about 148 pg, about 149 pg, about 150 pg, about 151 pg, about 152 pg, about 153 pg, about 154 pg, about 155 pg, about 156 pg, about 157 pg, about 158 pg, about 159 pg, about 160 pg, about 161 pg, about 162 pg, about 163 pg, about 164 pg, about 165 pg, about 166 pg, about 167 pg, about 168 pg, about 169 pg, about 170 pg, about 171 pg, about 172 pg, about 173 pg, about 174 pg, about 175 pg, about 176 pg, about 177 pg, about 178 pg, about 179 pg, about 180 pg, about 181 pg, about 182 pg, about 183 pg, about 184 pg, about 185 pg, about 186 pg, about 187 pg, about 188 pg, about 189 pg, about 190 pg, about 191 pg, about 192 pg, about 193 pg, about 194 pg, about 195 pg, about 196 pg, about 197 pg, about 198 pg, about 199 pg, about 200 pg, about 201 pg, about 202 pg, about 203 pg, about 204 pg, about 205 pg, about 206 pg, about 207 pg, about 208 pg, about 209 pg, about 210 pg, about 211 pg, about 212 pg, about 213 pg, about 214 pg, about 215 pg, about 216 pg, about 217 pg, about 218 pg, about 219 pg, about 220 pg, about 221 pg, about 222 pg, about 223 pg, about 224 pg, about 225 pg, about 226 pg, about 227 pg, about 228 pg, about 229 pg, about 230 pg, about 231 pg, about 232 pg, about 233 pg, about 234 pg, about 235 pg, about 236 pg, about 237 pg, about 238 pg, about 239 pg, about 240 pg, about 241 pg, about 242 pg, about 243 pg, about 244 pg, about 245 pg, about 246 pg, about 247 pg, about 248 pg, about 249 pg, about 250 pg, about 251 pg, about 252 pg, about 253 pg, about 254 pg, about 255 pg, about 256 pg, about 257 pg, about 258 pg, about 259 pg, about 260 pg, about 261 pg, about 262 pg, about 263 pg, about 264 pg, about 265 pg, about 266 pg, about 267 pg, about 268 pg, about 269 pg, about 270 pg, about 271 pg, about 272 pg, about 273 pg, about 274 pg, about 275 pg, about 276 pg, about 277 pg, about 278 pg, about 279 pg, about 280 pg, about 281 pg, about 282 pg, about 283 pg, about 284 pg, about 285 pg, about 286 pg, about 287 pg, about 288 pg, about 289 pg, about 290 pg, about 291 pg, about 292 pg, about 293 pg, about 294 pg, about 295 pg, about 296 pg, about 297 pg, about 298 pg, about 299 pg, about 300 pg, about 301 pg, about 302 pg, about 303 pg, about 304 pg, about 305 pg, about 306 pg, about 307 pg, about 308 pg, about 309 pg, about 310 pg, about 311 pg, about 312 pg, about 313 pg, about 314 pg, about 315 pg, about 316 pg, about 317 pg, about 318 pg, about 319 pg, about 320 pg, about 321 pg, about 322 pg, about 323 pg, about 324 pg, about 325 pg, about 326 pg, about 327 pg, about 328 pg, about 329 pg, about 330 pg, about 331 pg, about 332 pg, about 333 pg, about 334 pg, about 335 pg, about 336 pg, about 337 pg, about 338 pg, about 339 pg, about 340 pg, about 341 pg, about 342 pg, about 343 pg, about 344 pg, about 345 pg, about 346 pg, about 347 pg, about 348 pg, about 349 pg, about 350 pg, about 351 pg, about 352 pg, about 353 pg, about 354 pg, about 355 pg, about 356 pg, about 357 pg, about 358 pg, about 359 pg, about 360 pg, about 361 pg, about 362 pg, about 363 pg, about 364 pg, about 365 pg, about 366 pg, about 367 pg, about 368 pg, about 369 pg, about 370 pg, about 371 pg, about 372 pg, about 373 pg, about 374 pg, about 375 pg, about 376 pg, about 377 pg, about 378 pg, about 379 pg, about 380 pg, about 381 pg, about 382 pg, about 383 pg, about 384 pg, about 385 pg, about 386 pg, about 387 pg, about 388 pg, about 389 pg, about 390 pg, about 391 pg, about 392 pg, about 393 pg, about 394 pg, about 395 pg, about 396 pg, about 397 pg, about 398 pg, about 399 pg, or about 400 pg of adjuvant. In embodiments, the immunogenic composition comprises about 50 pg of adjuvant. In embodiments, the immunogenic composition comprises about 75 pg of adjuvant.

Methods of Stimulating an Immune Response against RSV, Influenza, SARS- CoV-2 or a variant thereof, or a combination thereof

[00232] In embodiments, the disclosure provides a method for eliciting an immune response against one or more of RSV, influenza, and SARS-CoV-2 or a variant thereof, comprising administering an immunogenic composition 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 a RSV F glycoprotein, a CoV S glycoprotein, and an influenza HA glycoprotein.

[00233] In embodiments, the immunogenic compositions lack an adjuvant. In embodiments, the immunogenic compositions comprise an adjuvant. Non-limiting examples of adjuvants are described herein.

[00234] In embodiments, the immunogenic compositions described herein have an efficacy against a virus (e.g., RSV, influenza, SARS-CoV-2, a SARS-CoV-2 variant thereof, or a combination thereof) that is 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 %.

[00235] Compositions disclosed herein may 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 kidney dialytic infusion techniques. Typically, the systemic, parenteral administration is intramuscular injection. As used herein, the term “mucosal administration” includes oral, intranasal, intravaginal, intra-rectal, intra-tracheal, intestinal and ophthalmic administration. In embodiments, the methods comprise administering immunogenic compositions described herein intramuscularly. In embodiments, the methods comprise administering immunogenic compositions described herein intranasally.

[00236] Compositions may be administered on a single dose schedule or a multiple dose schedule. Multiple doses may be used in a primary immunization schedule or in a booster immunization schedule. 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 administered. In a multiple dose schedule the various doses may be given by the same or different routes e.g., a parenteral prime and mucosal boost, a mucosal prime and parenteral boost, etc. In aspects, a boost dose is administered about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months (1 year), about 2 years, about 3 years, about 4 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, or about 10 years after the first dose. In embodiments, a boost dose is administered every year after administration of the initial dose. In embodiments, the follow-on boost dose is administered 3 weeks or 4 weeks after administration of the prior dose. In embodiments, the first dose is administered at day 0, and the boost dose is administered at day 21 . In embodiments, the first dose is administered at day 0, and the boost dose is administered at day 28. In embodiments, the first dose is administered at day 0, a boost dose is administered at day 21 , and a second boost dose is administered about six months after administration of the first dose or second dose. In embodiments, the first dose is administered at day 0, and the boost dose is administered at day 28, and a second boost dose is administered about six months after administration of the first dose. In embodiments, the first dose is administered at day 0, a boost dose is administered at day 21 , and a second boost dose is administered about six months after administration of the second dose. In embodiments, the first dose is administered at day 0, and the boost dose is administered at day 28, and a second boost dose is administered about six months after administration of the second dose.

[00237] In embodiments, the first dose is administered at day 0, a boost dose is administered at day 21 , and a second boost dose is administered about 1 year after administration of the first dose or the first boost dose. In embodiments, the first dose is administered at day 0, a first boost dose is administered at day 28, and a second boost dose is administered about 1 year after administration of the first dose. In embodiments, the first dose is administered at day 0, a boost dose is administered at day 21 , and a second boost dose is administered about 1 year after administration of the second dose. In embodiments, the first dose is administered at day 0, a first boost dose is administered at day 28, and a second boost dose is administered about 1 year after administration of the second dose. In embodiments, the second boost dose is administered from 6 months to 24 months or from 12 to 24 months after the first boost dose.

[00238] In embodiments, the dose, as measured in pg, may be the total weight of the dose including the solute, or the weight of the RSV F glycoprotein nanoparticles, or the weight of the RSV F glycoprotein. Dose is measured using protein concentration assay either A280 or ELISA.

[00239] Certain populations may be administered with or without adjuvants. In certain aspects, compositions may be free of added adjuvant. In such circumstances, the dose may be increased by about 10%.

[00240] In embodiments, the immunogenic compositions described herein are provided in pre-filled syringes. When the immunogenic composition is prepared in a pre-filled syringe, the RSV F glycoproteins and adjuvant are combined in advance of administration.

[00241] In embodiments, the dose is administered in a volume of about 0.1 mL to about 1 .5 mL, for example, 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.

[00242] In embodiments, the concentration of viral glycoprotein nanoparticles (e.g., RSV F, CoV S, or HA glycoprotein nanoparticles) in the immunogenic compositions or vaccine compositions described herein is from about 1 pg/mL to about 50 pg/mL, 10 pg/mL to about 100 pg/mL, about 10 pg/mL to about 50 pg/mL, about 175 pg/mL to about 325 pg/mL, about 200 pg/mL to about 300 pg/mL, about 220 pg/mL to about 280 pg/mL, or about 240 pg/mL to about 260 pg/mL.

[00243] In embodiments, the methods comprise administering from about 1 pg to about 300 pg, from about 5 pg to about 25 pg, from about 1 pg to about 300 pg, from about 90 pg to about 270 pg, from about 100 pg to about 160 pg, from about 110 pg to about 150 pg, from about 120 pg to about 140 pg, or from about 140 pg to about 160 of each RSV F glycoprotein. In embodiments, the methods comprise administering from about 5 pg to about 60 pg of each RSV F glycoprotein. In embodiments, the methods comprise administering from about 24 pg to about 40 pg of each RSV F glycoprotein. In embodiments, the methods comprise administering from about 30 pg to about 40 pg of each RSV F glycoprotein. In embodiments, the methods comprise administering from about 30 pg to about 60 pg of each RSV F glycoprotein. In embodiments, the methods comprise administering from about 33 pg to about 39 pg of each RSV F glycoprotein. In embodiments, the methods comprise administering from about 20 pg to about 240 pg of each RSV F glycoprotein. In embodiments, the methods comprise administering from about 30 pg to about 60 pg of each RSV F glycoprotein. In embodiments, the methods comprise administering from about 24 pg to about 40 pg of each RSV F glycoprotein. In embodiments, the methods comprise administering about 1 pg, about 2 pg, about 3 pg, about 4 pg, about 5 pg, about 6 pg, about 7 pg, about 8 pg, about 9 pg, about 10 pg, about 11 pg, about 12 pg, about 13 pg, about 14 pg, about 15 pg, about 16 pg, about 17 pg, about 18 pg, about 19 pg, about 20 pg, about 21 pg, about 22 pg, about 23 pg, about 24 pg, about 25 pg, about 26 pg, about 27 pg, about 28 pg, about 29 pg, about 30 pg, about 31 pg, about 32 pg, about 33 pg, about 34 pg, about 35 pg, about 36 pg, about 37 pg, about 38 pg, about 39 pg, about 40 pg, about 41 pg, about 42 pg, about 43 pg, about 44 pg, about 45 pg, about 46 pg, about 47 pg, about 48 pg, about 49 pg, about 50 pg, about 51 pg, about 52 pg, about 53 pg, about 54 pg, about 55 pg, about 56 pg, about 57 pg, about 58 pg, about 59 pg, about 60 pg, about 61 pg, about 62 pg, about 63 pg, about 64 pg, about 65 pg, about 66 pg, about 67 pg, about 68 pg, about 69 pg, about 70 pg, about 71 pg, about 72 pg, about 73 pg, about 74 pg, about 75 pg, about 76 pg, about 77 pg, about 78 pg, about 79 pg, about 80 pg, about 81 pg, about 82 pg, about 83 pg, about 84 pg, about 85 pg, about 86 pg, about 87 pg, about 88 pg, about 89 pg, about 90 pg, about 91 pg, about 92 pg, about 93 pg, about 94 pg, about 95 pg, about 96 pg, about 97 pg, about 98 pg, about 99 pg, about 100 pg, about 101 pg, about 102 pg, about 103 pg, about 104 pg, about 105 pg, about 106 pg, about 107 pg, about 108 pg, about 109 pg, about 110 pg, about 111 pg, about 112 pg, about 113 pg, about 114 pg, about 115 pg, about 116 pg, about 117 pg, about 118 pg, about 119 pg, about 120 pg, about 121 pg, about 122 pg, about 123 pg, about 124 pg, about 125 pg, about 126 pg, about 127 pg, about 128 pg, about 129 pg, about 130 pg, about 131 pg, about 132 pg, about 133 pg, about 134 pg, about 135 pg, about 136 pg, about 137 pg, about 138 pg, about 139 pg, about 140 pg, about 141 pg, about 142 pg, about 143 pg, about 144 pg, about 145 pg, about 146 pg, about 147 pg, about 148 pg, about 149 pg, about 150 pg, about 151 pg, about 152 pg, about 153 pg, about 154 pg, about 155 pg, about 156 pg, about 157 pg, about 158 pg, about 159 pg, about 160 pg, about 161 pg, about 162 pg, about 163 pg, about 164 pg, about 165 pg, about 166 pg, about 167 pg, about 168 pg, about 169 pg, about 170 pg, about 171 pg, about 172 pg, about 173 pg, about 174 pg, about 175 pg, about 176 pg, about 177 pg, about 178 pg, about 179 pg, about 180 pg, about 181 pg, about 182 pg, about 183 pg, about 184 pg, about 185 pg, about 186 pg, about 187 pg, about 188 pg, about 189 pg, about 190 pg, about 191 pg, about 192 pg, about 193 pg, about 194 pg, about 195 pg, about 196 pg, about 197 pg, about 198 pg, about 199 pg, about 200 pg, about 201 pg, about 202 pg, about 203 pg, about 204 pg, about 205 pg, about 206 pg, about 207 pg, about 208 pg, about 209 pg, about 210 pg, about 211 pg, about 212 pg, about 213 pg, about 214 pg, about 215 pg, about 216 pg, about 217 pg, about 218 pg, about 219 pg, about 220 pg, about 221 pg, about 222 pg, about 223 pg, about 224 pg, about 225 pg, about 226 pg, about 227 pg, about 228 pg, about 229 pg, about 230 pg, about 231 pg, about 232 pg, about 233 pg, about 234 pg, about 235 pg, about 236 pg, about 237 pg, about 238 pg, about 239 pg, about 240 pg, about 241 pg, about 242 pg, about 243 pg, about 244 pg, about 245 pg, about 246 pg, about 247 pg, about 248 pg, about 249 pg, about 250 pg, about 251 pg, about 252 pg, about 253 pg, about 254 pg, about 255 pg, about 256 pg, about 257 pg, about 258 pg, about 259 pg, about 260 pg, about 261 pg, about 262 pg, about 263 pg, about 264 pg, about 265 pg, about 266 pg, about 267 pg, about 268 pg, about 269 pg, about 270 pg, about 271 pg, about 272 pg, about 273 pg, about 274 pg, about 275 pg, about 276 pg, about 277 pg, about 278 pg, about 279 pg, about 280 pg, about 281 pg, about 282 pg, about 283 pg, about 284 pg, about 285 pg, about 286 pg, about 287 pg, about 288 pg, about 289 pg, about 290 pg, about 291 pg, about 292 pg, about 293 pg, about 294 pg, about 295 pg, about 296 pg, about 297 pg, about 298 pg, about 299 pg, or about 300 pg of each RSV F glycoprotein, including all values and ranges therebetween. In embodiments, the methods comprise administering about 60 pg of each RSV F glycoprotein. In embodiments, the methods comprise administering about 240 pg of each RSV F glycoprotein. [00244] In embodiments, the methods comprise administering from about 1 pg to about 1000 pg of total RSV F glycoproteins. In embodiments, the methods comprise administering about 1 pg, about 2 pg, about 3 pg, about 4 pg, about 5 pg, about 6 pg, about 7 pg, about 8 pg, about 9 pg, about 10 pg, about 11 pg, about 12 pg, about 13 pg, about 14 pg, about 15 pg, about 16 pg, about 17 pg, about 18 pg, about 19 pg, about 20 pg, about 21 pg, about 22 pg, about 23 pg, about 24 pg, about 25 pg, about 26 pg, about 27 pg, about 28 pg, about 29 pg, about 30 pg, about 31 pg, about 32 pg, about 33 pg, about 34 pg, about 35 pg, about 36 pg, about 37 pg, about 38 pg, about 39 pg, about 40 pg, about 41 pg, about 42 pg, about 43 pg, about 44 pg, about 45 pg, about 46 pg, about 47 pg, about 48 pg, about 49 pg, about 50 pg, about 51 pg, about 52 pg, about 53 pg, about 54 pg, about 55 pg, about 56 pg, about 57 pg, about 58 pg, about 59 pg, about 60 pg, about 61 pg, about 62 pg, about 63 pg, about 64 pg, about 65 pg, about 66 pg, about 67 pg, about 68 pg, about 69 pg, about 70 pg, about 71 pg, about 72 pg, about 73 pg, about 74 pg, about 75 pg, about 76 pg, about 77 pg, about 78 pg, about 79 pg, about 80 pg, about 81 pg, about 82 pg, about 83 pg, about 84 pg, about 85 pg, about 86 pg, about 87 pg, about 88 pg, about 89 pg, about 90 pg, about 91 pg, about 92 pg, about 93 pg, about 94 pg, about 95 pg, about 96 pg, about 97 pg, about 98 pg, about 99 pg, about 100 pg, about 101 pg, about 102 pg, about 103 pg, about 104 pg, about 105 pg, about 106 pg, about 107 pg, about 108 pg, about 109 pg, about 110 pg, about 111 pg, about 112 pg, about 113 pg, about 114 pg, about 115 pg, about 116 pg, about 117 pg, about 118 pg, about 119 pg, about 120 pg, about 121 pg, about 122 pg, about 123 pg, about 124 pg, about 125 pg, about 126 pg, about 127 pg, about 128 pg, about 129 pg, about 130 pg, about 131 pg, about 132 pg, about 133 pg, about 134 pg, about 135 pg, about 136 pg, about 137 pg, about 138 pg, about 139 pg, about 140 pg, about 141 pg, about 142 pg, about 143 pg, about 144 pg, about 145 pg, about 146 pg, about 147 pg, about 148 pg, about 149 pg, about 150 pg, about 151 pg, about 152 pg, about 153 pg, about 154 pg, about 155 pg, about 156 pg, about 157 pg, about 158 pg, about 159 pg, about 160 pg, about 161 pg, about 162 pg, about 163 pg, about 164 pg, about 165 pg, about 166 pg, about 167 pg, about 168 pg, about 169 pg, about 170 pg, about 171 pg, about 172 pg, about 173 pg, about 174 pg, about 175 pg, about 176 pg, about 177 pg, about 178 pg, about 179 pg, about 180 pg, about 181 pg, about 182 pg, about 183 pg, about 184 pg, about 185 pg, about 186 pg, about 187 pg, about 188 pg, about 189 pg, about 190 pg, about 191 pg, about 192 pg, about 193 pg, about 194 pg, about 195 pg, about 196 pg, about 197 pg, about 198 pg, about 199 pg, about 200 pg, about 201 pg, about 202 pg, about 203 pg, about 204 pg, about 205 pg, about 206 pg, about 207 pg, about 208 pg, about 209 pg, about 210 pg, about 211 pg, about 212 pg, about 213 pg, about 214 pg, about 215 pg, about 216 pg, about 217 pg, about 218 pg, about 219 pg, about 220 pg, about 221 pg, about 222 pg, about 223 pg, about 224 pg, about 225 pg, about 226 pg, about 227 pg, about 228 pg, about 229 pg, about 230 pg, about 231 pg, about 232 pg, about 233 pg, about 234 pg, about 235 pg, about 236 pg, about 237 pg, about 238 pg, about 239 pg, about 240 pg, about 241 pg, about 242 pg, about 243 pg, about 244 pg, about 245 pg, about 246 pg, about 247 pg, about 248 pg, about 249 pg, about 250 pg, about 251 pg, about 252 pg, about 253 pg, about 254 pg, about 255 pg, about 256 pg, about 257 pg, about 258 pg, about 259 pg, about 260 pg, about 261 pg, about 262 pg, about 263 pg, about 264 pg, about 265 pg, about 266 pg, about 267 pg, about 268 pg, about 269 pg, about 270 pg, about 271 pg, about 272 pg, about 273 pg, about 274 pg, about 275 pg, about 276 pg, about 277 pg, about 278 pg, about 279 pg, about 280 pg, about 281 pg, about 282 pg, about 283 pg, about 284 pg, about 285 pg, about 286 pg, about 287 pg, about 288 pg, about 289 pg, about 290 pg, about 291 pg, about 292 pg, about 293 pg, about 294 pg, about 295 pg, about 296 pg, about 297 pg, about 298 pg, about 299 pg, about 300 pg, about 301 pg, about 302 pg, about 303 pg, about 304 pg, about 305 pg, about 306 pg, about 307 pg, about 308 pg, about 309 pg, about 310 pg, about 311 pg, about 312 pg, about 313 pg, about 314 pg, about 315 pg, about 316 pg, about 317 pg, about 318 pg, about 319 pg, about 320 pg, about 321 pg, about 322 pg, about 323 pg, about 324 pg, about 325 pg, about 326 pg, about 327 pg, about 328 pg, about 329 pg, about 330 pg, about 331 pg, about 332 pg, about 333 pg, about 334 pg, about 335 pg, about 336 pg, about 337 pg, about 338 pg, about 339 pg, about 340 pg, about 341 pg, about 342 pg, about 343 pg, about 344 pg, about 345 pg, about 346 pg, about 347 pg, about 348 pg, about 349 pg, about 350 pg, about 351 pg, about 352 pg, about 353 pg, about 354 pg, about 355 pg, about 356 pg, about 357 pg, about 358 pg, about 359 pg, about 360 pg, about 361 pg, about 362 pg, about 363 pg, about 364 pg, about 365 pg, about 366 pg, about 367 pg, about 368 pg, about 369 pg, about 370 pg, about 371 pg, about 372 pg, about 373 pg, about 374 pg, about 375 pg, about 376 pg, about 377 pg, about 378 pg, about 379 pg, about 380 pg, about 381 pg, about 382 pg, about 383 pg, about 384 pg, about 385 pg, about 386 pg, about 387 pg, about 388 pg, about 389 pg, about 390 pg, about 391 pg, about 392 pg, about 393 pg, about 394 pg, about 395 pg, about 396 pg, about 397 pg, about 398 pg, about 399 pg, about 400 pg, about 401 pg, about 402 pg, about 403 pg, about 404 pg, about 405 pg, about 406 pg, about 407 pg, about 408 pg, about 409 pg, about 410 pg, about 411 pg, about 412 pg, about 413 pg, about 414 pg, about 415 pg, about 416 pg, about 417 pg, about 418 pg, about 419 pg, about 420 pg, about 421 pg, about 422 pg, about 423 pg, about 424 pg, about 425 pg, about 426 pg, about 427 pg, about 428 pg, about 429 pg, about 430 pg, about 431 pg, about 432 pg, about 433 pg, about 434 pg, about 435 pg, about 436 pg, about 437 pg, about 438 pg, about 439 pg, about 440 pg, about 441 pg, about 442 pg, about 443 pg, about 444 pg, about 445 pg, about 446 pg, about 447 pg, about 448 pg, about 449 pg, about 450 pg, about 451 pg, about 452 pg, about 453 pg, about 454 pg, about 455 pg, about 456 pg, about 457 pg, about 458 pg, about 459 pg, about 460 pg, about 461 pg, about 462 pg, about 463 pg, about 464 pg, about 465 pg, about 466 pg, about 467 pg, about 468 pg, about 469 pg, about 470 pg, about 471 pg, about 472 pg, about 473 pg, about 474 pg, about 475 pg, about 476 pg, about 477 pg, about 478 pg, about 479 pg, about 480 pg, about 481 pg, about 482 pg, about 483 pg, about 484 pg, about 485 pg, about 486 pg, about 487 pg, about 488 pg, about 489 pg, about 490 pg, about 491 pg, about 492 pg, about 493 pg, about 494 pg, about 495 pg, about 496 pg, about 497 pg, about 498 pg, about 499 pg, about 500 pg, about 501 pg, about 502 pg, about 503 pg, about 504 pg, about 505 pg, about 506 pg, about 507 pg, about 508 pg, about 509 pg, about 510 pg, about 511 pg, about 512 pg, about 513 pg, about 514 pg, about 515 pg, about 516 pg, about 517 pg, about 518 pg, about 519 pg, about 520 pg, about 521 pg, about 522 pg, about 523 pg, about 524 pg, about 525 pg, about 526 pg, about 527 pg, about 528 pg, about 529 pg, about 530 pg, about 531 pg, about 532 pg, about 533 pg, about 534 pg, about 535 pg, about 536 pg, about 537 pg, about 538 pg, about 539 pg, about 540 pg, about 541 pg, about 542 pg, about 543 pg, about 544 pg, about 545 pg, about 546 pg, about 547 pg, about 548 pg, about 549 pg, about 550 pg, about 551 pg, about 552 pg, about 553 pg, about 554 pg, about 555 pg, about 556 pg, about 557 pg, about 558 pg, about 559 pg, about 560 pg, about 561 pg, about 562 pg, about 563 pg, about 564 pg, about 565 pg, about 566 pg, about 567 pg, about 568 pg, about 569 pg, about 570 pg, about 571 pg, about 572 pg, about 573 pg, about 574 pg, about 575 pg, about 576 pg, about 577 pg, about 578 pg, about 579 pg, about 580 pg, about 581 pg, about 582 pg, about 583 pg, about 584 pg, about 585 pg, about 586 pg, about 587 pg, about 588 pg, about 589 pg, about 590 pg, about 591 pg, about 592 pg, about 593 pg, about 594 pg, about 595 pg, about 596 pg, about 597 pg, about 598 pg, about 599 pg, about 600 pg, about 601 pg, about 602 pg, about 603 pg, about 604 pg, about 605 pg, about 606 pg, about 607 pg, about 608 pg, about 609 pg, about 610 pg, about 611 pg, about 612 pg, about 613 pg, about 614 pg, about 615 pg, about 616 pg, about 617 pg, about 618 pg, about 619 pg, about 620 pg, about 621 pg, about 622 pg, about 623 pg, about 624 pg, about 625 pg, about 626 pg, about 627 pg, about 628 pg, about 629 pg, about 630 pg, about 631 pg, about 632 pg, about 633 pg, about 634 pg, about 635 pg, about 636 pg, about 637 pg, about 638 pg, about 639 pg, about 640 pg, about 641 pg, about 642 pg, about 643 pg, about 644 pg, about 645 pg, about 646 pg, about 647 pg, about 648 pg, about 649 pg, about 650 pg, about 651 pg, about 652 pg, about 653 pg, about 654 pg, about 655 pg, about 656 pg, about 657 pg, about 658 pg, about 659 pg, about 660 pg, about 661 pg, about 662 pg, about 663 pg, about 664 pg, about 665 pg, about 666 pg, about 667 pg, about 668 pg, about 669 pg, about 670 pg, about 671 pg, about 672 pg, about 673 pg, about 674 pg, about 675 pg, about 676 pg, about 677 pg, about 678 pg, about 679 pg, about 680 pg, about 681 pg, about 682 pg, about 683 pg, about 684 pg, about 685 pg, about 686 pg, about 687 pg, about 688 pg, about 689 pg, about 690 pg, about 691 pg, about 692 pg, about 693 pg, about 694 pg, about 695 pg, about 696 pg, about 697 pg, about 698 pg, about 699 pg, about 700 pg, about 701 pg, about 702 pg, about 703 pg, about 704 pg, about 705 pg, about 706 pg, about 707 pg, about 708 pg, about 709 pg, about 710 pg, about 711 pg, about 712 pg, about 713 pg, about 714 pg, about 715 pg, about 716 pg, about 717 pg, about 718 pg, about 719 pg, about 720 pg, about 721 pg, about 722 pg, about 723 pg, about 724 pg, about 725 pg, about 726 pg, about 727 pg, about 728 pg, about 729 pg, about 730 pg, about 731 pg, about 732 pg, about 733 pg, about 734 pg, about 735 pg, about 736 pg, about 737 pg, about 738 pg, about 739 pg, about 740 pg, about 741 pg, about 742 pg, about 743 pg, about 744 pg, about 745 pg, about 746 pg, about 747 pg, about 748 pg, about 749 pg, about 750 pg, about 751 pg, about 752 pg, about 753 pg, about 754 pg, about 755 pg, about 756 pg, about 757 pg, about 758 pg, about 759 pg, about 760 pg, about 761 pg, about 762 pg, about 763 pg, about 764 pg, about 765 pg, about 766 pg, about 767 pg, about 768 pg, about 769 pg, about 770 pg, about 771 pg, about 772 pg, about 773 pg, about 774 pg, about 775 pg, about 776 pg, about 777 pg, about 778 pg, about 779 pg, about 780 pg, about 781 pg, about 782 pg, about 783 pg, about 784 pg, about 785 pg, about 786 pg, about 787 pg, about 788 pg, about 789 pg, about 790 pg, about 791 pg, about 792 pg, about 793 pg, about 794 pg, about 795 pg, about 796 pg, about 797 pg, about 798 pg, about 799 pg, about 800 pg, about 801 pg, about 802 pg, about 803 pg, about 804 pg, about 805 pg, about 806 pg, about 807 pg, about 808 pg, about 809 pg, about 810 pg, about 811 pg, about 812 pg, about 813 pg, about 814 pg, about 815 pg, about 816 pg, about 817 pg, about 818 pg, about 819 pg, about 820 pg, about 821 pg, about 822 pg, about 823 pg, about 824 pg, about 825 pg, about 826 pg, about 827 pg, about 828 pg, about 829 pg, about 830 pg, about 831 pg, about 832 pg, about 833 pg, about 834 pg, about 835 pg, about 836 pg, about 837 pg, about 838 pg, about 839 pg, about 840 pg, about 841 pg, about 842 pg, about 843 pg, about 844 pg, about 845 pg, about 846 pg, about 847 pg, about 848 pg, about 849 pg, about 850 pg, about 851 pg, about 852 pg, about 853 pg, about 854 pg, about 855 pg, about 856 pg, about 857 pg, about 858 pg, about 859 pg, about 860 pg, about 861 pg, about 862 pg, about 863 pg, about 864 pg, about 865 pg, about 866 pg, about 867 pg, about 868 pg, about 869 pg, about 870 pg, about 871 pg, about 872 pg, about 873 pg, about 874 pg, about 875 pg, about 876 pg, about 877 pg, about 878 pg, about 879 pg, about 880 pg, about 881 pg, about 882 pg, about 883 pg, about 884 pg, about 885 pg, about 886 pg, about 887 pg, about 888 pg, about 889 pg, about 890 pg, about 891 pg, about 892 pg, about 893 pg, about 894 pg, about 895 pg, about 896 pg, about 897 pg, about 898 pg, about 899 pg, about 900 pg, about 901 pg, about 902 pg, about 903 pg, about 904 pg, about 905 pg, about 906 pg, about 907 pg, about 908 pg, about 909 pg, about 910 pg, about 911 pg, about 912 pg, about 913 pg, about 914 pg, about 915 pg, about 916 pg, about 917 pg, about 918 pg, about 919 pg, about 920 pg, about 921 pg, about 922 pg, about 923 pg, about 924 pg, about 925 pg, about 926 pg, about 927 pg, about 928 pg, about 929 pg, about 930 pg, about 931 pg, about 932 pg, about 933 pg, about 934 pg, about 935 pg, about 936 pg, about 937 pg, about 938 pg, about 939 pg, about 940 pg, about 941 pg, about 942 pg, about 943 pg, about 944 pg, about 945 pg, about 946 pg, about 947 pg, about 948 pg, about 949 pg, about 950 pg, about 951 pg, about 952 pg, about 953 pg, about 954 pg, about 955 pg, about 956 pg, about 957 pg, about 958 pg, about 959 pg, about 960 pg, about 961 pg, about 962 pg, about 963 pg, about 964 pg, about 965 pg, about 966 pg, about 967 pg, about 968 pg, about 969 pg, about 970 pg, about 971 pg, about 972 pg, about 973 pg, about 974 pg, about 975 pg, about 976 pg, about 977 pg, about 978 pg, about 979 pg, about 980 pg, about 981 pg, about 982 pg, about 983 pg, about 984 pg, about 985 pg, about 986 pg, about 987 pg, about 988 pg, about 989 pg, about 990 pg, about 991 pg, about 992 pg, about 993 pg, about 994 pg, about 995 pg, about 996 pg, about 997 pg, about 998 pg, about 999 pg, about 1000 pg, of total RSV F glycoproteins, including all values and ranges therebetween. [00245] In embodiments, the methods comprise administering from about 1 pg to about 100 pg of each CoV S glycoprotein. In embodiments, the methods comprise administering from about 2.5 pg to about 35 pg of each CoV S glycoprotein. In embodiments, the methods comprise administering from about 5 pg to about 25 pg of each CoV S glycoprotein. In embodiments, the methods comprise administering from about 14 pg to about 15 pg of each CoV S glycoprotein. In embodiments, the methods comprise administering from about 15 pg to about 35 pg of each CoV S glycoprotein. In embodiments, the methods comprise administering from about 1 pg to about 100 pg of total CoV S glycoproteins. In embodiments, the methods comprise administering about 1 pg, about 2 pg, about 3 pg, about 4 pg, about 5 pg, about 6 pg, about 7 pg, about 8 pg, about 9 pg, about 10 pg, about 11 pg, about 12 pg, about 13 pg, about 14 pg, about 15 pg, about 16 pg, about 17 pg, about 18 pg, about 19 pg, about 20 pg, about 21 pg, about 22 pg, about 23 pg, about 24 pg, about 25 pg, about 26 pg, about

27 pg, about 28 pg, about 29 pg, about 30 pg, about 31 pg, about 32 pg, about 33 pg, about 34 pg, about 35 pg, about 36 pg, about 37 pg, about 38 pg, about 39 pg, about

40 pg, about 41 pg, about 42 pg, about 43 pg, about 44 pg, about 45 pg, about 46 pg, about 47 pg, about 48 pg, about 49 pg, about 50 pg, about 51 pg, about 52 pg, about 53 pg, about 54 pg, about 55 pg, about 56 pg, about 57 pg, about 58 pg, about 59 pg, about 60 pg, about 61 pg, about 62 pg, about 63 pg, about 64 pg, about 65 pg, about 66 pg, about 67 pg, about 68 pg, about 69 pg, about 70 pg, about 71 pg, about 72 pg, about 73 pg, about 74 pg, about 75 pg, about 76 pg, about 77 pg, about 78 pg, about 79 pg, about 80 pg, about 81 pg, about 82 pg, about 83 pg, about 84 pg, about 85 pg, about 86 pg, about 87 pg, about 88 pg, about 89 pg, about 90 pg, about 91 pg, about 92 pg, about 93 pg, about 94 pg, about 95 pg, about 96 pg, about 97 pg, about 98 pg, about 99 pg, about 100 pg, including all values and ranges therebetween, of each CoV S glycoprotein. In embodiments, the methods comprise administering about 1 pg, about 2 pg, about 3 pg, about 4 pg, about 5 pg, about 6 pg, about 7 pg, about 8 pg, about 9 pg, about 10 pg, about 11 pg, about 12 pg, about 13 pg, about 14 pg, about 15 pg, about 16 pg, about 17 pg, about 18 pg, about 19 pg, about 20 pg, about 21 pg, about 22 pg, about 23 pg, about 24 pg, about 25 pg, about 26 pg, about 27 pg, about

28 pg, about 29 pg, about 30 pg, about 31 pg, about 32 pg, about 33 pg, about 34 pg, about 35 pg, about 36 pg, about 37 pg, about 38 pg, about 39 pg, about 40 pg, about

41 pg, about 42 pg, about 43 pg, about 44 pg, about 45 pg, about 46 pg, about 47 pg, about 48 pg, about 49 pg, about 50 pg, about 51 pg, about 52 pg, about 53 pg, about 54 pg, about 55 pg, about 56 pg, about 57 pg, about 58 pg, about 59 pg, about 60 pg, about 61 pg, about 62 pg, about 63 pg, about 64 pg, about 65 pg, about 66 pg, about 67 pg, about 68 pg, about 69 pg, about 70 pg, about 71 pg, about 72 pg, about 73 pg, about 74 pg, about 75 pg, about 76 pg, about 77 pg, about 78 pg, about 79 pg, about 80 pg, about 81 pg, about 82 pg, about 83 pg, about 84 pg, about 85 pg, about 86 pg, about 87 pg, about 88 pg, about 89 pg, about 90 pg, about 91 pg, about 92 pg, about 93 pg, about 94 pg, about 95 pg, about 96 pg, about 97 pg, about 98 pg, about 99 pg, about 100 pg of total CoV S glycoproteins, including all values and ranges therebetween. In embodiments, the methods comprise administering about 1 pg of a CoV S glycoprotein. In embodiments, the methods comprise administering about 13 pg of a CoV S glycoprotein. In embodiments, the immunogenic compositions comprise about 5 pg of a CoV S glycoprotein. In embodiments, the methods comprise administering about 25 pg of a CoV S glycoprotein.

[00246] In embodiments, the methods comprise administering from about 1 pg to about 300 pg of each HA glycoprotein. In embodiments, the methods comprise administering from about 5 pg to about 60 pg of each HA glycoprotein. In embodiments, the methods comprise administering from about 24 pg to about 40 pg of each HA glycoprotein. In embodiments, the methods comprise administering from about 30 pg to about 40 pg of each HA glycoprotein. In embodiments, the methods comprise administering from about 30 pg to about 60 pg of each HA glycoprotein. In embodiments, the methods comprise administering from about 33 pg to about 39 pg of each HA glycoprotein. In embodiments, the methods comprise administering from about 20 pg to about 240 pg of each HA glycoprotein. In embodiments, the methods comprise administering from about 30 pg to about 60 pg of each HA glycoprotein. In embodiments, the methods comprise administering from about 24 pg to about 40 pg of each HA glycoprotein. In embodiments, the methods comprise administering about 1 pg, about 2 pg, about 3 pg, about 4 pg, about 5 pg, about 6 pg, about 7 pg, about 8 pg, about 9 pg, about 10 pg, about 11 pg, about 12 pg, about 13 pg, about 14 pg, about 15 pg, about 16 pg, about 17 pg, about 18 pg, about 19 pg, about 20 pg, about 21 pg, about 22 pg, about 23 pg, about 24 pg, about 25 pg, about 26 pg, about 27 pg, about 28 pg, about 29 pg, about 30 pg, about 31 pg, about 32 pg, about 33 pg, about 34 pg, about 35 pg, about 36 pg, about 37 pg, about 38 pg, about 39 pg, about 40 pg, about 41 pg, about 42 pg, about 43 pg, about 44 pg, about 45 pg, about 46 pg, about 47 pg, about 48 pg, about 49 pg, about 50 pg, about 51 pg, about 52 pg, about 53 pg, about 54 pg, about 55 pg, about 56 pg, about 57 pg, about 58 pg, about 59 pg, about 60 pg, about 61 pg, about 62 pg, about 63 pg, about 64 pg, about 65 pg, about 66 pg, about 67 pg, about 68 pg, about 69 pg, about 70 pg, about 71 pg, about 72 pg, about 73 pg, about 74 pg, about 75 pg, about 76 pg, about 77 pg, about 78 pg, about 79 pg, about 80 pg, about 81 pg, about 82 pg, about 83 pg, about 84 pg, about 85 pg, about 86 pg, about 87 pg, about 88 pg, about 89 pg, about 90 pg, about 91 pg, about 92 pg, about 93 pg, about 94 pg, about 95 pg, about 96 pg, about 97 pg, about 98 pg, about 99 pg, about 100 pg, about 101 pg, about 102 pg, about 103 pg, about 104 pg, about 105 pg, about 106 pg, about 107 pg, about 108 pg, about 109 pg, about 110 pg, about

111 pg, about 112 pg, about 113 pg, about 114 pg, about 115 pg, about 116 pg, about 117 pg, about 118 pg, about 119 pg, about 120 pg, about 121 pg, about 122 pg, about 123 pg, about 124 pg, about 125 pg, about 126 pg, about 127 pg, about 128 pg, about 129 pg, about 130 pg, about 131 pg, about 132 pg, about 133 pg, about 134 pg, about 135 pg, about 136 pg, about 137 pg, about 138 pg, about 139 pg, about 140 pg, about 141 pg, about 142 pg, about 143 pg, about 144 pg, about 145 pg, about 146 pg, about 147 pg, about 148 pg, about 149 pg, about 150 pg, about 151 pg, about 152 pg, about 153 pg, about 154 pg, about 155 pg, about 156 pg, about 157 pg, about 158 pg, about 159 pg, about 160 pg, about 161 pg, about 162 pg, about 163 pg, about 164 pg, about 165 pg, about 166 pg, about 167 pg, about 168 pg, about 169 pg, about 170 pg, about 171 pg, about 172 pg, about 173 pg, about 174 pg, about 175 pg, about 176 pg, about 177 pg, about 178 pg, about 179 pg, about 180 pg, about 181 pg, about 182 pg, about 183 pg, about 184 pg, about 185 pg, about 186 pg, about 187 pg, about 188 pg, about 189 pg, about 190 pg, about 191 pg, about 192 pg, about 193 pg, about 194 pg, about 195 pg, about 196 pg, about 197 pg, about 198 pg, about 199 pg, about 200 pg, about 201 pg, about 202 pg, about 203 pg, about 204 pg, about 205 pg, about 206 pg, about 207 pg, about 208 pg, about 209 pg, about 210 pg, about 211 pg, about 212 pg, about 213 pg, about 214 pg, about 215 pg, about 216 pg, about 217 pg, about 218 pg, about 219 pg, about 220 pg, about 221 pg, about 222 pg, about 223 pg, about 224 pg, about 225 pg, about 226 pg, about 227 pg, about 228 pg, about 229 pg, about 230 pg, about 231 pg, about 232 pg, about 233 pg, about 234 pg, about 235 pg, about 236 pg, about 237 pg, about 238 pg, about 239 pg, about 240 pg, about 241 pg, about 242 pg, about 243 pg, about 244 pg, about 245 pg, about 246 pg, about 247 pg, about 248 pg, about 249 pg, about 250 pg, about 251 pg, about 252 pg, about 253 pg, about 254 pg, about 255 pg, about 256 pg, about 257 pg, about 258 pg, about 259 pg, about 260 pg, about 261 pg, about 262 pg, about 263 pg, about 264 pg, about 265 pg, about 266 pg, about

267 pg, about 268 pg, about 269 pg, about 270 pg, about 271 pg, about 272 pg, about

273 pg, about 274 pg, about 275 pg, about 276 pg, about 277 pg, about 278 pg, about

279 pg, about 280 pg, about 281 pg, about 282 pg, about 283 pg, about 284 pg, about

285 pg, about 286 pg, about 287 pg, about 288 pg, about 289 pg, about 290 pg, about

291 pg, about 292 pg, about 293 pg, about 294 pg, about 295 pg, about 296 pg, about

297 pg, about 298 pg, about 299 pg, or about 300 pg of each HA glycoprotein, including all values and ranges therebetween. In embodiments, the methods comprise administering about 30 pg of each HA glycoprotein. In embodiments, the methods comprise administering about 33 pg of each HA glycoprotein. In embodiments, the methods comprise administering about 39 pg of each HA glycoprotein. In embodiments, the methods comprise administering about 54 pg of each HA glycoprotein. In embodiments, the methods comprise administering about 60 pg of each HA glycoprotein. In embodiments, the methods comprise administering about 240 pg of each HA glycoprotein.

[00247] In embodiments, the methods comprise administering from about 1 pg to about 1000 pg of total HA glycoproteins. In embodiments, the methods comprise administering about 1 pg, about 2 pg, about 3 pg, about 4 pg, about 5 pg, about 6 pg, about 7 pg, about 8 pg, about 9 pg, about 10 pg, about 11 pg, about 12 pg, about 13 pg, about 14 pg, about 15 pg, about 16 pg, about 17 pg, about 18 pg, about 19 pg, about 20 pg, about 21 pg, about 22 pg, about 23 pg, about 24 pg, about 25 pg, about 26 pg, about 27 pg, about 28 pg, about 29 pg, about 30 pg, about 31 pg, about 32 pg, about 33 pg, about 34 pg, about 35 pg, about 36 pg, about 37 pg, about 38 pg, about 39 pg, about 40 pg, about 41 pg, about 42 pg, about 43 pg, about 44 pg, about 45 pg, about 46 pg, about 47 pg, about 48 pg, about 49 pg, about 50 pg, about 51 pg, about 52 pg, about 53 pg, about 54 pg, about 55 pg, about 56 pg, about 57 pg, about 58 pg, about 59 pg, about 60 pg, about 61 pg, about 62 pg, about 63 pg, about 64 pg, about 65 pg, about 66 pg, about 67 pg, about 68 pg, about 69 pg, about 70 pg, about 71 pg, about 72 pg, about 73 pg, about 74 pg, about 75 pg, about 76 pg, about 77 pg, about 78 pg, about 79 pg, about 80 pg, about 81 pg, about 82 pg, about 83 pg, about 84 pg, about 85 pg, about 86 pg, about 87 pg, about 88 pg, about 89 pg, about 90 pg, about 91 pg, about 92 pg, about 93 pg, about 94 pg, about 95 pg, about 96 pg, about 97 pg, about 98 pg, about 99 pg, about 100 pg, about 101 pg, about 102 pg, about 103 pg, about 104 pg, about 105 pg, about 106 pg, about 107 pg, about 108 pg, about 109 pg, about 110 pg, about 111 pg, about 112 pg, about 113 pg, about 114 pg, about 115 pg, about 116 pg, about 117 pg, about 118 pg, about 119 pg, about 120 pg, about 121 pg, about 122 pg, about 123 pg, about 124 pg, about 125 pg, about 126 pg, about 127 pg, about 128 pg, about 129 pg, about 130 pg, about 131 pg, about 132 pg, about 133 pg, about 134 pg, about 135 pg, about 136 pg, about 137 pg, about 138 pg, about 139 pg, about 140 pg, about 141 pg, about 142 pg, about 143 pg, about 144 pg, about 145 pg, about 146 pg, about 147 pg, about 148 pg, about 149 pg, about 150 pg, about 151 pg, about 152 pg, about 153 pg, about 154 pg, about 155 pg, about 156 pg, about 157 pg, about 158 pg, about 159 pg, about 160 pg, about 161 pg, about 162 pg, about 163 pg, about 164 pg, about 165 pg, about 166 pg, about 167 pg, about 168 pg, about 169 pg, about 170 pg, about 171 pg, about 172 pg, about 173 pg, about 174 pg, about 175 pg, about 176 pg, about 177 pg, about 178 pg, about 179 pg, about 180 pg, about 181 pg, about 182 pg, about 183 pg, about 184 pg, about 185 pg, about 186 pg, about 187 pg, about 188 pg, about 189 pg, about 190 pg, about 191 pg, about 192 pg, about 193 pg, about 194 pg, about 195 pg, about 196 pg, about 197 pg, about 198 pg, about 199 pg, about 200 pg, about 201 pg, about 202 pg, about 203 pg, about 204 pg, about 205 pg, about 206 pg, about 207 pg, about 208 pg, about 209 pg, about 210 pg, about 211 pg, about 212 pg, about 213 pg, about 214 pg, about 215 pg, about 216 pg, about 217 pg, about 218 pg, about 219 pg, about 220 pg, about 221 pg, about 222 pg, about 223 pg, about 224 pg, about 225 pg, about 226 pg, about 227 pg, about 228 pg, about 229 pg, about 230 pg, about 231 pg, about 232 pg, about 233 pg, about 234 pg, about 235 pg, about 236 pg, about 237 pg, about 238 pg, about 239 pg, about 240 pg, about 241 pg, about 242 pg, about 243 pg, about 244 pg, about 245 pg, about 246 pg, about 247 pg, about 248 pg, about 249 pg, about 250 pg, about 251 pg, about 252 pg, about 253 pg, about 254 pg, about 255 pg, about 256 pg, about 257 pg, about 258 pg, about 259 pg, about 260 pg, about 261 pg, about 262 pg, about 263 pg, about 264 pg, about 265 pg, about 266 pg, about 267 pg, about 268 pg, about 269 pg, about 270 pg, about 271 pg, about 272 pg, about 273 pg, about 274 pg, about 275 pg, about 276 pg, about 277 pg, about 278 pg, about 279 pg, about 280 pg, about 281 pg, about 282 pg, about 283 pg, about 284 pg, about 285 pg, about 286 pg, about 287 pg, about 288 pg, about 289 pg, about 290 pg, about 291 pg, about 292 pg, about 293 pg, about 294 pg, about 295 pg, about 296 pg, about 297 pg, about 298 pg, about 299 pg, about 300 pg, about 301 pg, about 302 pg, about 303 pg, about 304 pg, about 305 pg, about 306 pg, about 307 pg, about 308 pg, about 309 pg, about 310 pg, about 311 pg, about 312 pg, about 313 pg, about 314 pg, about 315 pg, about 316 pg, about 317 pg, about 318 pg, about 319 pg, about 320 pg, about 321 pg, about 322 pg, about 323 pg, about 324 pg, about 325 pg, about 326 pg, about 327 pg, about 328 pg, about 329 pg, about 330 pg, about 331 pg, about 332 pg, about 333 pg, about 334 pg, about 335 pg, about 336 pg, about 337 pg, about 338 pg, about 339 pg, about 340 pg, about 341 pg, about 342 pg, about 343 pg, about 344 pg, about 345 pg, about 346 pg, about 347 pg, about 348 pg, about 349 pg, about 350 pg, about 351 pg, about 352 pg, about 353 pg, about 354 pg, about 355 pg, about 356 pg, about 357 pg, about 358 pg, about 359 pg, about 360 pg, about 361 pg, about 362 pg, about 363 pg, about 364 pg, about 365 pg, about 366 pg, about 367 pg, about 368 pg, about 369 pg, about 370 pg, about 371 pg, about 372 pg, about 373 pg, about 374 pg, about 375 pg, about 376 pg, about 377 pg, about 378 pg, about 379 pg, about 380 pg, about 381 pg, about 382 pg, about 383 pg, about 384 pg, about 385 pg, about 386 pg, about 387 pg, about 388 pg, about 389 pg, about 390 pg, about 391 pg, about 392 pg, about 393 pg, about 394 pg, about 395 pg, about 396 pg, about 397 pg, about 398 pg, about 399 pg, about 400 pg, about 401 pg, about 402 pg, about 403 pg, about 404 pg, about 405 pg, about 406 pg, about 407 pg, about 408 pg, about 409 pg, about 410 pg, about 411 pg, about 412 pg, about 413 pg, about 414 pg, about 415 pg, about 416 pg, about 417 pg, about 418 pg, about 419 pg, about 420 pg, about 421 pg, about 422 pg, about 423 pg, about 424 pg, about 425 pg, about 426 pg, about 427 pg, about 428 pg, about 429 pg, about 430 pg, about 431 pg, about 432 pg, about 433 pg, about 434 pg, about 435 pg, about 436 pg, about 437 pg, about 438 pg, about 439 pg, about 440 pg, about 441 pg, about 442 pg, about 443 pg, about 444 pg, about 445 pg, about 446 pg, about 447 pg, about 448 pg, about 449 pg, about 450 pg, about 451 pg, about 452 pg, about 453 pg, about 454 pg, about 455 pg, about 456 pg, about 457 pg, about 458 pg, about 459 pg, about 460 pg, about 461 pg, about 462 pg, about 463 pg, about 464 pg, about 465 pg, about 466 pg, about 467 pg, about 468 pg, about 469 pg, about 470 pg, about 471 pg, about 472 pg, about 473 pg, about 474 pg, about 475 pg, about 476 pg, about 477 pg, about 478 pg, about 479 pg, about 480 pg, about 481 pg, about 482 pg, about 483 pg, about 484 pg, about 485 pg, about 486 pg, about 487 pg, about 488 pg, about 489 pg, about 490 pg, about 491 pg, about 492 pg, about 493 pg, about 494 pg, about 495 pg, about 496 pg, about 497 pg, about 498 pg, about 499 pg, about 500 pg, about 501 pg, about 502 pg, about 503 pg, about 504 pg, about 505 pg, about 506 pg, about 507 pg, about 508 pg, about 509 pg, about 510 pg, about 511 pg, about 512 pg, about 513 pg, about 514 pg, about 515 pg, about 516 pg, about 517 pg, about 518 pg, about 519 pg, about 520 pg, about 521 pg, about 522 pg, about 523 pg, about 524 pg, about 525 pg, about 526 pg, about 527 pg, about 528 pg, about 529 pg, about 530 pg, about 531 pg, about 532 pg, about 533 pg, about 534 pg, about 535 pg, about 536 pg, about 537 pg, about 538 pg, about 539 pg, about 540 pg, about 541 pg, about 542 pg, about 543 pg, about 544 pg, about 545 pg, about 546 pg, about 547 pg, about 548 pg, about 549 pg, about 550 pg, about 551 pg, about 552 pg, about 553 pg, about 554 pg, about 555 pg, about 556 pg, about 557 pg, about 558 pg, about 559 pg, about 560 pg, about 561 pg, about 562 pg, about 563 pg, about 564 pg, about 565 pg, about 566 pg, about 567 pg, about 568 pg, about 569 pg, about 570 pg, about 571 pg, about 572 pg, about 573 pg, about 574 pg, about 575 pg, about 576 pg, about 577 pg, about 578 pg, about 579 pg, about 580 pg, about 581 pg, about 582 pg, about 583 pg, about 584 pg, about 585 pg, about 586 pg, about 587 pg, about 588 pg, about 589 pg, about 590 pg, about 591 pg, about 592 pg, about 593 pg, about 594 pg, about 595 pg, about 596 pg, about 597 pg, about 598 pg, about 599 pg, about 600 pg, about 601 pg, about 602 pg, about 603 pg, about 604 pg, about 605 pg, about 606 pg, about 607 pg, about 608 pg, about 609 pg, about 610 pg, about 611 pg, about 612 pg, about 613 pg, about 614 pg, about 615 pg, about 616 pg, about 617 pg, about 618 pg, about 619 pg, about 620 pg, about 621 pg, about 622 pg, about 623 pg, about 624 pg, about 625 pg, about 626 pg, about 627 pg, about 628 pg, about 629 pg, about 630 pg, about 631 pg, about 632 pg, about 633 pg, about 634 pg, about 635 pg, about 636 pg, about 637 pg, about 638 pg, about 639 pg, about 640 pg, about 641 pg, about 642 pg, about 643 pg, about 644 pg, about 645 pg, about 646 pg, about 647 pg, about 648 pg, about 649 pg, about 650 pg, about 651 pg, about 652 pg, about 653 pg, about 654 pg, about 655 pg, about 656 pg, about 657 pg, about 658 pg, about 659 pg, about 660 pg, about 661 pg, about 662 pg, about 663 pg, about 664 pg, about 665 pg, about 666 pg, about 667 pg, about 668 pg, about 669 pg, about 670 pg, about 671 pg, about 672 pg, about 673 pg, about 674 pg, about 675 pg, about 676 pg, about 677 pg, about 678 pg, about 679 pg, about 680 pg, about 681 pg, about 682 pg, about 683 pg, about 684 pg, about 685 pg, about 686 pg, about 687 pg, about 688 pg, about 689 pg, about 690 pg, about 691 pg, about 692 pg, about 693 pg, about 694 pg, about 695 pg, about 696 pg, about 697 pg, about 698 pg, about 699 pg, about 700 pg, about 701 pg, about 702 pg, about 703 pg, about 704 pg, about 705 pg, about 706 pg, about 707 pg, about 708 pg, about 709 pg, about 710 pg, about 711 pg, about 712 pg, about 713 pg, about 714 pg, about 715 pg, about 716 pg, about 717 pg, about 718 pg, about 719 pg, about 720 pg, about 721 pg, about 722 pg, about 723 pg, about 724 pg, about 725 pg, about 726 pg, about 727 pg, about 728 pg, about 729 pg, about 730 pg, about 731 pg, about 732 pg, about 733 pg, about 734 pg, about 735 pg, about 736 pg, about 737 pg, about 738 pg, about 739 pg, about 740 pg, about 741 pg, about 742 pg, about 743 pg, about 744 pg, about 745 pg, about 746 pg, about 747 pg, about 748 pg, about 749 pg, about 750 pg, about 751 pg, about 752 pg, about 753 pg, about 754 pg, about 755 pg, about 756 pg, about 757 pg, about 758 pg, about 759 pg, about 760 pg, about 761 pg, about 762 pg, about 763 pg, about 764 pg, about 765 pg, about 766 pg, about 767 pg, about 768 pg, about 769 pg, about 770 pg, about 771 pg, about 772 pg, about 773 pg, about 774 pg, about 775 pg, about 776 pg, about 777 pg, about 778 pg, about 779 pg, about 780 pg, about 781 pg, about 782 pg, about 783 pg, about 784 pg, about 785 pg, about 786 pg, about 787 pg, about 788 pg, about 789 pg, about 790 pg, about 791 pg, about 792 pg, about 793 pg, about 794 pg, about 795 pg, about 796 pg, about 797 pg, about 798 pg, about 799 pg, about 800 pg, about 801 pg, about 802 pg, about 803 pg, about 804 pg, about 805 pg, about 806 pg, about 807 pg, about 808 pg, about 809 pg, about 810 pg, about 811 pg, about 812 pg, about 813 pg, about 814 pg, about 815 pg, about 816 pg, about 817 pg, about 818 pg, about 819 pg, about 820 pg, about 821 pg, about 822 pg, about 823 pg, about 824 pg, about 825 pg, about 826 pg, about 827 pg, about 828 pg, about 829 pg, about 830 pg, about 831 pg, about 832 pg, about 833 pg, about 834 pg, about 835 pg, about 836 pg, about 837 pg, about 838 pg, about 839 pg, about 840 pg, about 841 pg, about 842 pg, about 843 pg, about 844 pg, about 845 pg, about 846 pg, about 847 pg, about 848 pg, about 849 pg, about 850 pg, about 851 pg, about 852 pg, about 853 pg, about 854 pg, about 855 pg, about 856 pg, about 857 pg, about 858 pg, about 859 pg, about 860 pg, about 861 pg, about 862 pg, about 863 pg, about 864 pg, about 865 pg, about 866 pg, about 867 pg, about 868 pg, about 869 pg, about 870 pg, about 871 pg, about 872 pg, about 873 pg, about 874 pg, about 875 pg, about 876 pg, about 877 pg, about 878 pg, about 879 pg, about 880 pg, about 881 pg, about 882 pg, about 883 pg, about 884 pg, about 885 pg, about 886 pg, about 887 pg, about 888 pg, about 889 pg, about 890 pg, about 891 pg, about 892 pg, about 893 pg, about 894 pg, about 895 pg, about 896 pg, about 897 pg, about 898 pg, about 899 pg, about 900 pg, about 901 pg, about 902 pg, about 903 pg, about 904 pg, about 905 pg, about 906 pg, about 907 pg, about 908 pg, about 909 pg, about 910 pg, about 911 pg, about 912 pg, about 913 pg, about 914 pg, about 915 pg, about 916 pg, about 917 pg, about 918 pg, about 919 pg, about 920 pg, about 921 pg, about 922 pg, about 923 pg, about 924 pg, about 925 pg, about 926 pg, about 927 pg, about 928 pg, about 929 pg, about 930 pg, about 931 pg, about 932 pg, about 933 pg, about 934 pg, about 935 pg, about 936 pg, about 937 pg, about 938 pg, about 939 pg, about 940 pg, about 941 pg, about 942 pg, about 943 pg, about 944 pg, about 945 pg, about 946 pg, about 947 pg, about 948 pg, about 949 pg, about 950 pg, about 951 pg, about 952 pg, about 953 pg, about 954 pg, about 955 pg, about 956 pg, about 957 pg, about 958 pg, about 959 pg, about 960 pg, about 961 pg, about 962 pg, about 963 pg, about 964 pg, about 965 pg, about 966 pg, about 967 pg, about 968 pg, about 969 pg, about 970 pg, about 971 pg, about 972 pg, about 973 pg, about 974 pg, about 975 pg, about 976 pg, about 977 pg, about 978 pg, about 979 pg, about 980 pg, about 981 pg, about 982 pg, about 983 pg, about 984 pg, about 985 pg, about 986 pg, about 987 pg, about 988 pg, about 989 pg, about 990 pg, about 991 pg, about 992 pg, about 993 pg, about 994 pg, about 995 pg, about 996 pg, about 997 pg, about 998 pg, about 999 pg, about 1000 pg, of total HA glycoproteins, including all values and ranges therebetween.

[00248] In embodiments, the methods comprise administering from about 1 pg to about 400 pg, from 1 pg to about 350 pg, from 1 pg to about 300 pg, from 1 pg to about 250 pg, from 1 pg to about 200 pg, from 1 pg to about 150 pg, from 1 pg to about 100 pg, from 1 pg to about 50 pg, or from 1 pg to about 25 pg of each viral glycoprotein. In embodiments, the methods comprise administering about 1 pg, about 2 pg, about 3 pg, about 4 pg, about 5 pg, about 6 pg, about 7 pg, about 8 pg, about 9 pg, about 10 pg, about 11 pg, about 12 pg, about 13 pg, about 14 pg, about 15 pg, about 16 pg, about 17 pg, about 18 pg, about 19 pg, about 20 pg, about 21 pg, about 22 pg, about 23 pg, about 24 pg, about 25 pg, about 26 pg, about 27 pg, about 28 pg, about 29 pg, about 30 pg, about 31 pg, about 32 pg, about 33 pg, about 34 pg, about 35 pg, about 36 pg, about 37 pg, about 38 pg, about 39 pg, about 40 pg, about 41 pg, about 42 pg, about 43 pg, about 44 pg, about 45 pg, about 46 pg, about 47 pg, about 48 pg, about 49 pg, about 50 pg, about 51 pg, about 52 pg, about 53 pg, about 54 pg, about 55 pg, about 56 pg, about 57 pg, about 58 pg, about 59 pg, about 60 pg, about 61 pg, about 62 pg, about 63 pg, about 64 pg, about 65 pg, about 66 pg, about 67 pg, about 68 pg, about 69 pg, about 70 pg, about 71 pg, about 72 pg, about 73 pg, about 74 pg, about 75 pg, about 76 pg, about 77 pg, about 78 pg, about 79 pg, about 80 pg, about 81 pg, about 82 pg, about 83 pg, about 84 pg, about 85 pg, about 86 pg, about 87 pg, about 88 pg, about 89 pg, about 90 pg, about 91 pg, about 92 pg, about 93 pg, about 94 pg, about 95 pg, about 96 pg, about 97 pg, about 98 pg, about 99 pg, about pg, about 101 pg, about 102 pg, about 103 pg, about 104 pg, about 105 pg, about pg, about 107 pg, about 108 pg, about 109 pg, about 110 pg, about 111 pg, about pg, about 113 pg, about 114 pg, about 115 pg, about 116 pg, about 117 pg, about pg, about 119 pg, about 120 pg, about 121 pg, about 122 pg, about 123 pg, about pg, about 125 pg, about 126 pg, about 127 pg, about 128 pg, about 129 pg, about pg, about 131 pg, about 132 pg, about 133 pg, about 134 pg, about 135 pg, about pg, about 137 pg, about 138 pg, about 139 pg, about 140 pg, about 141 pg, about pg, about 143 pg, about 144 pg, about 145 pg, about 146 pg, about 147 pg, about pg, about 149 pg, about 150 pg, about 151 pg, about 152 pg, about 153 pg, about pg, about 155 pg, about 156 pg, about 157 pg, about 158 pg, about 159 pg, about pg, about 161 pg, about 162 pg, about 163 pg, about 164 pg, about 165 pg, about pg, about 167 pg, about 168 pg, about 169 pg, about 170 pg, about 171 pg, about pg, about 173 pg, about 174 pg, about 175 pg, about 176 pg, about 177 pg, about pg, about 179 pg, about 180 pg, about 181 pg, about 182 pg, about 183 pg, about pg, about 185 pg, about 186 pg, about 187 pg, about 188 pg, about 189 pg, about pg, about 191 pg, about 192 pg, about 193 pg, about 194 pg, about 195 pg, about pg, about 197 pg, about 198 pg, about 199 pg, about 200 pg, about 201 pg, about pg, about 203 pg, about 204 pg, about 205 pg, about 206 pg, about 207 pg, about pg, about 209 pg, about 210 pg, about 211 pg, about 212 pg, about 213 pg, about pg, about 215 pg, about 216 pg, about 217 pg, about 218 pg, about 219 pg, about pg, about 221 pg, about 222 pg, about 223 pg, about 224 pg, about 225 pg, about pg, about 227 pg, about 228 pg, about 229 pg, about 230 pg, about 231 pg, about pg, about 233 pg, about 234 pg, about 235 pg, about 236 pg, about 237 pg, about pg, about 239 pg, about 240 pg, about 241 pg, about 242 pg, about 243 pg, about pg, about 245 pg, about 246 pg, about 247 pg, about 248 pg, about 249 pg, about pg, about 251 pg, about 252 pg, about 253 pg, about 254 pg, about 255 pg, about pg, about 257 pg, about 258 pg, about 259 pg, about 260 pg, about 261 pg, about pg, about 263 pg, about 264 pg, about 265 pg, about 266 pg, about 267 pg, about pg, about 269 pg, about 270 pg, about 271 pg, about 272 pg, about 273 pg, about pg, about 275 pg, about 276 pg, about 277 pg, about 278 pg, about 279 pg, about pg, about 281 pg, about 282 pg, about 283 pg, about 284 pg, about 285 pg, about pg, about 287 pg, about 288 pg, about 289 pg, about 290 pg, about 291 pg, about pg, about 293 pg, about 294 pg, about 295 pg, about 296 pg, about 297 pg, about pg, about 299 pg, about 300 pg, about 301 pg, about 302 pg, about 303 pg, about 304 pg, about 305 pg, about 306 pg, about 307 pg, about 308 pg, about 309 pg, about

310 pg, about 311 pg, about 312 pg, about 313 pg, about 314 pg, about 315 pg, about

316 pg, about 317 pg, about 318 pg, about 319 pg, about 320 pg, about 321 pg, about 322 pg, about 323 pg, about 324 pg, about 325 pg, about 326 pg, about 327 pg, about

328 pg, about 329 pg, about 330 pg, about 331 pg, about 332 pg, about 333 pg, about

334 pg, about 335 pg, about 336 pg, about 337 pg, about 338 pg, about 339 pg, about

340 pg, about 341 pg, about 342 pg, about 343 pg, about 344 pg, about 345 pg, about

346 pg, about 347 pg, about 348 pg, about 349 pg, about 350 pg, about 351 pg, about

352 pg, about 353 pg, about 354 pg, about 355 pg, about 356 pg, about 357 pg, about

358 pg, about 359 pg, about 360 pg, about 361 pg, about 362 pg, about 363 pg, about

364 pg, about 365 pg, about 366 pg, about 367 pg, about 368 pg, about 369 pg, about

370 pg, about 371 pg, about 372 pg, about 373 pg, about 374 pg, about 375 pg, about

376 pg, about 377 pg, about 378 pg, about 379 pg, about 380 pg, about 381 pg, about

382 pg, about 383 pg, about 384 pg, about 385 pg, about 386 pg, about 387 pg, about

388 pg, about 389 pg, about 390 pg, about 391 pg, about 392 pg, about 393 pg, about

394 pg, about 395 pg, about 396 pg, about 397 pg, about 398 pg, about 399 pg, or about 400 pg of each viral glycoprotein.

[00249] In embodiments, the methods comprise administering from about 1 pg to about 400 pg, from 1 pg to about 350 pg, from 1 pg to about 300 pg, from 1 pg to about 250 pg, from 1 pg to about 200 pg, from 1 pg to about 150 pg, from 1 pg to about 100 pg, from 1 pg to about 50 pg, or from 1 pg to about 25 pg of adjuvant. In embodiments, the immunogenic compositions comprise about 1 pg, about 2 pg, about 3 pg, about 4 pg, about 5 pg, about 6 pg, about 7 pg, about 8 pg, about 9 pg, about 10 pg, about 11 pg, about 12 pg, about 13 pg, about 14 pg, about 15 pg, about 16 pg, about 17 pg, about 18 pg, about 19 pg, about 20 pg, about 21 pg, about 22 pg, about 23 pg, about 24 pg, about 25 pg, about 26 pg, about 27 pg, about 28 pg, about 29 pg, about 30 pg, about 31 pg, about 32 pg, about 33 pg, about 34 pg, about 35 pg, about 36 pg, about 37 pg, about 38 pg, about 39 pg, about 40 pg, about 41 pg, about 42 pg, about 43 pg, about 44 pg, about 45 pg, about 46 pg, about 47 pg, about 48 pg, about 49 pg, about 50 pg, about 51 pg, about 52 pg, about 53 pg, about 54 pg, about 55 pg, about 56 pg, about 57 pg, about 58 pg, about 59 pg, about 60 pg, about 61 pg, about 62 pg, about 63 pg, about 64 pg, about 65 pg, about 66 pg, about 67 pg, about 68 pg, about 69 pg, about 70 pg, about 71 pg, about 72 pg, about 73 pg, about 74 pg, about 75 pg, about 76 pg, about 77 pg, about 78 pg, about 79 pg, about 80 pg, about 81 pg, about 82 pg, about 83 pg, about 84 pg, about 85 pg, about 86 pg, about 87 pg, about 88 pg, about 89 pg, about 90 pg, about 91 pg, about 92 pg, about 93 pg, about 94 pg, about 95 pg, about 96 pg, about 97 pg, about 98 pg, about 99 pg, about 100 pg, about 101 pg, about 102 pg, about 103 pg, about 104 pg, about 105 pg, about 106 pg, about

107 pg, about 108 pg, about 109 pg, about 110 pg, about 111 pg, about 112 pg, about 113 pg, about 114 pg, about 115 pg, about 116 pg, about 117 pg, about 118 pg, about 119 pg, about 120 pg, about 121 pg, about 122 pg, about 123 pg, about 124 pg, about 125 pg, about 126 pg, about 127 pg, about 128 pg, about 129 pg, about 130 pg, about 131 pg, about 132 pg, about 133 pg, about 134 pg, about 135 pg, about 136 pg, about 137 pg, about 138 pg, about 139 pg, about 140 pg, about 141 pg, about 142 pg, about 143 pg, about 144 pg, about 145 pg, about 146 pg, about 147 pg, about 148 pg, about 149 pg, about 150 pg, about 151 pg, about 152 pg, about 153 pg, about 154 pg, about 155 pg, about 156 pg, about 157 pg, about 158 pg, about 159 pg, about 160 pg, about 161 pg, about 162 pg, about 163 pg, about 164 pg, about 165 pg, about 166 pg, about 167 pg, about 168 pg, about 169 pg, about 170 pg, about 171 pg, about 172 pg, about 173 pg, about 174 pg, about 175 pg, about 176 pg, about 177 pg, about 178 pg, about 179 pg, about 180 pg, about 181 pg, about 182 pg, about 183 pg, about 184 pg, about 185 pg, about 186 pg, about 187 pg, about 188 pg, about 189 pg, about 190 pg, about 191 pg, about 192 pg, about 193 pg, about 194 pg, about 195 pg, about 196 pg, about 197 pg, about 198 pg, about 199 pg, about 200 pg, about 201 pg, about 202 pg, about 203 pg, about 204 pg, about 205 pg, about 206 pg, about 207 pg, about 208 pg, about 209 pg, about 210 pg, about 211 pg, about 212 pg, about 213 pg, about 214 pg, about 215 pg, about 216 pg, about 217 pg, about 218 pg, about 219 pg, about 220 pg, about 221 pg, about 222 pg, about 223 pg, about 224 pg, about 225 pg, about 226 pg, about 227 pg, about 228 pg, about 229 pg, about 230 pg, about 231 pg, about 232 pg, about 233 pg, about 234 pg, about 235 pg, about 236 pg, about 237 pg, about 238 pg, about 239 pg, about 240 pg, about 241 pg, about 242 pg, about 243 pg, about 244 pg, about 245 pg, about 246 pg, about 247 pg, about 248 pg, about 249 pg, about 250 pg, about 251 pg, about 252 pg, about 253 pg, about 254 pg, about 255 pg, about 256 pg, about 257 pg, about 258 pg, about 259 pg, about 260 pg, about 261 pg, about 262 pg, about 263 pg, about 264 pg, about 265 pg, about 266 pg, about 267 pg, about 268 pg, about 269 pg, about 270 pg, about 271 pg, about 272 pg, about 273 pg, about 274 pg, about 275 pg, about 276 pg, about 277 pg, about 278 pg, about 279 pg, about 280 pg, about 281 pg, about 282 pg, about 283 pg, about 284 pg, about 285 pg, about 286 pg, about 287 pg, about 288 pg, about 289 pg, about 290 pg, about 291 pg, about 292 pg, about

293 pg, about 294 pg, about 295 pg, about 296 pg, about 297 pg, about 298 pg, about

299 pg, about 300 pg, about 301 pg, about 302 pg, about 303 pg, about 304 pg, about

305 pg, about 306 pg, about 307 pg, about 308 pg, about 309 pg, about 310 pg, about

311 pg, about 312 pg, about 313 pg, about 314 pg, about 315 pg, about 316 pg, about

317 pg, about 318 pg, about 319 pg, about 320 pg, about 321 pg, about 322 pg, about

323 pg, about 324 pg, about 325 pg, about 326 pg, about 327 pg, about 328 pg, about

329 pg, about 330 pg, about 331 pg, about 332 pg, about 333 pg, about 334 pg, about

335 pg, about 336 pg, about 337 pg, about 338 pg, about 339 pg, about 340 pg, about

341 pg, about 342 pg, about 343 pg, about 344 pg, about 345 pg, about 346 pg, about

347 pg, about 348 pg, about 349 pg, about 350 pg, about 351 pg, about 352 pg, about

353 pg, about 354 pg, about 355 pg, about 356 pg, about 357 pg, about 358 pg, about

359 pg, about 360 pg, about 361 pg, about 362 pg, about 363 pg, about 364 pg, about

365 pg, about 366 pg, about 367 pg, about 368 pg, about 369 pg, about 370 pg, about

371 pg, about 372 pg, about 373 pg, about 374 pg, about 375 pg, about 376 pg, about

377 pg, about 378 pg, about 379 pg, about 380 pg, about 381 pg, about 382 pg, about

383 pg, about 384 pg, about 385 pg, about 386 pg, about 387 pg, about 388 pg, about

389 pg, about 390 pg, about 391 pg, about 392 pg, about 393 pg, about 394 pg, about

395 pg, about 396 pg, about 397 pg, about 398 pg, about 399 pg, or about 400 pg of adjuvant. In embodiments, the methods comprise administering about 50 pg of adjuvant. In embodiments, the methods comprise administering about 75 pg of adjuvant.

[00250] In another embodiment, the disclosure provides a method of formulating a vaccine composition that induces immunity to an infection or at least one disease symptom thereof to a mammal, comprising adding to the composition an effective dose of a RSV F glycoprotein or RSV F glycoprotein nanoparticles. The disclosed RSV F glycoprotein and RSV F glycoprotein nanoparticles are useful for preparing compositions that stimulate an immune response that confers immunity or substantial immunity to infectious agents. Thus, in one embodiment, the disclosure provides a method of inducing immunity to infections or at least one disease symptom thereof in a subject, comprising administering at least one effective dose of a RSV F glycoprotein or RSV F glycoprotein nanoparticles.

[00251] In embodiments, the present disclosure provides a method of producing one or more of high affinity anti-RSV antibodies. The high affinity antibodies produced by immunization with the nanoparticles disclosed herein are produced by administering an immunogenic composition comprising an RSV F glycoprotein or RSV F glycoprotein nanoparticle to an animal, collecting the serum and/or plasma from the animal, and purifying the antibody from the serum/ and or plasma. In one embodiment, the animal is a human. In embodiments, the animal is a chicken, mouse, guinea pig, rat, rabbit, goat, human, horse, sheep, or cow. In one embodiment, the animal is bovine or equine. In another embodiment, the bovine or equine animal is transgenic. In yet a further embodiment, the transgenic bovine or equine animal produces human antibodies. In embodiments, the animal produces monoclonal antibodies. In embodiments, the animal produces polyclonal antibodies. In one embodiment, the method further comprises administration of an adjuvant or immune stimulating compound. In a further embodiment, the purified high affinity antibody is administered to a human subject. In one embodiment, the human subject is at risk for infection with RSV.

[00252] In embodiments, the composition is administered intranasally. In embodiments, intranasal administration of the composition includes administration of a liquid drop of the composition directly onto the nasal epithelium. In embodiments, the methods comprise administering from 1 to 100, from 1 to 90, from 1 to 80, from 1 to 70, from 1 to 60, from 1 to 50, from 1 to 40, from 1 to 30, from 1 to 20, from 1 to 10, orfrom 1 to 5 drops of the immunogenic composition are intranasally. In embodiments, the liquid drop is delivered by an intranasal delivery device described herein.

[00253] In embodiments, the methods comprise administering an aerosol of an immunogenic composition described herein to the nasal epithelium. In embodiments, the aerosol is a liquid aerosol. In embodiments, the aerosol is a powder aerosol. A powder aerosol comprises a dry powder composition. In embodiments, the methods comprise administering from 1 to 100, from 1 to 90, from 1 to 80, from 1 to 70, from 1 to 60, from 1 to 50, from 1 to 40, from 1 to 30, from 1 to 20, from 1 to 10, or from 1 to 5 sprays of the 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 by reference herein in their entireties for all purposes. An intranasal delivery device may also be a prefilled syringe, as described herein. [00254] In embodiments, the methods comprise administering an intranasal immunogenic composition that is in solution. In embodiments, the methods comprise administering an intranasal immunogenic composition that is in suspension.

[00255] In embodiments, the methods comprise intranasally administering a mist or puff of an intranasal immunogenic composition to a patient’s nostril. In embodiments, the methods comprise intranasally administering from 1 to about 100 mists or puffs of an intranasal immunogenic composition to a patient’s nostril. In embodiments, the methods comprise intranasally administering about 1 , about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11 , about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21 , about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31 , about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41 , about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 51 , about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, about 60, about 61 , about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71 , about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 79, about 80, about 81 , about 82, about 83, about 84, about 85, about 86, about 87, about 88, about 89, about 90, about 91 , about 92, about 93, about 94, about 95, about 96, about 97, about 98, about 99, or about 100 puffs or mists, including all values and ranges therebetween, of an intranasal immunogenic composition to a patient’s nostril. In embodiments, a mist or puff is delivered to each of the patient’s nostrils.

[00256] 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.

[00257] In embodiments, the methods comprise intranasally administering from 0.05 mL to about 1 mL of an intranasal immunogenic composition described herein. In embodiments, the methods comprise intranasally administering from 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 described herein, including all values and ranges therebetween. In embodiments the methods comprise intranasally administering 0.2 mL of an intranasal immunogenic composition described herein. In embodiments the methods comprise intranasally administering 0.5 mL of an intranasal immunogenic composition described herein. In embodiments the methods comprise intranasally administering 0.25 mL of an intranasal immunogenic composition described herein to each nostril of a patient.

[00258] In embodiments, provided herein are methods of 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 of inducing mucosal immunity against SARS-CoV-2, influenza, RSV, or a combination thereof in a patient comprising intranasally administering the immunogenic composition described herein. In embodiments, the antibodies are located in the bronchoalveolar lavage of the subject.

[00259] In some embodiments, the disclosure provides co-formulation (i.e. , prefilled syringes or pre-mix) strategies for immunogenic compositions comprising a RSV F glycoprotein and an adjuvant (e.g., a saponin adjuvant). Typical vaccine administration strategies currently being utilized are bedside mix formulations. That is, vaccine compositions and adjuvants are stored separately and are mixed prior to administration. Pre-mix, co-formulation, or pre-filled syringe strategies for vaccine are less common due to the concerns of the stability of the antigens (e.g., a a RSV F glycoprotein) and their subsequent immunogenic capabilities. The present disclosure provides immunogenic compositions that can be pre-mixed and stored in advance. The disclosed vaccination strategies and formulations may improve the efficiency of vaccination and may reduce the risks of bedside mixing errors, while maintaining the overall safety and immunogenicity.

[00260] A variety of containers may be used to store and transport the pre-mix formulations, including syringes for single administrations and plastic ampules. In some instances, plastic ampules can be manufactured using the blow-fill-seal manufacturing technique or method. In general, the blow-fill-seal (BFS) manufacturing method includes extruding a plastic material (e.g., resin) to form a parison, which is then placed into a mold and cut to size. A filling needle or mandrel is then used to inflate the plastic, which in turn, results in a hollow ampule that substantially conforms to the shape of the mold. Once inflated, a desired volume of liquid can be injected into the ampule, the filling needle or mandrel can be removed, and the ampule can be sealed. Accordingly, BFS can be an automated process that can be performed in a sterile environment without direct human intervention.

[00261] In some instances, the ability to aseptically manufacture sterile ampules containing a desired liquid can make BFS manufactured ampules particularly well suited for the pharmaceutical industry. BFS technology, however, has not been compatible with all pharmaceutical liquids, products, etc. For example, some known BFS manufacturing methods include delivering the liquid or product into the ampule while the plastic is still relatively hot, which can result in adverse effects to temperature sensitive liquids and/or products such as vaccines, biologies, etc. Advances in cool BFS technology, however, have increased the variety of suitable products, liquids, etc. allowing some vaccines, biologies, and/or other temperature sensitive pharmaceuticals to be contained in BFS ampules.

[00262] In some instances, a BFS ampule can have a size, shape, and/or configuration that is at least partially based on a desired use and/or a desired pharmaceutical liquid or dosage that the ampule is configured to contain. For example, some known BFS ampules can include a pierce through top, a twist-off top, a top including a male orfemale luer, and/or the like. Some known BFS ampules can have a size and/or shape based on volume of the liquid or dosage configured to be disposed therein. In addition, some known BFS ampules can be manufactured in a strip of multiple, temporarily connected ampules, which can increase manufacturing, packaging, and/or storing efficiencies and/or the like.

[00263] In embodiments, the immunogenic compositions described herein are provided in pre-filled syringes. When the immunogenic composition is prepared in a pre-filled syringe, an antigen and adjuvant is combined in advance of administration. In embodiments, the pre-filled syringe contains hemagglutinin, a SARS-CoV-2 S glycoprotein, an RSV F glycoprotein, or a combination thereof. In embodiments, a subject is administered an intranasal immunogenic composition from a pre-filled syringe.

[00264] In embodiments, the pre-filled syringe contains a RSV F glycoprotein, a HA glycoprotein, a 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 the first iscom particle comprises fraction A of Quillaja Saponaria Molina and not fraction C of Quillaja Saponaria Molina; and the second iscom particle comprises fraction C of Quillaja Saponaria Molina and not fraction A of Quillaja Saponaria Molina; wherein fraction A of Quillaja Saponaria Molina and fraction C of Quillaja Saponaria Molina account for about 85 % by weight and about 15 % by weight, respectively, of the sum of weights of fraction A of Quillaja Saponaria Molina and fraction C of Quillaja Saponaria Molina in the adjuvant. In embodiments, the pre-filled syringe contains a RSV F glycoprotein and a saponin adjuvant, wherein the adjuvant comprises at least two iscom particles, wherein the first iscom particle comprises fraction A of Quillaja Saponaria Molina and not fraction C of Quillaja Saponaria Molina; and the second iscom particle comprises fraction C of Quillaja Saponaria Molina and not fraction A of Quillaja Saponaria Molina; wherein fraction A of Quillaja Saponaria Molina and fraction C of Quillaja Saponaria Molina account for about 92 % by weight and about 8 % by weight, respectively, of the sum of weights of fraction A of Quillaja Saponaria Molina and fraction C of Quillaja Saponaria Molina in the adjuvant. In embodiments, the pre-filled syringe contains a RSV F glycoprotein and a saponin adjuvant, wherein the adjuvant comprises at least two iscom particles, wherein the first iscom particle comprises fraction A of Quillaja Saponaria Molina and not fraction C of Quillaja Saponaria Molina; and the second iscom particle comprises fraction C of Quillaja Saponaria Molina and not fraction A of Quillaja Saponaria Molina; wherein fraction A of Quillaja Saponaria Molina accounts for at least about 75 % by weight and fraction C of Quillaja Saponaria Molina accounts for the remainder of the sum of weights of fraction A of Quillaja Saponaria Molina and fraction C of Quillaja Saponaria Molina in the adjuvant.

EXAMPLES

Example 1

Expression and Purification of RSV F Polypeptide Nanoparticles

[00265] The native RSV F glycoprotein (SEQ ID NO: 329 and SEQ ID NO: 330) and RSV F glycoproteins which have amino acid sequences corresponding to SEQ ID NOS: 329-336 were expressed in a baculovirus expression system and recombinant plaques expressing the RSV F glycoproteins were picked and confirmed. In each case the signal peptide is SEQ ID NO: 360.

Table A: Selected RSV F glycoproteins

Protein and Nanoparticle Production

[0266]The recombinant virus is amplified by infection of Sf9 insect cells. A culture of insect cells is infected at ~0.6 MOI (Multiplicity of infection = virus ffu or pfu/cell) with baculovirus. The culture and supernatant is harvested 48-72 hrs post-infection. The crude cell harvest, approximately 30 mL, is clarified by centrifugation for 15 minutes at approximately 800 x g. The resulting crude cell harvests containing the RSV F glycoprotein are purified as nanoparticles as described below.

[0267]To produce nanoparticles, non-ionic surfactant TERGITOL® nonylphenol ethoxylate NP-9 is used in the membrane protein extraction protocol. Crude extraction is further purified by passing through anion exchange chromatography, lentil lectin affinity/HIC and cation exchange chromatography. The washed cells are lysed by detergent treatment and then subjected to low pH treatment which leads to precipitation of BV and Sf9 host cell DNA and protein. The neutralized low pH treatment lysate is clarified and further purified on anion exchange and affinity chromatography before a second low pH treatment is performed.

[0268] Affinity chromatography is used to remove Sf9/BV proteins, DNA and NP-9, as well as to concentrate the RSV F glycoprotein. Briefly, lentil lectin is a metalloprotein containing calcium and manganese, which reversibly binds polysaccharides and glycosylated proteins containing glucose or mannose. The RSV F glycoprotein - containing anion exchange flow through fraction is loaded onto the lentil lectin affinity chromatography resin (Capto Lentil Lectin, GE Healthcare). The glycosylated RSV F protein is selectively bound to the resin while non-glycosylated proteins and DNA are removed in the column flow through. Weakly bound glycoproteins are removed by buffers containing high salt and low molar concentration of methyl alpha-D- mannopyranoside (MMP). [0269]The column washes are also used to detergent exchange the NP-9 detergent with the surfactant polysorbate 80 (PS80). The RSV F glycoproteins are eluted in nanoparticle structure from the lentil lectin column with a high concentration of MMP. After elution, the RSV F glycoproteins are assembled into nanoparticles composed of RSV F glycoprotein trimers and PS80 contained in a detergent core.

Example 2

Immunogenicity of RSV F Glycoproteins Nanoparticle Vaccines in Cotton Rats [0270] Methods: RSV F glycoprotein nanoparticles were produced as described in Example 1. Cotton rats were immunized intramuscularly (IM) with a composition of Table B. Each composition contained 0.01-0.0001 pg RSV F glycoprotein and 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 pg saponin adjuvant. Table B. Compositions

[0271] Animals were immunized on days 0 and 21 . Blood was collected on Days 0, 21 , and 42 to evaluate immunogenicity. Animals were intranasally challenged with 105 pfu RSV A/Long on Day 42, followed by tissue collection four days later.

[0272] Immune responses to RSV F compositions were measured by anti-RSV F IgG ELISA and RSVA/RSVB Neutralization Assays. Protective efficacy was evaluated by measuring lung and nose tissue viral titers using an RSV plaque assay in HEp2 cells. [0273] Neutralization assay, and prefusion F specificity of antibodies was determined by Octet QK384 competition binning assays using antigenic site-specific antibodies to pre- and post-fusion epitopes. F protein bound to amine-reactive biosensor tips was presented in consecutive Association and Competition steps with polyclonal antibodies in sera and competing mAbs to RSV F sites II, IV, , V and p27. Competitive antibody equivalents (CAE) were calculated based on percentage competition, concentration of competing antibody, and dilution of serum. [0274] Protective efficacy was evaluated by measuring lung and nose tissue viral titers using an RSV plaque assay in HEp2 cells.

[0275] Results. Octet kinetic analysis of antigenic site-specific monoclonal antibodies confirmed all pre- and post-fusion antigenic sites and neutralizing epitopes on the RSV F glycoproteins were intact in the BV2279 glycoprotein. Immunization with BV1184 or BV2279 elicited comparable anti-F IgG responses and RSV A/B neutralizing responses. Immunization with BV2279 generated greater antibody responses toward Site (|), IV, and VIII compared to immunization with BV1184. The BV 2279 nanoparticle vaccine provided sterilizing immunity in the lungs at the 0.01 pg dose and reduced viral load in upper airways post RSV live virus challenge in cotton rats. In contrast, the BV1184 vaccine did not reduce vaccine load in the upper airways. Together, these data show that the BV2279 vaccine is a potent immunogen eliciting a robust RSV F- specific immune response to pre- and post-fusion F epitopes.

[0276] The table below shows that the BV2279 protein retains both pre-fusion and post-fusion epitopes.

[0277] Fig. 1 shows the binding of the BV2279 glycoprotein to antibodies recognizing the site II (palivizumab), site IV (RSV.42, referred to as “RSV.42.2”), site <|) (D25), and site VIII (hRSV90) epitopes. Fig. 2 shows anti-RSV F IgG titers in cotton rat sera 42 days after immunization with either the BV2279 (labeled “prefusion RSV F Cav1”) or BV1184 (labeled “prefusogenic RSV F”) vaccine compositions of Example 2. Fig. 3 shows the RSV neutralization titers against the RSV A (Long) and RSV B (18537) strains after immunization with either the BV2279 (labeled “prefusion RSV F Cav1”) or BV1184 (labeled “prefusogenic RSV F”) vaccine compositions of Example 2. Fig. 4 shows competing antibody equivalent titers induced by immunization with either the BV2279 (labeled “prefusion RSV F Cav1”) or BV1184 (labeled “prefusogenic RSV F”) vaccine compositions of Example 2. Fig. 5 shows the RSV viral titers in cotton rat tissue four days post viral challenge. The rats were immunized with either the BV2279 (labeled “prefusion RSV F Cav1”) or BV1184 (labeled “prefusogenic RSV F”) vaccine compositions of Example 2.

INCORPORATION BY REFERENCE

[0278] All references, articles, publications, patents, patent publications, and patent applications cited herein are incorporated by reference in their entireties for all purposes. However, mention of any reference, article, publication, patent, patent publication, and patent application cited herein is not, and should not be taken as, an acknowledgment 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 by reference herein in their entireties 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) 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.

2. The RSV F glycoprotein of claim 1 , comprising (a) deletion of one or more of amino acids 137-146; and (b) an inactivated primary 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 an 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 a N116Q mutation.

10. The RSV F glycoprotein of any one of claims 1-9, wherein the RSV F glycoprotein comprises an inactivated primary furin cleavage site having the amino acid sequence of KKQKQQ (SEQ ID NO: 342), an S190F mutation, a V207 mutation, and 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 primary furin cleavage site having the amino acid sequence of KKQKQQ (SEQ ID NO: 342), an S190F mutation, a V207 mutation, and deletion of amino acids 137-146, wherein the RSV F glycoprotein has at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 %, or 100 % identity to an 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 with at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 %, or 100 % identity to the F1 domain of SEQ ID NO: 352; and (ii) an F2 domain having an amino acid sequence with at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 %, or 100 % identity to the F2 domain of SEQ ID NO: 357.

13. The RSV F glycoprotein of any one of claims 1-12, wherein the F1 domain is at least 80 %, at least 81 %, at least 82 %, at least 83 %, at least 84 % at least 85 %, at least 86 %, at least 87 %, at least 88 %, at least 89 %, at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 %, or 100 % identical to a F1 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 a 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 a 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 a RSV F glycoprotein of 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 a 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 domain form a single subunit called p27-F1 , and wherein p27-F1 and the F2 subunit are connected 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 MELLIHRLSAIFLTL (SEQ ID NO: 367).

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

23. 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 non-ionic 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 non-ionic detergent is PS80.

27. A cell expressing the RSV F glycoprotein of any 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 a 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 a

(i) a first RSV F glycoprotein with 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 an RSV F glycoprotein of any one of SEQ ID NOS: 337-340; and

(ii) second RSV F glycoprotein with 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 an RSV F glycoprotein of any one of SEQ ID NOS: 364-366.

32. The immunogenic composition of any one of claims 29-30, comprising a

(i) a first RSV F glycoprotein with 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 an RSV F glycoprotein of SEQ ID NO: 337; and

(ii) second RSV F glycoprotein with 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 an RSV F glycoprotein of any one of SEQ ID NOs: 364.

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

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

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

37. The immunogenic composition of any one of claims 29-36, comprising from 1 to about 20, from 1 to about 19, from 1 to about 18, from 1 to about 17, from 1 to about 16, from 1 to about 15, from 1 to about 14, from 1 to about 13, from 1 to about 12, from 1 to about 11 , from 1 to about 10, from 1 to about 9, from 1 to about 8, from 1 to about 7, from 1 to about 6, from 1 to about 5, from 1 to about 4, from 1 to about 3, from 1 to about 2, from about 2 to about 20, from about 2 to about 19, from about 2 to about 18, from about 2 to about 17, from about 2 to about 16, from about 2 to about 15, from about 2 to about 14, from about 2 to about 13, from about 2 to about 12, from about 2 to about 11 , from about 2 to about 10, from about 2 to about 9, from about 2 to about 8, from about 2 to about 7, from about 2 to about 6, from about 2 to about 5, from about 2 to about 4, from about 2 to about 3, from about 3 to about 20, from about 3 to about 19, from about 3 to about 18, from about 3 to about 17, from about 3 to about 16, from about 3 to about 15, from about 3 to about 14, from about 3 to about 13, from about 3 to about 12, from about 3 to about 11 , from about 3 to about 10, from about 3 to about 9, from about 3 to about 8, from about 3 to about 7, from about 3 to about 6, from about 3 to about 5, from about 3 to about 4, from about 4 to about 20, from about 4 to about 19, from about 4 to about 18, from about 4 to about 17, from about 4 to about 16, from about 4 to about 15, from about 4 to about 14, from about 4 to about 13, from about 4 to about 12, from about 4 to about 11 , from about 4 to about 10, from about 4 to about 9, from about 4 to about 8, from about 4 to about 7, from about 4 to about 6, from about 4 to about 5, from about 5 to about 20, from about 5 to about 19, from about 5 to about 18, from about 5 to about 17, from about 5 to about 16, from about 5 to about 15, from about 5 to about 14, from about 5 to about 13, from about 5 to about 12, from about 5 to about 11 , from about 5 to about 10, from about 5 to about 9, from about 5 to about 8, from about 5 to about 7, from about 5 to about 6, from about 6 to about 20, from about 6 to about 19, from about 6 to about 18, from about 6 to about 17, from about 6 to about 16, from about 6 to about 15, from about 6 to about 14, from about 6 to about 13, from about 6 to about 12, from about 6 to about 11 , from about 6 to about 10, from about 6 to about 9, from about 6 to about 8, from about 6 to about 7, from about 7 to about 20, from about 7 to about 19, from about 7 to about 18, from about 7 to about 17, from about 7 to about 16, from about 7 to about 15, from about 7 to about 14, from about 7 to about 13, from about 7 to about 12, from about 7 to about 11 , from about 7 to about 10, from about 7 to about 9, from about 7 to about 8, from about 8 to about 20, from about 8 to about 19, from about 8 to about 18, from about 8 to about 17, from about 8 to about 16, from about 8 to about 15, from about 8 to about 14, from about 8 to about 13, from about 8 to about 12, from about 8 to about 11 , from about 8 to about 10, from about 8 to about 9, from about 9 to about 20, from about 9 to about 19, from about 9 to about 18, from about 9 to about 17, from about 9 to about 16, from about 9 to about 15, from about 9 to about 14, from about 9 to about 13, from about 9 to about 12, from about 9 to about 11 , from about 9 to about 10, from about 10 to about 20, from about 10 to about 19, from about 10 to about 18, from about 10 to about 17, from about 10 to about 16, from about 10 to about 15, from about 10 to about 14, from about 10 to about 13, from about 10 to about 12, from 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, from about 13 to about 14, from about 14 to about 20, from about 14 to about 19, from about 14 to about 18, from about 14 to about 17, from about 14 to about 16, from about 14 to about 15, from about 15 to about 20, from about 15 to about 19, from about 15 to about 18, from about 15 to about 17, from about 15 to about 16, from about 16 to about 20, from about 16 to about 19, from about 16 to about 18, from about 16 to about 17, from about 17 to about 20, from about 17 to about 19, from about 17 to about 18, from about 18 to about 20, from about 18 to about 19, or from 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 an RSV F glycoprotein is an influenza hemagglutinin (HA) glycoprotein or a SARS-CoV-2 Spike (CoV S) glycoprotein.

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

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

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

42. The immunogenic composition of any one of claims 39-41 , comprising a first, second, and 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 glycoprotein.

44. The immunogenic composition of any one of claims 41-43, comprising: (i) a detergent-core nanoparticle comprising the first HA glycoprotein and a nonionic detergent; and

(ii) a 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 non-ionic 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 non-ionic 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 non-ionic 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 HaSMaN comprising the fourth HA glycoprotein.

49. The immunogenic composition of any one of claims 44-48, wherein the HA glycoproteins of the detergent-core nanoparticles is from a Type A influenza strain.

50. The immunogenic composition of any one of claims 44-48, wherein the HA glycoproteins of the detergent-core nanoparticles is from a Type B influenza strain.

51 . The immunogenic composition of any one of claims 44 and 46-48, wherein the HA glycoproteins of the HaSMaNs are from a Type A influenza strain.

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

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

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

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

56. The immunogenic composition of claim 55, wherein the SARS-CoV-2 S glycoprotein contains (i) an inactive furin cleavage site, and (ii) proline 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 the 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 has at least 80 %, at least 81 %, at least 82 %, at least 83 %, at least 84

% at least 85 %, at least 86 %, at least 87 %, at least 88 %, at least 89 %, at least 90

%, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96

%, at least 97 %, at least 98 %, at least 99 %, or 100 % 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 has at least 80 %, at least 81 %, at least 82 %, at least 83 %, at least 84

% at least 85 %, at least 86 %, at least 87 %, at least 88 %, at least 89 %, at least 90

%, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96

%, at least 97 %, at least 98 %, at least 99 %, or 100 % 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 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 to a polypeptide of any one of SEQ ID NOS: 87, 89, 106, 112, 113, 114, 1 15, 132, 144, 151 , 153, 156,

158, 174, 175, 176, 181 , 182, 183, 184, 186, 188, 190, 192, 195, 217, 218, 219, 220,

221 , 222, 223, 224, 225, 226, 227, 228, 233, 234, 235, 236, 260, 261 , 262, 263, 264,

274, 276, 278, 280, 284, 288, 292, 296, 300, 304, 308, 312, 316, 320, 324, and 328.

62. The immunogenic composition of any one of claims 29-61 , comprising:

(i) a first CoV S glycoprotein comprising (a) an inactive furin cleavage site, and (b) proline 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) proline 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) from 1 to about 2, from 1 to about 3, from 1 to about 4, from 1 to about 5, from 2 to about 5, from 1 to about 6, from 1 to about 7, from 1 to about 8, from 1 to about 9, from 1 to about 10, from 1 to about 11 , from 1 to about 12, from 1 to about 13, from 1 to about 14, from 1 to about 15, from 1 to about 16, from 1 to about 17, from 1 to about 18, from 1 to about 18, from 1 to about 19, or from 1 to about 20, from 1 to about 25, from 1 to about 30, from 1 to about 35, from 1 to about 40, from 1 to about 45, or from 1 to about 50 modifications compared to the first CoV S glycoprotein.

63. The immunogenic composition of any one of claims 29-61 , comprising:

(i) a first CoV S glycoprotein comprising (a) an inactive furin cleavage site, and (b) proline 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) proline 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) from 1 to about 2, from 1 to about 3, from 1 to about 4, from 1 to about 5, from 2 to about 5, from 1 to about 6, from 1 to about 7, from 1 to about 8, from 1 to about 9, from 1 to about 10, from 1 to about 11 , from 1 to about 12, from 1 to about 13, from 1 to about 14, from 1 to about 15, from 1 to about 16, from 1 to about 17, from 1 to about 18, from 1 to about 18, from 1 to about 19, or from 1 to about 20, from 1 to about 25, from 1 to about 30, from 1 to about 35, from 1 to about 40, from 1 to about 45, or from 1 to about 50 modifications compared to the first CoV S glycoprotein; and

(iii) a third CoV S glycoprotein comprising (a) an inactive furin cleavage site, and (b) proline 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) from 1 to about 2, from 1 to about 3, from 1 to about 4, from 1 to about 5, from 2 to about 5, from 1 to about 6, from 1 to about 7, from 1 to about 8, from 1 to about 9, from 1 to about 10, from 1 to about 11 , from 1 to about 12, from 1 to about 13, from 1 to about 14, from 1 to about 15, from 1 to about 16, from 1 to about 17, from 1 to about 18, from 1 to about 18, from 1 to about 19, or from 1 to about 20, from 1 to about 25, from 1 to about 30, from 1 to about 35, from 1 to about 40, from 1 to about 45, or from 1 to about 50 modifications compared to the first CoV S glycoprotein.

64. The immunogenic composition of any one of claims 29-61 , comprising: (i) a first CoV S glycoprotein comprising (a) an inactive furin cleavage site, and (b) proline 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) proline 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) from 1 to about 2, from 1 to about 3, from 1 to about 4, from 1 to about 5, from 2 to about 5, from 1 to about 6, from 1 to about 7, from 1 to about 8, from 1 to about 9, from 1 to about 10, from 1 to about 11 , from 1 to about 12, from 1 to about 13, from 1 to about 14, from 1 to about 15, from 1 to about 16, from 1 to about 17, from 1 to about 18, from 1 to about 18, from 1 to about 19, or from 1 to about 20, from 1 to about 25, from 1 to about 30, from 1 to about 35, from 1 to about 40, from 1 to about 45, or from 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) proline 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) from 1 to about 2, from 1 to about 3, from 1 to about 4, from 1 to about 5, from 2 to about 5, from 1 to about 6, from 1 to about 7, from 1 to about 8, from 1 to about 9, from 1 to about 10, from 1 to about 11 , from 1 to about 12, from 1 to about 13, from 1 to about 14, from 1 to about 15, from 1 to about 16, from 1 to about 17, from 1 to about 18, from 1 to about 18, from 1 to about 19, or from 1 to about 20, from 1 to about 25, from 1 to about 30, from 1 to about 35, from 1 to about 40, from 1 to about 45, or from 1 to about 50 modifications compared to the first CoV S glycoprotein; and

(iv) a fourth CoV S glycoprotein comprising (a) an inactive furin cleavage site, and (b) proline 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) from 1 to about 2, from 1 to about 3, from 1 to about 4, from 1 to about 5, from 2 to about 5, from 1 to about 6, from 1 to about 7, from 1 to about 8, from 1 to about 9, from 1 to about 10, from 1 to about 11 , from 1 to about 12, from 1 to about 13, from 1 to about 14, from 1 to about 15, from 1 to about 16, from 1 to about 17, from 1 to about 18, from 1 to about 18, from 1 to about 19, or from 1 to about 20, from 1 to about 25, from 1 to about 30, from 1 to about 35, from 1 to about 40, from 1 to about 45, or from 1 to about 50 modifications compared to the first CoV S glycoprotein.

65. The immunogenic composition of any one of claims 29-61 , comprising:

(i) a first CoV S glycoprotein comprising (a) an inactive furin cleavage site, and (b) proline 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) proline 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) from 1 to about 2, from 1 to about 3, from 1 to about 4, from 1 to about 5, from 2 to about 5, from 1 to about 6, from 1 to about 7, from 1 to about 8, from 1 to about 9, from 1 to about 10, from 1 to about 11 , from 1 to about 12, from 1 to about 13, from 1 to about 14, from 1 to about 15, from 1 to about 16, from 1 to about 17, from 1 to about 18, from 1 to about 18, from 1 to about 19, or from 1 to about 20, from 1 to about 25, from 1 to about 30, from 1 to about 35, from 1 to about 40, from 1 to about 45, or from 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) proline 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) from 1 to about 2, from 1 to about 3, from 1 to about 4, from 1 to about 5, from 2 to about 5, from 1 to about 6, from 1 to about 7, from 1 to about 8, from 1 to about 9, from 1 to about 10, from 1 to about 11 , from 1 to about 12, from 1 to about 13, from 1 to about 14, from 1 to about 15, from 1 to about 16, from 1 to about 17, from 1 to about 18, from 1 to about 18, from 1 to about 19, or from 1 to about 20, from 1 to about 25, from 1 to about 30, from 1 to about 35, from 1 to about 40, from 1 to about 45, or from 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) proline 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) from 1 to about 2, from 1 to about 3, from 1 to about 4, from 1 to about 5, from 2 to about 5, from 1 to about 6, from 1 to about 7, from 1 to about 8, from 1 to about 9, from 1 to about 10, from 1 to about 11 , from 1 to about 12, from 1 to about 13, from 1 to about 14, from 1 to about 15, from 1 to about 16, from 1 to about 17, from 1 to about 18, from 1 to about 18, from 1 to about 19, or from 1 to about 20, from 1 to about 25, from 1 to about 30, from 1 to about 35, from 1 to about 40, from 1 to about 45, or from 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) proline 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) from 1 to about 2, from 1 to about 3, from 1 to about 4, from 1 to about 5, from 2 to about 5, from 1 to about 6, from 1 to about 7, from 1 to about 8, from 1 to about 9, from 1 to about 10, from 1 to about 11 , from 1 to about 12, from 1 to about 13, from 1 to about 14, from 1 to about 15, from 1 to about 16, from 1 to about 17, from 1 to about 18, from 1 to about 18, from 1 to about 19, or from 1 to about 20, from 1 to about 25, from 1 to about 30, from 1 to about 35, from 1 to about 40, from 1 to about 45, or from 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 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 to a polypeptide of any one of SEQ ID NOS: 87, 89, 106, 112, 113, 114, 115, 132, 144,

151 , 153, 156, 158, 174, 175, 176, 181 , 182, 183, 184, 186, 188, 190, 192, 195, 217,

218, 219, 220, 221 , 222, 223, 224, 225, 226, 227, 228, 233, 234, 235, 236, 260, 261 ,

262, 263, 264, 274, 276, 278, 280, 284, 288, 292, 296, 300, 304, 308, 312, 316, 320,

324, and 328.

67. The immunogenic composition of claim 65 or 66, comprising about 5 pg of the first CoV S glycoprotein and from 1 ng to about 4.9 pg 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 glycoprotein 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 glycoprotein occur in the N-terminal domain (NTD).

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 glycoprotein 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 glycoprotein occur in the S2 subunit.

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

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

74. The immunogenic composition of any one of claims 55-71 , comprising about 25 pg 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 fraction A of Quillaja Saponaria Molina and not fraction C of Quillaja Saponaria Molina; and the second iscom particle comprises fraction C of Quillaja Saponaria Molina and not fraction A of Quillaja Saponaria Molina.

77. The immunogenic composition of claim 76, wherein: fraction A of Quillaja Saponaria Molina and fraction C of Quillaja Saponaria Molina account for about 85 % by weight and about 15 % by weight, respectively, of the sum of weights of fraction A of Quillaja Saponaria Molina and fraction C of Quillaja Saponaria Molina in the adjuvant.

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

79. The immunogenic composition of claim 76, wherein fraction A of Quillaja Saponaria Molina accounts for at least about 85 % by weight, and fraction C of Quillaja Saponaria Molina accounts for the remainder, respectively, of the sum of the weights of fraction A of Quillaja Saponaria Molina and fraction C of Quillaja Saponaria Molina in the adjuvant.

80. The immunogenic composition of claim 76, wherein fraction A of Quillaja Saponaria Molina accounts for 50-96% by weight and fraction C of Quillaja Saponaria Molina accounts for the remainder, respectively, of the sum of the weights of fraction A of Quillaja Saponaria Molina and fraction C of Quillaja Saponaria Molina in the adjuvant.

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

82. The immunogenic composition of any one of claims 75-81 , comprising about 20 pg or 50 pg 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 pre-filled syringe comprising the immunogenic composition of any one of claims 29-85.

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

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 pre-filled syringe.

94. The method of any one of claims 87-93, wherein the method prevents one or more of RSV, influenza, and SARS-CoV-2 with an efficacy from about 50 % to about 99 %, from about 50 % to about 95 %, from about 50 % to about 90 %, from about 50 % to about 85 %, from about 50 % to about 80 %, from about 60 % to about 99 %, from about 65 % to about 95 %, from about 65 % to about 90 %, from about 65 % to about 85 %, from about 69 % to about 81 %, from about 60 % to about 95 %, from about 60 % to about 90 %, from about 60 % to about 85 %, from about 60 % to about 80 %, from about 40 % to about 99 %, from about 40 % to about 95 %, from about 40 % to about 90 %, from about 40 % to about 85 %, from about 40 % to about 80 %, from about 40 % to about 75 %, from about 40 % to about 70 %, from about 40 % to about 65 %, from about 40 % to about 55 %, or from about 40 % to about 50 % for 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 after administration of the immunogenic composition.

95. The method of any one of claims 87-93, wherein the method prevents one or more of RSV, influenza, and SARS-CoV-2 with an efficacy from about 50 % to about 99 %, from about 50 % to about 95 %, from about 50 % to about 90 %, from about 50 % to about 85 %, from about 50 % to about 80 %, from about 60 % to about 99 %, from about 65 % to about 95 %, from about 65 % to about 90 %, from about 65 % to about 85 %, from about 69 % to about 81 %, from about 60 % to about 95 %, from about 60 % to about 90 %, from about 60 % to about 85 %, from about 60 % to about 80 %, from about 40 % to about 99 %, from about 40 % to about 95 %, from about 40 % to about 90 %, from about 40 % to about 85 %, from about 40 % to about 80 %, from about 40 % to about 75 %, from about 40 % to about 70 %, from about 40 % to about 65 %, from about 40 % to about 55 %, or from about 40 % to about 50 % 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 after administration of the immunogenic composition.

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 that has a gestational age from about 32 weeks to about 36 weeks.

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

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

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