RSV vaccine compositions, methods and uses thereof

Abstract

Provided are immunogenic compositions comprising respiratory syncytial virus (RSV) viral antigens and immunogens, such as recombinant peptides and proteins comprising RSV F protein peptides. The immunogenic compositions comprise secreted fusion proteins comprising soluble RSV viral antigens linked in-frame to a disulfide-linked trimeric fusion protein. The immunogenic compositions are useful, for example, for generating immune responses to treat or prevent RSV infection. The immunogenic compositions can be used in vaccine compositions, for example, as part of prophylactic and / or therapeutic vaccines. Also provided herein are methods for producing recombinant peptides and proteins, prophylactic, therapeutic, and / or diagnostic methods, and related kits.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of International Application No. PCT / CN2020 / 095295, filed June 10, 2020, and International Application No. PCT / CN2021 / 087045, filed April 13, 2021, the disclosures of which are incorporated herein by reference in their entireties.

[0002] Submission of sequence listing as an ASCII text file The contents of the following ASCII text file submission are incorporated herein by reference in their entirety: Sequence Listing in Computer Readable Form (CRF) (Filename: 165762000242SEQLIST.TXT, Recorded Date: June 9, 2021, Size: 229KB).

[0003] In some aspects, the present disclosure relates to immunogenic compositions for treating and / or preventing respiratory syncytial virus (RSV) infection, including RSV viral antigens and immunogens, such as recombinant peptides and proteins including RSV F protein peptides. [Background technology]

[0004] Respiratory syncytial virus (RSV) causes respiratory tract infections in adults and children and is a leading cause of lower respiratory tract infections and hospitalization during infancy and childhood. Despite the high infection rate of RSV, treatments, including prophylactics, therapeutics, and vaccines, are limited or unavailable. Improved approaches for the treatment of RSV are needed. Provided herein are compositions, methods, uses, and products that meet these and other needs. Summary of the Invention [Problem to be solved by the invention]

[0005] Despite the high infection rate of RSV, treatments, including prophylactics, therapeutics, and vaccines, are limited or unavailable, and improved approaches for the treatment of RSV are needed. The present invention provides compositions, methods, uses, and products that meet these and other needs. [Means for solving the problem]

[0006] In one aspect, provided herein is a protein comprising a plurality of recombinant polypeptides, each comprising a respiratory syncytial virus (RSV) F protein peptide, or a fragment or epitope thereof, linked to a C-terminal propeptide of collagen, wherein the C-terminal propeptides of the recombinant polypeptides form an inter-polypeptide disulfide bond. In some embodiments, the RSV is subtype A or subtype B. In some embodiments, the epitope is a linear epitope or a conformational epitope.

[0007] In some embodiments, disclosed herein are recombinant subunit vaccines comprising an ectodomain of the RSV F protein or a fragment thereof (e.g., without the transmembrane and cytoplasmic domains) fused in-frame to a collagen C-propeptide capable of forming a disulfide-linked homotrimer. The resulting subunit vaccine, such as a recombinant F trimer, can be expressed and purified from transgenic cells and is predicted to have a native-like conformation in a trimer form. This solves the problem of misfolding of viral antigens, which often occurs when they are expressed as soluble recombinant peptides or proteins without the transmembrane and / or cytoplasmic domains. Such misfolded viral antigens often do not retain the native conformation of the viral antigen and are unable to elicit neutralizing antibodies.

[0008] In some embodiments, the F protein peptide comprises an F1 subunit peptide, an F2 subunit peptide, or any combination thereof, and the protein comprises three recombinant polypeptides. In some embodiments, the F protein peptide comprises a signal peptide, a heptad repeat C (HRC) peptide, a pep27 peptide, a fusion peptide (FP), a heptad repeat A (HRA) peptide, a domain I peptide, a domain II peptide, or a heptad repeat B (HRB) peptide, or any combination thereof. In some embodiments, the F protein peptide comprises the F1 subunit of an F protein but not the F2 subunit, or vice versa. In some embodiments, the F protein peptide comprises the F1 and F2 subunits of an F protein, optionally lacking pep27, and optionally the F1 and F2 subunits are linked by a disulfide bond or an artificially introduced linker. In some embodiments, the F protein peptide does not comprise a transmembrane (TM) domain peptide and / or a cytoplasmic (CP) domain peptide. In some embodiments, the F protein peptide comprises a protease cleavage site, and the protease is optionally furin, trypsin, Factor Xa, thrombin, or cathepsin L. In some embodiments, the F protein peptide does not comprise a protease cleavage site, and the protease is optionally furin, trypsin, Factor Xa, thrombin, or cathepsin L.

[0009] In some embodiments, the F protein peptide is soluble or does not directly bind to a lipid bilayer, e.g., a membrane or viral envelope. In some embodiments, the F protein peptide is the same or different between recombinant polypeptides of the protein. In some embodiments, the F protein peptide is fused directly to the C-terminal propeptide or is linked to the C-terminal propeptide via a linker, such as a linker comprising a glycine-XY repeat, where X and Y are independently any amino acid, optionally proline or hydroxyproline.

[0010] In some embodiments, the protein is soluble or does not directly bind to a lipid bilayer, e.g., a membrane or viral envelope. In some embodiments, the protein can form rosette-like oligomers comprising F protein peptide trimers. In some embodiments, the protein can bind to a cell surface adhesion factor or receptor of a subject, and optionally the subject is a mammal, such as a primate, e.g., a human.

[0011] In some embodiments, the C-terminal propeptide is human collagen. In some embodiments, the C-terminal propeptide comprises the C-terminal polypeptide of proα1(I), proα1(II), proα1(III), proα1(V), proα1(XI), proα2(I), proα2(V), proα2(XI), or proα3(XI), or a fragment thereof. In some embodiments, the C-terminal propeptides are the same or different between the recombinant polypeptides. In some embodiments, the C-terminal propeptide comprises any of SEQ ID NOs: 48-63, or an amino acid sequence at least 90% identical thereto, capable of forming an inter-polypeptide disulfide bond to trimerize the recombinant polypeptides.

[0012] In some embodiments, the F protein peptide in each recombinant polypeptide is in a pre-fusion conformation or a post-fusion conformation, and optionally, the protein comprises a rosette-like oligomer comprising F protein peptide trimers as a crutch-shaped rod. In any of the foregoing embodiments, the F protein peptide in each recombinant polypeptide can comprise any of SEQ ID NOs: 17-47, or an amino acid sequence at least 80% identical thereto.

[0013] In any of the foregoing embodiments, the recombinant polypeptide may comprise any of SEQ ID NOs: 1-16, or an amino acid sequence at least 80% identical thereto. In any of the foregoing embodiments, the recombinant polypeptide may comprise any of SEQ ID NOs: 17-47, or an amino acid sequence at least 80% identical thereto, linked directly or indirectly to SEQ ID NOs: 48-63, or an amino acid sequence at least 90% identical thereto.

[0014] Also provided herein are immunogens comprising the proteins provided herein. Provided herein are protein nanoparticles comprising the proteins provided herein directly or indirectly bound to nanoparticles. Provided herein are virus-like particles (VLPs) comprising the proteins provided herein.

[0015] Also provided herein are isolated nucleic acids encoding recombinant polypeptides of one, two, three, or more of the proteins provided herein. In some embodiments, a polypeptide encoding an F protein peptide is fused in-frame to a polypeptide encoding a C-terminal propeptide of collagen. In some embodiments, the isolated nucleic acids provided herein are operably linked to a promoter.

[0016] In some embodiments, the isolated nucleic acids provided herein are DNA molecules. In some embodiments, the isolated nucleic acids provided herein are RNA molecules, optionally mRNA molecules such as nucleoside-modified mRNA, non-amplified mRNA, self-amplified mRNA, or trans-amplified mRNA.

[0017] Also provided herein are vectors comprising the isolated nucleic acids provided herein. In some embodiments, the vector is a viral vector.

[0018] In some aspects, provided herein are viruses, pseudoviruses, or cells comprising the vectors provided herein, optionally wherein the viruses or cells have a recombinant genome. In some aspects, provided herein are immunogenic compositions comprising a protein, immunogen, protein nanoparticle, VLP, isolated nucleic acid, vector, virus, pseudovirus, or cell provided herein and a pharmaceutically acceptable carrier.

[0019] Also provided herein are vaccines comprising the immunogenic compositions provided herein and, optionally, an adjuvant, wherein the vaccine is optionally a subunit vaccine. In some embodiments, the vaccine is a prophylactic and / or therapeutic vaccine.

[0020] In some aspects, provided herein are methods of producing a protein, the methods comprising expressing an isolated nucleic acid or vector provided herein in a host cell to produce a protein provided herein; and purifying the protein. Provided herein are proteins produced by the methods provided herein.

[0021] Provided herein are methods for generating an immune response in a subject against a RSV F protein peptide or a fragment or epitope thereof, the method comprising administering to a subject an effective amount of a protein, immunogen, protein nanoparticle, VLP, isolated nucleic acid, vector, virus, pseudovirus, cell, immunogenic composition, or vaccine provided herein to generate an immune response. In some embodiments, the methods provided herein are for treating or preventing infection with RSV. In some embodiments, the generation of an immune response inhibits or reduces RSV replication in the subject. In some embodiments, the immune response comprises a cell-mediated response and / or a humoral response, and optionally includes the production of one or more neutralizing antibodies, such as polyclonal or monoclonal antibodies. In some embodiments, the immune response is directed against a RSV F protein peptide or a fragment or epitope thereof, but not against the C-terminal propeptide. In some embodiments, administration to a subject does not result in antibody-dependent enhancement of infection (ADE) in the subject due to one or more prior exposures to RSV. In some embodiments, the administration does not result in antibody-dependent enhancement (ADE) in the subject upon one or more subsequent exposures to RSV. In some embodiments, the method further comprises a priming step and / or a boosting step. In some embodiments, the administering step is carried out via topical, transdermal, subcutaneous, intradermal, oral, intranasal (e.g., intranasal spray), intratracheal, sublingual, buccal, rectal, vaginal, inhalation, intravenous (e.g., intravenous injection), intraarterial, intramuscular (e.g., intramuscular injection), intracardiac, intraosseous, intraperitoneal, transmucosal delivery, intravitreal, subretinal, intra-articular, periarticular, topical, or epicutaneous administration. In some embodiments, the effective amount is administered in a single dose or in a series of doses separated by one or more intervals. In some embodiments, the effective amount is administered without an adjuvant. In some embodiments, the effective amount is administered with an adjuvant.

[0022] The present invention provides a method for producing neutralizing antibodies or neutralizing antisera against RSV in a subject by administering an effective amount of a protein provided herein to the subject. In some embodiments, the subject is a mammal, optionally a human or non-human primate. In some embodiments, the method further comprises isolating a neutralizing antibody or neutralizing antisera from the subject. In some embodiments, the method further comprises administering an effective amount of the isolated neutralizing antibody or neutralizing antisera to a human subject via passive immunization to prevent or treat RSV infection. In some embodiments, the neutralizing antibody or neutralizing antisera against RSV comprises a polyclonal antibody against a RSV F protein peptide or a fragment or epitope thereof, and optionally, the neutralizing antibody or neutralizing antisera is free or substantially free of antibodies against the C-terminal propeptide of collagen. In some embodiments, the neutralizing antibody comprises a monoclonal antibody against a RSV F protein peptide or a fragment or epitope thereof, and optionally, the neutralizing antibody is free or substantially free of antibodies against the C-terminal propeptide of collagen.

[0023] In some embodiments, the proteins, immunogens, protein nanoparticles, VLPs, isolated nucleic acids, vectors, viruses, pseudoviruses, cells, immunogenic compositions, or vaccines provided herein are for use in eliciting an immune response to RSV in a subject and / or in treating or preventing infection by RSV.

[0024] In some embodiments, provided herein is the use of a protein, immunogen, protein nanoparticle, VLP, isolated nucleic acid, vector, virus, pseudovirus, cell, immunogenic composition, or vaccine for inducing an immune response against RSV in a subject and / or for treating or preventing infection by RSV. In some embodiments, provided herein is the use of a protein, immunogen, protein nanoparticle, VLP, isolated nucleic acid, vector, virus, pseudovirus, cell, immunogenic composition, or vaccine for the manufacture of a medicament or prophylactic for inducing an immune response against RSV in a subject and / or for treating or preventing infection by RSV.

[0025] Also provided herein are methods for analyzing a sample, the methods comprising contacting the sample with a protein provided herein and detecting binding between the protein and an analyte capable of specifically binding to an RSV F protein peptide, or a fragment or epitope thereof. In some embodiments, the analyte is an antibody, receptor, or cell that recognizes the F protein peptide, or a fragment or epitope thereof. In some embodiments, binding indicates the presence of the analyte in the sample and / or RSV infection in the subject from which the sample was derived.

[0026] Provided herein are kits that include a protein provided herein and a substrate, pad, or vial that contains or immobilizes the protein, and optionally, the kit is an ELISA or lateral flow assay kit. [Brief explanation of the drawings]

[0027] [Figure 1A] 1 shows the expression levels and purification of exemplary fusion peptides containing RSV F protein peptides. This figure is a schematic diagram of an exemplary fusion peptide containing an extracellular F domain fused to a trimerization peptide. [Figure 1B]Figure 1 shows the expression levels and purification of an exemplary fusion peptide containing an RSV F protein peptide. This figure shows an 8% SDS-PAGE analysis of exemplary fusion peptide expression from serum-free fed-batch cell cultures. 10 μL of cell-free conditioned medium from days 1 to 12 was separated under non-reducing conditions and subsequently stained with Coomassie blue. [Figure 1C] This figure shows the expression level and purification of an exemplary fusion peptide containing an RSV F protein peptide. This figure shows the purity assessment of an exemplary fusion peptide by SEC-HPLC, and the peak area of ​​the exemplary protein was 94.6% as detected by OD280. [Figure 2A] Figure 1 shows the characterization of an exemplary purified fusion peptide containing an RSV F protein peptide. This figure shows SDS-PAGE and Western blot analysis of an exemplary purified fusion peptide containing an RSV F protein peptide under non-reducing and reducing conditions. Using antibodies specific to collagen F and propeptides, 2 μg of purified protein was loaded for Coomassie blue staining by 8% SDS-PAGE, and 0.1 μg of purified protein was loaded for Western blot. [Figure 2B] Figure 1 shows the characterization of an exemplary purified fusion peptide comprising an RSV F protein peptide. This figure shows a negative staining electron micrograph of an exemplary fusion peptide protein comprising an RSV F protein peptide, showing individual or mostly rosette-like oligomeric crutch-shaped molecules. Examples of individual and rosette-like molecules are shown below, along with diagrams of their structures. [Figure 2C]1 shows the characterization of an exemplary purified fusion peptide containing an RSV F protein peptide. This figure shows the results of a biolayer interferometry binding test of an exemplary fusion peptide containing palivizumab and an RSV F protein peptide. 5 μg / mL of palivizumab was first immobilized on a Protein A sensor, and then the sensor was immersed in various concentrations of exemplary fusion peptides to measure binding kinetics. The buffer reference solution was subtracted, and the resulting curve was fitted to a 1:1 binding model to obtain Kon and Kdis, and the values ​​are shown in the table below. The KD obtained for an exemplary fusion peptide containing palivizumab and an RSV F protein peptide is less than 1 picomole. [Figure 3A] Immunization with an exemplary fusion peptide containing an RSV F protein peptide protects against RSV infection. Figure 3A shows a schematic diagram of the experimental procedure: mice were immunized on days 0 and 21, followed by intranasal (in) RSV challenge on day 49 after serum collection. [Figure 3B] Figure 3B shows that immunization with exemplary fusion peptides containing RSV F protein peptides protects against RSV infection. Figure 3B is a graph showing serum anti-F IgG ELISA titers against purified exemplary fusion peptides containing RSV F protein peptides. [Figure 3C] Figure 3C shows that immunization with an exemplary fusion peptide containing an RSV F protein peptide protects against RSV infection. Figure 3C is a graph of a virus microneutralization assay showing serum neutralizing antibody titers that result in a 50% inhibition of CPE formation in response to RSV infection. [Figure 3D] Figure 3D shows that immunization with an exemplary fusion peptide containing an RSV F protein peptide protects against RSV infection. Figure 3D is a graph showing RSV titers in the lungs of immunized mice 5 days after challenge by plaque assay. Values ​​represent plaques per gram of lung tissue. [Figure 3E]Figure 3E shows that immunization with an exemplary fusion peptide containing an RSV F protein peptide protects against RSV infection. Figure 3E is a graph showing the competitive IgG titer of palivizumab measured by the dilution of serum samples that results in 50% inhibition of palivizumab binding to heat-inactivated RSV (HI-RSV) particles. Values ​​are expressed as log2 and mean ± SEM. [Figure 4] Photographs showing that immunization with an exemplary fusion peptide containing an RSV F protein peptide protects against vaccine-induced disease enhancement. Five days after challenge, lung tissues were collected, fixed in 10% neutral buffered formalin, embedded in paraffin, sectioned at 5 μm, and stained with H&E, and photographs were taken at 200x magnification. DETAILED DESCRIPTION OF THE INVENTION

[0028] In some embodiments, compositions and methods of use of recombinant soluble surface antigens from RNA viruses in covalently trimeric form are disclosed. In some embodiments, the resulting fusion proteins are secreted as disulfide-linked homotrimers, which are structurally more stable while preserving the conformation of native-like trimeric viral antigens, thereby enabling their use as more effective vaccines against these dangerous pathogens.

[0029] In some embodiments, disclosed herein are methods of using viral antigen trimers, optionally via intramuscular injection or intranasal administration, without an adjuvant, with an adjuvant, or with two or more adjuvants, as part of a vaccine or multivalent vaccine to prevent viral infection.

[0030] In some embodiments, disclosed herein are methods of using viral antigen trimers as antigens for the diagnosis of viral infections by detection of antibodies, e.g., IgM or IgG, such as neutralizing antibodies that recognize viral antigens.

[0031] In some embodiments, disclosed herein are methods of using viral antigen trimers as antigens to generate polyclonal or monoclonal antibodies that can be used for passive immunization, e.g., neutralizing mAbs for treating RSV infection in infants.

[0032] In some embodiments, disclosed herein are viral antigen trimers as part of a vaccine or multivalent vaccine, which includes multiple trimeric subunit vaccines that contain viral antigens of the same protein from a virus, or that contain two or more different proteins from one or more viruses or one or more strains of the same virus.

[0033] In some embodiments, disclosed herein are monovalent vaccines comprising the viral antigen trimers disclosed herein. In some embodiments, disclosed herein are bivalent vaccines comprising the viral antigen trimers disclosed herein. In some embodiments, disclosed herein are trivalent vaccines comprising the viral antigen trimers disclosed herein. In some embodiments, disclosed herein are tetravalent vaccines comprising the viral antigen trimers disclosed herein.

[0034] In some embodiments, disclosed herein are monovalent vaccines comprising the F trimers disclosed herein. In some embodiments, disclosed herein are bivalent vaccines comprising the F trimers disclosed herein. In some embodiments, disclosed herein are bivalent vaccines comprising at least one F trimer comprising a first F protein antigen and at least one F trimer comprising a second F protein antigen. In some embodiments, the first and second F protein antigens are derived from the same F protein of one or more viral species or strains / subtypes, or from two or more different F proteins of one or more viral species or one or more strains / subtypes of the same viral species. In some embodiments, disclosed herein are trivalent vaccines comprising the F trimers disclosed herein. In some embodiments, disclosed herein are trivalent vaccines comprising at least one F trimer comprising a first F protein antigen, at least one F trimer comprising a second F protein antigen, and at least one F trimer comprising a third F protein antigen. In some embodiments, the first, second, and third F protein antigens are derived from the same F protein of one or more viral species or strains / subtypes, or are derived from two, three, or more different F proteins of one or more viral species or one or more strains / subtypes of the same viral species. In some embodiments, disclosed herein are tetravalent vaccines comprising the F trimers disclosed herein. In some embodiments, disclosed herein are tetravalent vaccines comprising at least one F trimer comprising a first F protein antigen, at least one F trimer comprising a second F protein antigen, at least one F trimer comprising a third F protein antigen, and at least one F trimer comprising a fourth F protein antigen. In some embodiments, the first, second, third, and fourth F protein antigens are derived from the same F protein of one or more viral species or strains / subtypes, or are derived from two, three, four, or more different F proteins of one or more viral species or one or more strains / subtypes of the same viral species.

[0035] Provided herein are immunogenic compositions, methods, and uses of fusion peptides and proteins comprising RSV viral antigens or immunogens for the treatment of RSV infection, e.g., prophylactic and therapeutic agents. Respiratory syncytial virus (RSV) is considered a leading cause of acute lower respiratory tract infections (ALRTIs) among infants and young children, accounting for over 7,000 to 20,000 child deaths annually. RSV infection is the second most common cause of infant mortality in the developing world. In addition, RSV can lead to severe disease in elderly and immunocompromised populations. Despite the disease burden caused by RSV, there is currently no approved vaccine. Palivizumab (Synagis®), a highly potent prophylactic humanized mAb, is the only available passive immunization measure for these infants at high risk of RSV infection.

[0036] Despite decades of research, RSV vaccine development has not been successful for a number of reasons: manufacturing, stability, and efficacy issues of RSV vaccine candidates have been difficult to overcome, and safety has been a major concern, particularly since formalin-inactivated RSV (FI-RSV) vaccines have been found to mediate vaccine-induced disease enhancement (VED).

[0037] The proteins, including recombinant polypeptides and fusion proteins, comprising RSV viral antigens and immunogens provided herein are useful for effectively and safely treating (e.g., therapeutically, prophylactically) RSV infection. For example, the proteins, including RSV viral antigens and immunogens provided herein, treat RSV infection without mediation of VED and / or antibody-dependent enhancement (ADE). In addition, the proteins, including RSV viral antigens and immunogens provided herein, are easily manufactured and exhibit stability under highly stressful conditions, such as high temperature, extreme pH, and high and low osmolality. Thus, the proteins and immunogenic compositions provided herein avoid and satisfy the manufacturing, stability, safety, and efficacy issues that have hindered RSV vaccine development.

[0038] In some embodiments, the RSV viral antigens and immunogens provided herein comprise the RSV glycoprotein (F), also referred to herein as RSV F protein peptides or peptides. The RSV F protein peptide is a homotrimeric type I transmembrane protein that mediates membrane and viral entry into host cells. The RSV F protein peptide is synthesized as an F0 proprotein precursor and is converted to disulfide-linked F1 and F2 mature forms after cleavage at two sites by furin. The RSV F protein peptide is highly conserved between RSV A and B strains. Neutralizing antibodies, such as palivizumab, target the antigenic site of F and provide protection against respiratory disease caused by RSV infection.

[0039] In some embodiments, proteins comprising a RSV viral antigen or immunogen, such as a RSV F protein peptide, can generate an immune response, e.g., an immune response against a RSV F peptide protein. In some embodiments, the immune response inhibits or reduces RSV replication in a subject, e.g., a patient. In some embodiments, the immune response includes the production of one or more neutralizing antibodies, such as polyclonal and / or monoclonal antibodies. In some embodiments, the neutralizing antibodies inhibit or reduce RSV replication in a subject, e.g., a patient. In some embodiments, administration of the protein to a subject, e.g., as an immunogenic composition, does not result in antibody-dependent enhancement (ADE) in the subject due to prior exposure to RSV. In some aspects, proteins comprising a RSV viral antigen and immunogen, e.g., a RSV F protein peptide, are used as vaccines.

[0040] In some embodiments, the RSV viral antigen and immunogen, such as a RSV F protein peptide, are linked to a protein or peptide to form a fusion protein or recombinant polypeptide. In some embodiments, the protein or peptide to which the RSV viral antigen or immunogen is linked can be linked, for example, covalently or non-covalently, to a protein or peptide, such as a protein or peptide of a fusion protein or recombinant polypeptide. Thus, in some cases, the protein or peptide to which the RSV viral antigen or immunogen is linked is a multimerization domain.

[0041] In some embodiments, a RSV viral antigen and immunogen, e.g., a RSV F protein peptide, is linked to a collagen propeptide, e.g., at the C-terminus of the collagen propeptide, to form a fusion peptide or recombinant polypeptide. Thus, in some embodiments, the proteins provided herein comprise a RSV viral antigen and immunogen, e.g., a recombinant polypeptide comprising a RSV F protein peptide or a fragment or epitope thereof, linked to a collagen C-terminal propeptide. In some embodiments, the collagen propeptide is derived from the human C-propeptide of α1 collagen and is capable of self-trimerization.

[0042] In some embodiments, the binding of RSV viral antigens and immunogens, such as RSV F protein peptides, to collagen propeptides, for example, at the C-terminus of the collagen propeptide, supports the protein's ability to generate an immune response. For example, recombinant protein production can preserve the tertiary and quaternary structure of the RSV F protein peptide, which can be important for the stability of the native conformation of the RSV F protein peptide and the availability of antigenic sites on the surface of proteins capable of eliciting an immune response, such as neutralizing antibodies. Furthermore, binding of the RSV F protein peptide to a protein or peptide capable of self-trimerization allows the recombinant protein to aggregate, thus mimicking the native homotrimeric structure of the RSV F protein peptide on the viral envelope.

[0043] In some embodiments, binding of the RSV F protein peptide to the C-terminal propeptide of collagen results in a self-trimerizing recombinant polypeptide. In some embodiments, the proteins provided herein comprise a plurality of self-trimerizing RSV F protein peptides and a propeptide of a collagen recombinant polypeptide, optionally wherein the plurality of recombinant proteins form a structure, e.g., a rosette (see, e.g., FIG. 2B). In some embodiments, the trimeric nature of the recombinant protein contributes to protein stability. In some embodiments, a macrostructure, e.g., a rosette of a plurality of self-trimerizing recombinant proteins, contributes to protein stability. In some embodiments, the trimeric nature of the recombinant protein and the macrostructure, e.g., a rosette, of a plurality of self-trimerizing recombinant proteins contribute to protein stability. In some embodiments, the trimeric nature of the recombinant protein supports the protein's ability to generate an immune response. In some embodiments, a macrostructure, e.g., a rosette of a plurality of self-trimerizing recombinant proteins, contributes to protein stability. In some embodiments, the trimeric nature of the recombinant protein and the macrostructure of multiple self-trimerizing recombinant proteins aids the protein's ability to generate an immune response.

[0044] Also provided herein are immunogenic compositions comprising the proteins provided herein, methods for producing the proteins provided herein, methods for treating subjects with the proteins and compositions provided herein, and kits.

[0045] All publications, including patent documents, scientific articles, and databases, referred to in this application are incorporated by reference in their entirety in this application to the same extent as if each individual publication was individually incorporated by reference. To the extent that a definition set forth herein contradicts or is inconsistent with a definition set forth in a patent, application, application publication, or other publication incorporated by reference herein, the definition set forth herein takes precedence over the definition incorporated by reference.

[0046] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0047] I. Viral Antigens and Immunogens Respiratory syncytial virus (RSV) is the most common cause of acute lower respiratory tract infection in infants and young children and a major disease burden in the elderly. Despite the fact that the RSV virus was characterized half a century ago, no vaccine for RSV currently exists, and development has been hampered by vaccine-mediated disease enhancement with the administration of formalin-inactivated RSV to children in the 1960s. Challenges with antigen production, purity, stability, and efficacy of RSV vaccine candidates have also posed obstacles to development.

[0048] In some embodiments, the proteins provided herein comprise RSV viral antigens and / or immunogens. In some embodiments, the RSV viral antigens and / or immunogens provided herein can promote or stimulate a cell-mediated response and / or a humoral response. In some embodiments, the response, e.g., a cell-mediated or humoral response, comprises the production of antibodies, e.g., neutralizing antibodies. In some embodiments, neutralizing antibodies (NAbs) against viral antigens and / or immunogens provide adaptive immune protection against RSV exposure by blocking infection of susceptible cells. In some embodiments, the efficacy of vaccines against some viruses is attributed to and / or correlates with their ability to induce NAbs. In some embodiments, the RSV viral antigen or immunogen is a RSV F protein peptide disclosed herein.

[0049] The RSV F protein is the envelope glycoprotein of respiratory syncytial virus (RSV). It is translated as a single precursor polypeptide (termed F0). The RSV F protein mediates viral entry into cells and cell-cell fusion, is the target of neutralizing antibodies, and is highly conserved between RSV A and B strains. F0 can be cleaved into three fragments by cellular furin at Arg109 and Arg136, releasing a shorter F2 polypeptide covalently linked at the N-terminus by two disulfides to a longer F1 polypeptide with an 18-amino acid fusion domain at the N-terminus, and a hydrophobic transmembrane region near the C-terminus; an intervening 27-amino acid fragment. The neutralizing monoclonal antibodies palivizumab and motavizumab bind to RSV F antigenic site II (Asn258-Val278) and have been shown to protect against both lower and upper respiratory tract RSV disease in high-risk and term infants. The structure of the RSV F epitope polypeptide that binds to these neutralizing antibodies is larger than that of the linear peptide, with palivizumab binding to RSV F with nanomolar affinity and motavizumab binding to RSV F with picomolar affinity. Modeling predicts that full binding of palivizumab and motavizumab requires one or two amino acids from the RSV F promoter, respectively. Therefore, preservation of the RSV F tertiary and quaternary structure may be important in RSV F vaccine development to preserve the native conformation of this important neutralizing region.

[0050] In some embodiments, the F0 precursor polypeptide is 574 amino acids in length, as set forth in SEQ ID NO:31.

[0051] [Table 1]

[0052] In some embodiments, the F0 precursor polypeptide is 574 amino acids in length, as set forth in SEQ ID NO:32.

[0053] [Table 2]

[0054] In some embodiments, the RSV F protein peptide referred to herein comprises a proline or alanine at residue 102. In some embodiments, the RSV F protein peptide referred to herein comprises a substitution, deletion, and / or insertion at and / or near residue 102 of SEQ ID NO: 31 or 32. In some embodiments, the RSV F protein peptide referred to herein comprises a glutamic acid or alanine at residue 218. In some embodiments, the RSV F protein peptide referred to herein comprises a substitution, deletion, and / or insertion at and / or near residue 218 of SEQ ID NO: 31 or 32. In some embodiments, the RSV F protein peptide referred to herein comprises a valine or isoleucine at residue 379. In some embodiments, the RSV F protein peptide referred to herein comprises a substitution, deletion, and / or insertion at and / or near residue 379 of SEQ ID NO: 31 or 32. In some embodiments, the RSV F protein peptide referred to herein comprises a valine or methionine at residue 447. In some embodiments, the RSV F protein peptide referred to herein comprises a substitution, deletion, and / or insertion at and / or near residue 447 of SEQ ID NO: 31 or 32. In some embodiments, the RSV F protein peptide referred to herein comprises a substitution, deletion, and / or insertion at and / or near any one or more of proline or alanine at residue 102, glutamic acid or alanine at residue 218, valine or isoleucine at residue 379, and valine or methionine at residue 447. In some embodiments, the RSV F protein peptide referred to herein comprises a substitution, deletion, and / or insertion at and / or near any one of residues 102, 218, 379, and 447 of SEQ ID NO: 31 or 32. In some embodiments, the RSV F protein peptide referred to herein comprises a substitution, deletion, and / or insertion at and / or near any one or more other residues of SEQ ID NO: 31 or 32.

[0055] In some embodiments, the RSV F protein peptides referred to herein include substitutions, deletions, and / or insertions at and / or near residues 106, 107, 108, and / or 109 of SEQ ID NO: 31 or 32. In some embodiments, the RSV F protein peptides referred to herein include glutamine or asparagine at residues 108 and / or 109. In some embodiments, the RSV F protein peptides referred to herein include glutamine at residues 108 and 109. In some embodiments, the RSV F protein peptides referred to herein include asparagine at residues 108 and 109. In some embodiments, the RSV F protein peptides referred to herein include substitutions, deletions, and / or insertions at and / or near residues 131, 132, 133, 134, 135, and / or 136 of SEQ ID NO: 31 or 32. In some embodiments, the RSV F protein peptide herein comprises a glycine, arginine, glutamine, or asparagine at and / or near residues 131, 132, 133, 134, 135, and / or 136 of SEQ ID NO: 31 or 32. In some embodiments, the RSV F protein peptide herein comprises a glutamine at residues 131, 132, 133, 134, 135, and / or 136 of SEQ ID NO: 31 or 32. In some embodiments, the RSV F protein peptide herein comprises a glutamine at residues 133, 135, and 136 of SEQ ID NO: 31 or 32.

[0056] In some embodiments, the RSV F protein peptides referred to herein include substitutions, deletions, and / or insertions at and / or near residues 109, 136, 161, and / or 215 of SEQ ID NO: 31 or 32. In some embodiments, the RSV F protein peptides referred to herein include an alanine or proline at any one or more of residues 109, 136, 161, and / or 215 of SEQ ID NO: 31 or 32. In some embodiments, the RSV F protein peptides referred to herein include an alanine at residue 109 of SEQ ID NO: 31 or 32. In some embodiments, the RSV F protein peptides referred to herein include an alanine at residue 136 of SEQ ID NO: 31 or 32. In some embodiments, the RSV F protein peptides referred to herein include an alanine at residue 109 and 136 of SEQ ID NO: 31 or 32. In some embodiments, the RSV F protein peptides referred to herein include a proline at residue 161 of SEQ ID NO: 31 or 32. In some embodiments, the RSV F protein peptide herein comprises a proline at residue 215 of SEQ ID NO: 31 or 32. In some embodiments, the RSV F protein peptide herein comprises a proline at residues 161 and 215 of SEQ ID NO: 31 or 32. In some embodiments, the RSV F protein peptide herein comprises an alanine at residues 109 and 136 and a proline at residues 161 and 215 of SEQ ID NO: 31 or 32.

[0057] In some embodiments, the RSV F protein peptides referred to herein include substitutions, deletions, and / or insertions at and / or near any one or more of residues 131-154 of SEQ ID NO: 31 or 32. In some embodiments, the RSV F protein peptides referred to herein include 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or more deletions of residues 131-154 of SEQ ID NO: 31 or 32. In some embodiments, the RSV F protein peptides referred to herein include one or more deletions of residues 137-154 of SEQ ID NO: 31 or 32. In some embodiments, the RSV F protein peptides referred to herein include one or more deletions of residues 137-146 of SEQ ID NO: 31 or 32. In some embodiments, the RSV F protein peptide herein comprises a glutamine at residues 133, 135, and 136 of SEQ ID NO: 31 or 32, and a deletion at residues 137-146.

[0058] In some embodiments, the RSV F protein peptide herein comprises amino acids 1-25 of the F precursor, which is the signal peptide MELLILKANAITTILTAVTFCFASG (SEQ ID NO: 33). In some embodiments, the precursor polypeptide F forms a precursor trimer. In some embodiments, the RSV F protein peptide herein is proteolytically cleaved by one or more cellular proteases, for example, at a conserved furin consensus cleavage site, to produce a Pep27 polypeptide (also referred to as p27), an F1 polypeptide, and an F2 polypeptide. In some embodiments, the Pep27 polypeptide (e.g., amino acids 110-136 of the F precursor) is excised and, in some aspects, does not form part of the mature RSV F trimer. In some embodiments, the F2 polypeptide (alternatively referred to herein as "F2" or "F2 subunit peptide") comprises amino acid residues 26-109 of the F precursor. In some embodiments, the F1 polypeptide (alternatively, which may also be referred to herein as "F1" or "F1 subunit peptide") comprises amino acid residues 137-574 of the F0 precursor, and may include an extracellular region (e.g., residues 137-524), a transmembrane domain (e.g., residues 525-550), and a cytoplasmic domain (e.g., residues 551-574).

[0059] In some embodiments, the RSV F protein peptide herein comprises an F1 and F2 polypeptide linked by a disulfide bond to form a heterodimer referred to as an RSV F "protomer." In some embodiments, the RSV F protein peptide herein comprises three promoters that form an RSV F trimer (which is therefore a homotrimer of three promoters). In some embodiments, the RSV F protein peptide herein is a mature RSV F trimer. In some embodiments, the RSV F protein peptide herein is membrane-bound. In some embodiments, the RSV F protein peptide herein is not membrane-bound. In some embodiments, the RSV F protein peptide herein is soluble and lacks the transmembrane and cytoplasmic domains or fragments thereof. For example, conversion to a soluble form can be achieved by truncating the RSV F protein at amino acids 513 (by removing amino acids 514 and beyond), 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, or 524. In nature, the mature RSV F trimer mediates the fusion of the virus and cellular membranes. The pre-fusion conformation of the mature RSV F trimer (which may be referred to herein as "pre-F" or pre-fusion) is highly unstable (metastable). However, when the RSV virus binds to the cellular membrane, the RSV F protein trimer undergoes a series of conformational changes and transitions to a highly stable post-fusion ("post-F") conformation.

[0060] In some embodiments, the RSV viral antigen or immunogen is a signal peptide (SP) (e.g., amino acids 1-22 of SEQ ID NO: 31 or 32) or a fragment and / or variant sequence thereof, a heptad repeat C (HRC) (e.g., F2, which can be amino acids 23-109 of SEQ ID NO: 31 or 32) or a fragment and / or variant sequence thereof, a furin cleavage site (FCS) (e.g., at the junction between amino acids 109 / 110 of SEQ ID NO: 31 or 32) or a fragment and / or variant sequence thereof, a 27-mer fragment (pep27) (e.g., amino acids 110-136 of SEQ ID NO: 31 or 32) or a fragment and / or variant sequence thereof, a putative fusion peptide (FP) (e.g., amino acids 137-155 of SEQ ID NO: 31 or 32) or a fragment and / or variant sequence thereof, or a fragment and / or variant sequence thereof. or a fragment and / or variant sequence thereof, heptad repeat A (HRA) (e.g., amino acids 156-214 of SEQ ID NO: 31 or 32) or a fragment and / or variant sequence thereof, domain I and II (e.g., amino acids 215-476 of SEQ ID NO: 31 or 32) or a fragment and / or variant sequence thereof, heptad repeat B (HRB) (e.g., amino acids 477-524 of SEQ ID NO: 31 or 32) or a fragment and / or variant sequence thereof, transmembrane (TM) (e.g., amino acids 525-550 of SEQ ID NO: 31 or 32) or a fragment and / or variant sequence thereof, and / or cytoplasmic (CP) domain (e.g., amino acids 551-574 of SEQ ID NO: 31 or 32) or a fragment and / or variant sequence thereof, in any suitable combination.

[0061] In some embodiments, the RSV viral antigen or immunogen is a RSV F protein peptide of RSV subtype A. In some embodiments, the RSV viral antigen or immunogen is a RSV F protein peptide of RSV subtype A2. In some embodiments, the RSV viral antigen or immunogen is a RSV F protein peptide of RSV subtype B. In some cases, the RSV F protein peptide is conserved across RSV subtypes.

[0062] In some embodiments, the RSV viral antigen or immunogen is a fragment of a RSV F protein peptide. In some embodiments, the RSV viral antigen or immunogen is an epitope of a RSV F protein peptide. In some embodiments, the epitope is a linear epitope. In some embodiments, the epitope is a conformational epitope. In some embodiments, the epitope is a neutralizing epitope site, for example, site I, II, or IV. In some embodiments, all neutralizing epitopes of a RSV F protein peptide or fragment thereof are present as the RSV viral antigen or immunogen.

[0063] In some cases, for example, when the RSV viral antigen or immunogen is a fragment of a RSV F protein peptide, only a single subunit of the RSV F protein peptide is present.

[0064] In some embodiments, the RSV viral antigen or immunogen is or comprises an F1 subunit peptide. In some embodiments, the F1 subunit peptide is or comprises the amino acid sequence 137-574 of the wild-type F protein. In some embodiments, the RSV viral antigen or immunogen is or comprises an F2 subunit peptide. In some embodiments, the RSV viral antigen or immunogen comprises an RSV F protein peptide comprising a signal peptide, a heptad repeat C (HRC) peptide, a pep27 peptide, a fusion peptide (FP), a heptad repeat A (HRA) peptide, a domain I peptide, a domain II peptide, or a heptad repeat B (HRB) peptide, or any combination thereof. In some embodiments, the RSV viral antigen or immunogen comprises an RSV F protein peptide comprising a signal peptide. In some embodiments, the RSV viral antigen or immunogen comprises an RSV F protein peptide comprising a pep27 peptide. In some embodiments, the RSV viral antigen or immunogen comprises an RSV F protein peptide comprising a fusion peptide (FP) (also known as a fusion domain (FD)). In some embodiments, the RSV viral antigen or immunogen comprises a RSV F protein peptide, including a signal peptide, a pep27 peptide, and a fusion peptide (FP).

[0065] In some embodiments, the RSV viral antigen or immunogen comprises a RSV F protein peptide comprising the F1 subunit and F2 subunit of the F protein. In some embodiments, the RSV viral antigen or immunogen comprises a RSV F protein peptide comprising the F1 subunit and F2 subunit, lacking the pep27 peptide of the F protein. In some embodiments, the RSV viral antigen or immunogen comprises a RSV F protein peptide comprising the F1 subunit peptide, the F2 subunit peptide, and the pep27 peptide of the F protein. In some embodiments, the RSV viral antigen or immunogen comprises a RSV F protein peptide comprising the F1 subunit peptide, the F2 subunit peptide, the pep27 peptide, and the FP of the F protein.

[0066] In some embodiments, for example, when a viral antigen or immunogen comprises both an F1 subunit peptide and an F2 subunit peptide of an RSV F protein peptide, the F1 and F2 subunits are linked. In some embodiments, the F1 and F2 subunits are linked by a disulfide bond. In some embodiments, the F1 and F2 subunits are linked by an artificially introduced linker. In some embodiments, the F1 and F2 subunits are linked via a pep27 peptide. For example, in some embodiments, the N-to-C-terminal orientation is or includes F2-pep27-F1. In some embodiments, the N-to-C-terminal orientation is or includes F2-pep27-FP-F1 (F2-pep27-FD-F1). In some embodiments, FP is considered a structural feature of the F1 subunit peptide.

[0067] In some cases, the RSV viral antigen or immunogen is a RSV F protein peptide that does not include a transmembrane (TM) domain peptide. In some cases, the RSV F protein does not include a cytoplasmic (CP) domain peptide. In some cases, the RSV F protein does not include a TM domain peptide or a CP domain peptide.

[0068] In some embodiments, the RSV viral antigen or immunogen comprises an RSV F protein peptide comprising a protease cleavage site. In some embodiments, the protease cleavage site is specific for cleavage by the protease furin. In some embodiments, the protease cleavage site is specific for cleavage by the protease trypsin. In some embodiments, the protease cleavage site is specific for cleavage by the protease factor Xa. In some embodiments, the protease cleavage site is specific for cleavage by the protease cathepsin L.

[0069] In some cases, the RSV viral antigen or immunogen comprises a RSV F protein peptide that does not contain a protease cleavage site. In some cases, the RSV viral antigen or immunogen comprises a RSV F protein peptide that does not contain a protease cleavage site specific for cleavage by the protease furin. In some cases, the RSV viral antigen or immunogen comprises a RSV F protein peptide that does not contain a protease cleavage site specific for cleavage by the protease trypsin. In some cases, the RSV viral antigen or immunogen comprises a RSV F protein peptide that does not contain a protease cleavage site specific for cleavage by the protease factor Xa. In some cases, the RSV viral antigen or immunogen comprises a RSV F protein peptide that does not contain a protease cleavage site specific for cleavage by the protease cathepsin L.

[0070] In some embodiments, the RSV viral antigen or immunogen comprises a soluble RSV F protein peptide. In some embodiments, the soluble RSV F protein peptide lacks the TM domain peptide and the CP domain peptide. In some embodiments, the soluble RSV F protein peptide does not bind to a lipid bilayer, such as a membrane or viral envelope.

[0071] In some embodiments, the RSV F protein peptide is produced from a codon-optimized nucleic acid sequence. In some embodiments, the RSV F protein peptide is produced from a non-codon-optimized nucleic acid sequence.

[0072] In some embodiments, the RSV F protein peptide can include any F protein sequence known in the art, such as those disclosed in U.S. Patent No. 10,017,543, which is incorporated herein by reference in its entirety.

[0073] In some embodiments, the RSV viral antigen or immunogen is or comprises a RSV F protein peptide having the amino acid sequence 1-520 of SEQ ID NO: 31 or 32. In some embodiments, the RSV viral antigen or immunogen is or comprises a RSV F protein peptide having the amino acid sequence 26-520 of SEQ ID NO: 31 or 32.

[0074] In some embodiments, the RSV viral antigen or immunogen is or comprises an F2 sequence, a pep27 sequence, and an F1 sequence (e.g., F2-pep27-F1). In some embodiments, the RSV viral antigen or immunogen comprises an exposed fusion peptide and has a post-fusion conformation. In some embodiments, the RSV viral antigen or immunogen comprises a furin cleavage site mutation. In some embodiments, the RSV viral antigen or immunogen comprises a furin site I mutation (e.g., R109A) and / or a furin site II mutation (e.g., R136A); in some of these instances, the RSV viral antigen or immunogen has a post-fusion conformation, while in other instances, the RSV viral antigen or immunogen has a pre-fusion conformation. In some embodiments, the RSV viral antigen or immunogen comprises a furin site I mutation and a furin site II mutation (e.g., R109A / R136A), and in some of these instances, the RSV viral antigen or immunogen comprises a full-length F0 lacking an exposed fusion peptide and is in a prefusion conformation. In some embodiments, the RSV viral antigen or immunogen comprises one or more mutations that prevent long helix formation and / or stabilize the α4-α5 hinge loop. In some embodiments, the RSV viral antigen or immunogen comprises one or more mutations that preserve the prefusion conformation. In some embodiments, the RSV viral antigen or immunogen comprises one or more mutations that improve expression. In some embodiments, substitutions at positions 161, 182, and 215 (e.g., with proline) result in higher expression levels, and E161P and S215P also increase protein stability. In some embodiments, the RSV viral antigen or immunogen comprises E161P and / or S215P and is in a prefusion conformation. In some embodiments, the RSV viral antigen or immunogen comprises R109A, R136A, E161P and / or S215P and has a prefusion conformation.

[0075] In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 17. In some embodiments, the viral antigen or immunogen comprises an amino acid sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 17, including sequences containing substitutions, deletions, and / or insertions at one or more amino acid positions.

[0076] In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 18. In some embodiments, the viral antigen or immunogen comprises an amino acid sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 18, including sequences containing substitutions, deletions, and / or insertions at one or more amino acid positions.

[0077] In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 19. In some embodiments, the viral antigen or immunogen comprises an amino acid sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 19, including sequences containing substitutions, deletions, and / or insertions at one or more amino acid positions.

[0078] In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 20. In some embodiments, the viral antigen or immunogen comprises an amino acid sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 20, including sequences containing substitutions, deletions, and / or insertions at one or more amino acid positions.

[0079] In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 21. In some embodiments, the viral antigen or immunogen comprises an amino acid sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 21, including sequences containing substitutions, deletions, and / or insertions at one or more amino acid positions.

[0080] In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 22. In some embodiments, the viral antigen or immunogen comprises an amino acid sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 22, including sequences containing substitutions, deletions, and / or insertions at one or more amino acid positions.

[0081] In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 23. In some embodiments, the viral antigen or immunogen comprises an amino acid sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 23, including sequences containing substitutions, deletions, and / or insertions at one or more amino acid positions.

[0082] In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 24. In some embodiments, the viral antigen or immunogen comprises an amino acid sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 24, including sequences containing substitutions, deletions, and / or insertions at one or more amino acid positions.

[0083] In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 25. In some embodiments, the viral antigen or immunogen comprises an amino acid sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 25, including sequences containing substitutions, deletions, and / or insertions at one or more amino acid positions.

[0084] In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 26. In some embodiments, the viral antigen or immunogen comprises an amino acid sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 26, including sequences containing substitutions, deletions, and / or insertions at one or more amino acid positions.

[0085] In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 27. In some embodiments, the viral antigen or immunogen comprises an amino acid sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 27, including sequences containing substitutions, deletions, and / or insertions at one or more amino acid positions.

[0086] In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 28. In some embodiments, the viral antigen or immunogen comprises an amino acid sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 28, including sequences containing substitutions, deletions, and / or insertions at one or more amino acid positions.

[0087] In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 29. In some embodiments, the viral antigen or immunogen comprises an amino acid sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 29, including sequences containing substitutions, deletions, and / or insertions at one or more amino acid positions.

[0088] In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 30. In some embodiments, the viral antigen or immunogen comprises an amino acid sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 30, including sequences containing substitutions, deletions, and / or insertions at one or more amino acid positions.

[0089] In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 31. In some embodiments, the viral antigen or immunogen comprises an amino acid sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 31, including sequences containing substitutions, deletions, and / or insertions at one or more amino acid positions.

[0090] In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 32. In some embodiments, the viral antigen or immunogen comprises an amino acid sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 32, including sequences containing substitutions, deletions, and / or insertions at one or more amino acid positions.

[0091] In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 33. In some embodiments, the viral antigen or immunogen comprises an amino acid sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 33, including sequences containing substitutions, deletions, and / or insertions at one or more amino acid positions.

[0092] In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 34. In some embodiments, the viral antigen or immunogen comprises an amino acid sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 34, including sequences containing substitutions, deletions, and / or insertions at one or more amino acid positions.

[0093] In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 35. In some embodiments, the viral antigen or immunogen comprises an amino acid sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 35, including sequences containing substitutions, deletions, and / or insertions at one or more amino acid positions.

[0094] In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 36. In some embodiments, the viral antigen or immunogen comprises an amino acid sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 36, including sequences containing substitutions, deletions, and / or insertions at one or more amino acid positions.

[0095] In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 37. In some embodiments, the viral antigen or immunogen comprises an amino acid sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 37, including sequences containing substitutions, deletions, and / or insertions at one or more amino acid positions.

[0096] In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 38. In some embodiments, the viral antigen or immunogen comprises an amino acid sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 38, including sequences containing substitutions, deletions, and / or insertions at one or more amino acid positions.

[0097] In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 39. In some embodiments, the viral antigen or immunogen comprises an amino acid sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 39, including sequences containing substitutions, deletions, and / or insertions at one or more amino acid positions.

[0098] In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 40. In some embodiments, the viral antigen or immunogen comprises an amino acid sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 40, including sequences containing substitutions, deletions, and / or insertions at one or more amino acid positions.

[0099] In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 41. In some embodiments, the viral antigen or immunogen comprises an amino acid sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 41, including sequences containing substitutions, deletions, and / or insertions at one or more amino acid positions.

[0100] In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 42. In some embodiments, the viral antigen or immunogen comprises an amino acid sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 42, including sequences containing substitutions, deletions, and / or insertions at one or more amino acid positions.

[0101] In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 43. In some embodiments, the viral antigen or immunogen comprises an amino acid sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 43, including sequences containing substitutions, deletions, and / or insertions at one or more amino acid positions.

[0102] In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 44. In some embodiments, the viral antigen or immunogen comprises an amino acid sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 44, including sequences containing substitutions, deletions, and / or insertions at one or more amino acid positions.

[0103] In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 45. In some embodiments, the viral antigen or immunogen comprises an amino acid sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 45, including sequences containing substitutions, deletions, and / or insertions at one or more amino acid positions.

[0104] In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 46. In some embodiments, the viral antigen or immunogen comprises an amino acid sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 46, including sequences containing substitutions, deletions, and / or insertions at one or more amino acid positions.

[0105] In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 47. In some embodiments, the viral antigen or immunogen comprises an amino acid sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 47, including sequences containing substitutions, deletions, and / or insertions at one or more amino acid positions.

[0106] In some embodiments, the viral antigen or immunogen herein may comprise a RSV glycoprotein (G) or a fragment, variant, or mutant thereof; a RSV small hydrophobic protein (SH) or a fragment, variant, or mutant thereof; a RSV fusion protein (F) or a fragment, variant, or mutant thereof; a RSV matrix protein (M) or a fragment, variant, or mutant thereof; a RSV nucleoprotein (N) or a fragment, variant, or mutant thereof; a RSV phosphoprotein (P) or a fragment, variant, or mutant thereof; a RSV "large" protein (L) or a fragment, variant, or mutant thereof; an M2-1 protein or a fragment, variant, or mutant thereof; a RSV M2-2 protein or a fragment, variant, or mutant thereof; a RSV NS-1 protein or a fragment, variant, or mutant thereof; or a RSV Ns-2 protein or a fragment, variant, or mutant thereof; or any combination thereof.

[0107] In some embodiments, the viral antigen or immunogen is produced from a nucleic acid sequence that is codon optimized. In some embodiments, the viral antigen or immunogen is produced from a nucleic acid sequence that is not codon optimized.

[0108] In some embodiments, as provided herein, the RSV viral antigen or immunogen can comprise a recombinant polypeptide or fusion peptide comprising the viral antigen or immunogen. The term viral antigen or immunogen can be used to refer to a protein comprising a RSV viral antigen or immunogen. In certain instances, the RSV viral antigen or immunogen is a RSV protein peptide provided herein.

[0109] II. Recombinant Peptides and Proteins It is contemplated that the RSV viral antigens and immunogens provided herein, such as RSV F protein peptides (see Section I), can be combined with other proteins or peptides, e.g., linked, to form recombinant polypeptides, including fusion peptides. In some embodiments, individual recombinant polypeptides (e.g., monomers) provided herein are linked to form recombinant polypeptide multimers, e.g., trimers. In some embodiments, the linkage of individual recombinant polypeptide monomers occurs via covalent interactions. In some embodiments, the linkage of individual recombinant polypeptide monomers occurs via non-covalent interactions. In some embodiments, for example, covalent or non-covalent interactions are mediated by the protein or peptide to which the RSV viral antigen or immunogen, e.g., the RSV F protein peptide, is linked. In some embodiments, for example, when the RSV viral antigen or immunogen is a RSV F protein peptide disclosed herein, the protein or peptide to which it is linked can be selected to preserve the natural homotrimeric structure of the glycoprotein. This can be advantageous for eliciting a strong and effective immunogenic response to the RSV F protein peptide. For example, preserving and / or maintaining the native conformation of a RSV viral antigen or immunogen (e.g., RSV F protein peptide) can improve or enable access to antigenic sites capable of generating an immune response. In some cases, recombinant polypeptides containing the RSV F protein peptides described herein (see, e.g., Section I) are referred to herein alternatively as recombinant RSV F antigens, recombinant RSV F immunogens, or recombinant RSV F proteins.

[0110] It is further contemplated that, in some cases, the recombinant polypeptide or its multimerized recombinant polypeptide can aggregate or aggregate to form a protein containing multiple RSV viral antigens and / or immunogen recombinant polypeptides. The formation of such a protein can be advantageous for generating a strong and effective immunogenic response to the RSV viral antigen and / or immunogen. For example, the formation of a protein containing multiple recombinant polypeptides, and thus multiple RSV viral antigens, such as RSV F protein peptides, preserves the tertiary and / or quaternary structure of the viral antigen, allowing an immune response to be initiated against the native structure. In some cases, aggregation can provide structural stability to the RSV viral antigen or immunogen, which in turn provides access to potential antigenic sites that can promote an immune response.

[0111] 1. Fusion peptides and recombinant polypeptides In some embodiments, RSV viral antigens or immunogens can be linked to a trimerization domain at their C-terminus (C-terminal linkage) to promote trimerization of the monomers. In some embodiments, trimerization stabilizes the membrane-proximal side of the RSV viral antigen or immunogen, e.g., the RSV F protein peptide, in a trimeric configuration.

[0112] Non-limiting examples of exogenous multimerization domains that promote stable trimers of soluble recombinant proteins include the GCN4 leucine zipper (Harbury et al. 1993 Science 262:1401-1407), trimerization motifs from pulmonary surfactant proteins (Hoppe et al. 1994 FEBS Lett 344:191-195), collagen (McAlinden et al. 2003 J Biol Chem 278:42200-42207), and phage T4 fibritin foldon (Miroshnikov et al. 1998 Protein Eng 11:329-414), any of which can bind to the RSV viral antigens or immunogens described herein (e.g., by binding to the C-terminus of the RSV F peptide) and promote trimerization of the recombinant viral antigens or immunogens. See also U.S. Patent Nos. 7,268,116, 7,666,837, 7,691,815, 10,618,949, 10,906,944, and 10,960,070, and U.S. Patent Application Publication No. 2020 / 0009244, which are incorporated herein by reference in their entireties.

[0113] In some embodiments, the recombinant viral antigen or immunogen can be linked to the multimerization domain using one or more peptide linkers (such as a gly-ser linker, e.g., a 10 amino acid glycine-serine peptide linker). As long as the recombinant viral antigen or immunogen trimer retains the desired properties (e.g., prefusion conformation), the trimer can include any of the stabilizing mutations provided herein (or a combination thereof).

[0114] To be therapeutically viable, a desired trimerizing protein moiety for biologic drug design must meet the following criteria: Ideally, it should be part of a naturally secreted protein, such as immunoglobulin Fc, that is abundant in the circulation (non-toxic), of human origin (lacking immunogenicity), relatively stable (long half-life), and capable of efficient self-trimerization (which is reinforced by covalent interchain disulfide bonds, thereby providing structural stability to the trimerized RSV viral antigen or immunogen).

[0115] Collagen is a family of fibrous proteins that are major components of the extracellular matrix. It is the most abundant protein in mammals, constituting approximately 25% of the total protein in the body. Collagen plays a major structural role in the formation of bone, tendon, skin, cornea, cartilage, blood vessels, and teeth. The fibrillar types of collagen, I, II, III, IV, V, and XI, are all synthesized as larger trimeric precursors called procollagens, in which a central uninterrupted triple-helical domain consisting of several hundred "GXY" repeats (or glycine repeats) is flanked by noncollagenous domains (NC), N-propeptides, and C-propeptides. Both the C- and N-terminal extensions are proteolytically processed during procollagen secretion, an event that initiates the assembly of the mature protein into collagen fibers that form the insoluble cellular matrix. BMP-1 is a protease that recognizes a specific peptide sequence in procollagen near the junction between the collagen's glycine repeats and C-prodomain and is responsible for the removal of the propeptide. Shed trimeric C-propeptide of type I collagen is detected in normal adult human serum at concentrations ranging from 50 to 300 ng / mL, with children having much higher levels, indicating active bone formation. In individuals with familial elevated serum concentrations of type I collagen C-propeptide, levels can be as high as 1 to 6 μg / mL without apparent abnormalities, suggesting that C-propeptide is nontoxic. Structural investigation of the trimeric C-propeptide of collagen suggests that it is a trilobed structure in which all three subunits converge at a linkage region near their N-terminus and connect to the rest of the procollagen molecule. This configuration, with the protruding protein to be fused in one direction, is similar to that of an Fc dimer.

[0116] Collagen types I, IV, V, and XI assemble primarily into heterotrimeric forms, consisting of two α-1 chains and one α-2 chain (in types I, IV, and V) or three distinct α chains (in type XI), which are highly homologous in sequence. Both collagen types II and III are homotrimers of α-1 chains. In the case of type I collagen, the most abundant form of collagen, a stable α(I) homotrimer also forms, present at various levels in different tissues. The C-propeptide chains of most of these collagens can self-assemble into homotrimers when overexpressed alone in cells. Although the N-propeptide domain is synthesized first, molecular assembly into trimeric collagen begins with the in-register bond of the C-propeptide. The C-propeptide complex is stabilized by the formation of interchain disulfide bonds, although the necessity of disulfide bonds for proper chain registration is unclear. The triple helix of glycine repeats repeats, thus propagating the bond from the C-terminus to the N-terminus in a zipper-like fashion. This knowledge led to the generation of non-native collagen matrices by exchanging the C-propeptides of different collagen chains using recombinant DNA technology. Non-collagenous proteins, such as cytokines and growth factors, have also been fused to the N-terminus of procollagen or mature collagen to allow de novo collagen matrix formation, with the goal of enabling the sustained release of non-collagenous proteins from the cell matrix. However, in both circumstances, the C-propeptide must be cleaved prior to the assembly of recombinant collagen fibers into an insoluble cell matrix.

[0117] Other protein trimerization domains, such as those from bacterial phage T4 of Escherichia coli and GCN4 from yeast fibritin derived aspartate transcarbamoylase, have previously been described to enable the trimerization of heterologous proteins; however, none of these trimerization proteins are human in nature, nor are they naturally secreted proteins. Therefore, any trimeric fusion proteins must be produced intracellularly, which not only raises the possibility of misfolding in the case of naturally secreted proteins such as soluble receptors, but also makes it difficult to purify such fusion proteins from thousands of other intracellular proteins. Furthermore, a fatal drawback to the use of such non-human protein trimerization domains (e.g., from yeast, bacterial phage, and bacteria) for trimeric biological drugs is their potential immunogenicity in the human body, rendering such fusion proteins ineffective immediately after injection into the human body.

[0118] The use of collagen in the recombinant polypeptides described herein therefore has many advantages, including: (1) collagen is the most abundant protein secreted in the mammalian body, constituting approximately 25% of the total protein in the body; (2) the major form of collagen occurs naturally as a trimeric helix, and their globular C-propeptide is responsible for initiating trimerization; (3) the trimeric C-propeptide of collagen, which is proteolytically released from mature collagen, is naturally detected in mammalian blood at submicrogram / mL levels and is not recognized as harmful to the body; (4) the linear triple chain structure of collagen The helical region of can be included as a linker with a predicted spacing of 2.9 Å per residue or can be removed as part of the fusion protein, allowing precise control of the distance between the protein to be trimerized and the collagen C-propeptide for optimal biological activity; (5) the recognition site for BMP1, which cleaves the C-propeptide from procollagen, can be mutated or deleted to prevent disruption of the trimeric fusion protein; (6) the C-propeptide domain self-trimerizes via disulfide bonds, providing a universal affinity tag that can be used for purification of any secreted fusion proteins produced. In some embodiments, the collagen C-propeptide bound to RSV viral antigens and immunogens, such as RSV F protein peptides, allows for the recombinant production of soluble covalent homotrimeric fusion proteins.

[0119] In some embodiments, the RSV viral antigen or immunogen is linked to a C-terminal propeptide of collagen to form a recombinant polypeptide. In some embodiments, the C-terminal propeptide of the recombinant polypeptide forms an inter-polypeptide disulfide bond. In some embodiments, the recombinant protein forms a trimer. In some embodiments, the RSV viral antigen or immunogen is a RSV F protein peptide described in Section I.

[0120] In some embodiments, the C-terminal propeptide is human collagen. In some embodiments, the C-terminal propeptide comprises the C-terminal polypeptide of proα1(I), proα1(II), proα1(III), proα1(V), proα1(XI), proα2(I), proα2(V), proα2(XI), or proα3(XI), or a fragment thereof. In some embodiments, the C-terminal propeptide is or comprises the C-terminal polypeptide of proα1(I).

[0121] In some embodiments, the C-terminal propeptide is or comprises the amino acid sequence set forth in SEQ ID NO: 48. In some embodiments, the C-terminal propeptide is an amino acid sequence having at least or about 85%, 90%, 92%, 95%, or 97% sequence identity to the sequence of SEQ ID NO: 48. In some embodiments, the C-terminal propeptide is or comprises the amino acid sequence set forth in SEQ ID NO: 49. In some embodiments, the C-terminal propeptide is an amino acid sequence having at least or about 85%, 90%, 92%, 95%, or 97% sequence identity to the sequence of SEQ ID NO: 49. In some embodiments, the C-terminal propeptide is or comprises the amino acid sequence set forth in SEQ ID NO: 50. In some embodiments, the C-terminal propeptide exhibits an amino acid sequence having at least or about 85%, 90%, 92%, 95%, or 97% sequence identity to the sequence of SEQ ID NO: 50. In some embodiments, the C-terminal propeptide is or comprises the amino acid sequence set forth in SEQ ID NO: 51. In some embodiments, the C-terminal propeptide is an amino acid sequence having at least or about 85%, 90%, 92%, 95%, or 97% sequence identity to the sequence of SEQ ID NO: 51. In some embodiments, the C-terminal propeptide is or comprises the amino acid sequence set forth in SEQ ID NO: 52. In some embodiments, the C-terminal propeptide is an amino acid sequence having at least or about 85%, 90%, 92%, 95%, or 97% sequence identity to the sequence of SEQ ID NO: 52. In some embodiments, the C-terminal propeptide is or comprises the amino acid sequence set forth in SEQ ID NO: 53. In some embodiments, the C-terminal propeptide is an amino acid sequence having at least or about 85%, 90%, 92%, 95%, or 97% sequence identity to the sequence of SEQ ID NO: 53.

[0122] In some embodiments, the C-terminal propeptide is or comprises the amino acid sequence set forth in SEQ ID NO: 54. In some embodiments, the C-terminal propeptide is an amino acid sequence having at least or about 85%, 90%, 92%, 95%, or 97% sequence identity to the sequence of SEQ ID NO: 54. In some embodiments, the C-terminal propeptide is or comprises the amino acid sequence set forth in SEQ ID NO: 55. In some embodiments, the C-terminal propeptide is an amino acid sequence having at least or about 85%, 90%, 92%, 95%, or 97% sequence identity to the sequence of SEQ ID NO: 55. In some embodiments, the C-terminal propeptide is or comprises the amino acid sequence set forth in SEQ ID NO: 56. In some embodiments, the C-terminal propeptide is an amino acid sequence having at least or about 85%, 90%, 92%, 95%, or 97% sequence identity to the sequence of SEQ ID NO: 56. In some embodiments, the C-terminal propeptide is or comprises the amino acid sequence set forth in SEQ ID NO: 57. In some embodiments, the C-terminal propeptide is an amino acid sequence having at least or about 85%, 90%, 92%, 95%, or 97% sequence identity to the sequence of SEQ ID NO: 57. In some embodiments, the C-terminal propeptide is or comprises the amino acid sequence set forth in SEQ ID NO: 58. In some embodiments, the C-terminal propeptide is an amino acid sequence having at least or about 85%, 90%, 92%, 95%, or 97% sequence identity to the sequence of SEQ ID NO: 58. In some embodiments, the C-terminal propeptide is or comprises the amino acid sequence set forth in SEQ ID NO: 59. In some embodiments, the C-terminal propeptide is an amino acid sequence having at least or about 85%, 90%, 92%, 95%, or 97% sequence identity to the sequence of SEQ ID NO: 59.

[0123] In some embodiments, the C-terminal propeptide is or comprises the amino acid sequence set forth in SEQ ID NO: 60. In some embodiments, the C-terminal propeptide is an amino acid sequence having at least or about 85%, 90%, 92%, 95%, or 97% sequence identity to the sequence of SEQ ID NO: 60. In some embodiments, the C-terminal propeptide is or comprises the amino acid sequence set forth in SEQ ID NO: 61. In some embodiments, the C-terminal propeptide is an amino acid sequence having at least or about 85%, 90%, 92%, 95%, or 97% sequence identity to the sequence of SEQ ID NO: 61. In some embodiments, the C-terminal propeptide is or comprises the amino acid sequence set forth in SEQ ID NO: 62. In some embodiments, the C-terminal propeptide is an amino acid sequence having at least or about 85%, 90%, 92%, 95%, or 97% sequence identity to the sequence of SEQ ID NO: 62. In some embodiments, the C-terminal propeptide is or comprises the amino acid sequence set forth in SEQ ID NO: 63. In some embodiments, the C-terminal propeptide is an amino acid sequence having at least or about 85%, 90%, 92%, 95%, or 97% sequence identity to the sequence of SEQ ID NO:63.

[0124] In some embodiments, the C-terminal propeptide comprises an aspartic acid (D) to asparagine (N) substitution at the BMP-1 site (e.g., RA D is RA N In some embodiments, the C-terminal propeptide is or comprises the amino acid sequence of a collagen trimerization domain (e.g., the C-propeptide of human α1(I) collagen) with an alanine (A) to asparagine (N) substitution at the BMP-1 site (e.g., R A D is R NThe C-terminal propeptide may be or comprise the amino acid sequence of a collagen trimerization domain (e.g., the C-propeptide of human α1(I) collagen) having a C-terminal amino acid sequence mutated to D. In some embodiments, as used herein, the C-terminal propeptide may comprise a mutated BMP-1 site, e.g., RSAN rather than DDAN. In some embodiments, as used herein, the C-terminal propeptide may comprise a BMP-1 site, e.g., a sequence comprising a RAD (e.g., RADDAN) sequence rather than a RAN (e.g., RANDAN) or RND (e.g., RNDDAN) sequence may be used in the fusion polypeptides disclosed herein.

[0125] In some embodiments, the C-terminal propeptide is or comprises an amino acid sequence that is a fragment of any of SEQ ID NOs: 48-63.

[0126] In some embodiments, the C-terminal propeptide can comprise a sequence comprising a glycine-XY repeat, where X and Y are independently any amino acid, or an amino acid sequence at least 85%, 90%, 92%, 95%, or 97% identical thereto, capable of forming an inter-polypeptide disulfide bond and trimerizing the recombinant polypeptide. In some embodiments, X and Y are independently proline or hydroxyproline.

[0127] In some cases, when an RSV F peptide protein (e.g., a RSV viral antigen or immunogen, see Section I) is linked to a C-terminal propeptide to form a recombinant polypeptide, the recombinant polypeptide forms a trimer resulting in a homotrimer of the RSV F protein peptide. In some embodiments, the trimerized recombinant polypeptide comprises an F protein peptide trimer as a crutch-shaped rod. In some embodiments, the RSV F protein peptide of the trimerized recombinant polypeptide is in a pre-fusion conformation. In some embodiments, the RSV F protein peptide of the trimerized recombinant polypeptide is in a post-fusion conformation. In some embodiments, the confirmation state allows access to different antigenic sites on the F protein peptide. In some embodiments, the antigenic site is an epitope, such as a linear epitope or a conformational epitope. As described above, an advantage of having a trimerized recombinant polypeptide is that it can initiate an immune response against a variety of potential and diverse antigenic sites.

[0128] In some embodiments, trimerized recombinant polypeptides comprise individual recombinant polypeptides that comprise the same viral antigen or immunogen. In some embodiments, trimerized recombinant polypeptides comprise individual recombinant polypeptides that each comprise a different viral antigen or immunogen from the other recombinant polypeptides. In some embodiments, trimerized recombinant polypeptides comprise individual recombinant polypeptides, where one of the individual recombinant polypeptides comprises a different viral antigen or immunogen from the other recombinant polypeptides. In some embodiments, trimerized recombinant polypeptides comprise individual recombinant polypeptides, where two of the individual recombinant polypeptides comprise the same viral antigen or immunogen, where the viral antigen or immunogen is different from the viral antigen or immunogen contained in the remaining recombinant polypeptides.

[0129] In some embodiments, the recombinant polypeptide comprises any RSV viral antigen or immunogen described in Section I. In some embodiments, the recombinant polypeptide comprises any RSV viral antigen or immunogen described in Section I linked, as described herein, to a C-terminal propeptide of a collagen described herein.

[0130] In some embodiments, the recombinant polypeptide or fusion protein comprises a first sequence set forth in any of SEQ ID NOs: 17-47 linked to a second sequence set forth in any of SEQ ID NOs: 48-63, wherein the C-terminus of the first sequence is directly linked to the N-terminus of the second sequence.

[0131] In some embodiments, the recombinant polypeptide or fusion protein comprises a first sequence set forth in any of SEQ ID NOs: 17-47 linked to a second sequence set forth in any of SEQ ID NOs: 48-63, wherein the C-terminus of the first sequence is indirectly linked to the N-terminus of the second sequence, e.g., via a linker. In some embodiments, the linker comprises a sequence comprising a glycine-XY repeat.

[0132] In some embodiments, the recombinant polypeptide is or comprises the sequence set forth in SEQ ID NO: 1. In some embodiments, the recombinant polypeptide is or comprises 102, 106, 107, 108, 109, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 161, 215, 218, 379, or 447 (amino acid positions relative to SEQ ID NO: 31 or 32). ), or any combination thereof. In some embodiments, the recombinant polypeptide is or comprises a variant of SEQ ID NO: 1, wherein the variant comprises any one, two, three, four, five or more mutations selected from the group consisting of P102A, R109A, R136A, E161P, E218A, S215P, I379A, and M447V, or any combination thereof.

[0133] In some embodiments, the recombinant polypeptide is or comprises the sequence set forth in SEQ ID NO: 2. In some embodiments, the recombinant polypeptide is or comprises 102, 106, 107, 108, 109, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 161, 215, 218, 379, or 447 (amino acid positions relative to SEQ ID NO: 31 or 32). 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 2, including sequences containing substitutions, deletions, and / or insertions at one or more amino acid positions such as P102A, R109A, R136A, E161P, E218A, S215P, I379A, and M447V, or any combination thereof. In some embodiments, the recombinant polypeptide is or comprises a variant of SEQ ID NO: 2, wherein the variant comprises any one, two, three, four, five, or more mutations selected from the group consisting of P102A, R109A, R136A, E161P, E218A, S215P, I379A, and M447V, or any combination thereof.

[0134] In some embodiments, the recombinant polypeptide is or comprises the sequence set forth in SEQ ID NO: 3. In some embodiments, the recombinant polypeptide is or comprises 102, 106, 107, 108, 109, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 161, 215, 218, 379, or 447 (amino acid positions relative to SEQ ID NO: 31 or 32). 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 3, including sequences containing substitutions, deletions, and / or insertions at one or more amino acid positions such as P102A, R109A, R136A, E161P, E218A, S215P, I379A, and M447V, or any combination thereof. In some embodiments, the recombinant polypeptide is or comprises a variant of SEQ ID NO: 3, wherein the variant comprises any one, two, three, four, five, or more mutations selected from the group consisting of P102A, R109A, R136A, E161P, E218A, S215P, I379A, and M447V, or any combination thereof.

[0135] In some embodiments, the recombinant polypeptide is or comprises the sequence set forth in SEQ ID NO: 4. In some embodiments, the recombinant polypeptide is or comprises 102, 106, 107, 108, 109, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 161, 215, 218, 379, or 447 (amino acid positions relative to SEQ ID NO: 31 or 32). ), or any combination thereof. In some embodiments, the recombinant polypeptide is or comprises a variant of SEQ ID NO: 4, wherein the variant comprises any one, two, three, four, five or more mutations selected from the group consisting of P102A, R109A, R136A, E161P, E218A, S215P, I379A, and M447V, or any combination thereof.

[0136] In some embodiments, the recombinant polypeptide is or comprises the sequence set forth in SEQ ID NO: 5. In some embodiments, the recombinant polypeptide is or comprises 102, 106, 107, 108, 109, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 161, 215, 218, 379, or 447 (amino acid positions relative to SEQ ID NO: 31 or 32). ), or any combination thereof. In some embodiments, the recombinant polypeptide is or comprises a variant of SEQ ID NO: 5, wherein the variant comprises any one, two, three, four, five or more mutations selected from the group consisting of P102A, R109A, R136A, E161P, E218A, S215P, I379A, and M447V, or any combination thereof.

[0137] In some embodiments, the recombinant polypeptide is or comprises the sequence set forth in SEQ ID NO: 6. In some embodiments, the recombinant polypeptide is or comprises 102, 106, 107, 108, 109, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 161, 215, 218, 379, or 447 (amino acid positions relative to SEQ ID NO: 31 or 32). 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 6, including sequences containing substitutions, deletions, and / or insertions at one or more amino acid positions such as P102A, R109A, R136A, E161P, E218A, S215P, I379A, and M447V, or any combination thereof. In some embodiments, the recombinant polypeptide is or comprises a variant of SEQ ID NO: 6, wherein the variant comprises any one, two, three, four, five, or more mutations selected from the group consisting of P102A, R109A, R136A, E161P, E218A, S215P, I379A, and M447V, or any combination thereof.

[0138] In some embodiments, the recombinant polypeptide is or comprises the sequence set forth in SEQ ID NO: 7. In some embodiments, the recombinant polypeptide is or comprises 102, 106, 107, 108, 109, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 161, 215, 218, 379, or 447 (amino acid positions relative to SEQ ID NO: 31 or 32). 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 7, including sequences containing substitutions, deletions, and / or insertions at one or more amino acid positions such as P102A, R109A, R136A, E161P, E218A, S215P, I379A, and M447V, or any combination thereof. In some embodiments, the recombinant polypeptide is or comprises a variant of SEQ ID NO: 7, wherein the variant comprises any one, two, three, four, five, or more mutations selected from the group consisting of P102A, R109A, R136A, E161P, E218A, S215P, I379A, and M447V, or any combination thereof.

[0139] In some embodiments, the recombinant polypeptide is or comprises the sequence set forth in SEQ ID NO: 8. In some embodiments, the recombinant polypeptide is or comprises 102, 106, 107, 108, 109, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 161, 215, 218, 379, or 447 (amino acid positions relative to SEQ ID NO: 31 or 32). 8, including sequences containing substitutions, deletions, and / or insertions at one or more amino acid positions such as P102A, R109A, R136A, E161P, E218A, S215P, I379A, and M447V, or any combination thereof. In some embodiments, the recombinant polypeptide is or comprises a variant of SEQ ID NO:8, wherein the variant comprises any one, two, three, four, five, or more mutations selected from the group consisting of P102A, R109A, R136A, E161P, E218A, S215P, I379A, and M447V, or any combination thereof.

[0140] In some embodiments, the recombinant polypeptide is or comprises the sequence set forth in SEQ ID NO: 9. In some embodiments, the recombinant polypeptide is or comprises 102, 106, 107, 108, 109, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 161, 215, 218, 379, or 447 (amino acid positions relative to SEQ ID NO: 31 or 32). 9, including sequences containing substitutions, deletions, and / or insertions at one or more amino acid positions such as P102A, R109A, R136A, E161P, E218A, S215P, I379A, and M447V, or any combination thereof. In some embodiments, the recombinant polypeptide is or comprises a variant of SEQ ID NO:9, wherein the variant comprises any one, two, three, four, five, or more mutations selected from the group consisting of P102A, R109A, R136A, E161P, E218A, S215P, I379A, and M447V, or any combination thereof.

[0141] In some embodiments, the recombinant polypeptide is or comprises the sequence set forth in SEQ ID NO: 10. In some embodiments, the recombinant polypeptide is or comprises the sequence set forth in SEQ ID NO: 10. In some embodiments, the recombinant polypeptide is or comprises the sequence set forth in SEQ ID NO: 102, 106, 107, 108, 109, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 161, 215, 218, 379, or 447 (amino acid positions relative to SEQ ID NO: 31 or 32). or any combination thereof. In some embodiments, the recombinant polypeptide is or comprises an amino acid sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 10, including sequences containing substitutions, deletions, and / or insertions at one or more amino acid positions, such as, or any combination thereof. In some embodiments, the recombinant polypeptide is or comprises a variant of SEQ ID NO: 10, wherein the variant comprises any one, two, three, four, five, or more mutations selected from the group consisting of P102A, R109A, R136A, E161P, E218A, S215P, I379A, and M447V, or any combination thereof.

[0142] In some embodiments, the recombinant polypeptide is or comprises the sequence set forth in SEQ ID NO: 11. In some embodiments, the recombinant polypeptide is or comprises the sequence set forth in SEQ ID NO: 11. In some embodiments, the recombinant polypeptide is or comprises the sequence set forth in SEQ ID NO: 102, 106, 107, 108, 109, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 161, 215, 218, 379, or 447 (amino acid positions relative to SEQ ID NO: 31 or 32). or any combination thereof. In some embodiments, the recombinant polypeptide is or comprises an amino acid sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 11, including sequences containing substitutions, deletions, and / or insertions at one or more amino acid positions, such as, or any combination thereof. In some embodiments, the recombinant polypeptide is or comprises a variant of SEQ ID NO: 11, wherein the variant comprises any one, two, three, four, five, or more mutations selected from the group consisting of P102A, R109A, R136A, E161P, E218A, S215P, I379A, and M447V, or any combination thereof.

[0143] In some embodiments, the recombinant polypeptide is or comprises the sequence set forth in SEQ ID NO: 12. ..., 102, 106, 107, 108, 109, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 161, 215, 218, 379, or 447 (amino acid positions relative to SEQ ID NO: 31 or 32). or any combination thereof. In some embodiments, the recombinant polypeptide is or comprises an amino acid sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 12, including sequences containing substitutions, deletions, and / or insertions at one or more amino acid positions, such as, or any combination thereof. In some embodiments, the recombinant polypeptide is or comprises a variant of SEQ ID NO: 12, wherein the variant comprises any one, two, three, four, five, or more mutations selected from the group consisting of P102A, R109A, R136A, E161P, E218A, S215P, I379A, and M447V, or any combination thereof.

[0144] In some embodiments, the recombinant polypeptide is or comprises the sequence set forth in SEQ ID NO: 13. ..., 102, 106, 107, 108, 109, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 161, 215, 218, 379, or 447 (amino acid positions relative to SEQ ID NO: 31 or 32). or any combination thereof. In some embodiments, the recombinant polypeptide is or comprises an amino acid sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 13, including sequences containing substitutions, deletions, and / or insertions at one or more amino acid positions, such as, or any combination thereof. In some embodiments, the recombinant polypeptide is or comprises a variant of SEQ ID NO: 13, wherein the variant comprises any one, two, three, four, five, or more mutations selected from the group consisting of P102A, R109A, R136A, E161P, E218A, S215P, I379A, and M447V, or any combination thereof.

[0145] In some embodiments, the recombinant polypeptide is or comprises the sequence set forth in SEQ ID NO: 14. ..., 102, 106, 107, 108, 109, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 161, 215, 218, 379, or 447 (amino acid positions relative to SEQ ID NO: 31 or 32). or any combination thereof. In some embodiments, the recombinant polypeptide is or comprises an amino acid sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 14, including sequences containing substitutions, deletions, and / or insertions at one or more amino acid positions, such as, or any combination thereof. In some embodiments, the recombinant polypeptide is or comprises a variant of SEQ ID NO: 14, wherein the variant comprises any one, two, three, four, five, or more mutations selected from the group consisting of P102A, R109A, R136A, E161P, E218A, S215P, I379A, and M447V, or any combination thereof.

[0146] In some embodiments, the recombinant polypeptide is or comprises the sequence set forth in SEQ ID NO: 15. In some embodiments, the recombinant polypeptide is or comprises the sequence set forth in SEQ ID NO: 15. In some embodiments, the recombinant polypeptide is or comprises the sequence set forth in SEQ ID NO: 15. or any combination thereof. In some embodiments, the recombinant polypeptide is or comprises an amino acid sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 15, including sequences containing substitutions, deletions, and / or insertions at one or more amino acid positions, such as, or any combination thereof. In some embodiments, the recombinant polypeptide is or comprises a variant of SEQ ID NO: 15, wherein the variant comprises any one, two, three, four, five, or more mutations selected from the group consisting of P102A, R109A, R136A, E161P, E218A, S215P, I379A, and M447V, or any combination thereof.

[0147] In some embodiments, the recombinant polypeptide is or comprises the sequence set forth in SEQ ID NO: 16. ..., 102, 106, 107, 108, 109, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 161, 215, 218, 379, or 447 (amino acid positions relative to SEQ ID NO: 31 or 32). or any combination thereof. In some embodiments, the recombinant polypeptide is or comprises an amino acid sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 16, including sequences containing substitutions, deletions, and / or insertions at one or more amino acid positions, such as, or any combination thereof. In some embodiments, the recombinant polypeptide is or comprises a variant of SEQ ID NO: 16, wherein the variant comprises any one, two, three, four, five, or more mutations selected from the group consisting of P102A, R109A, R136A, E161P, E218A, S215P, I379A, and M447V, or any combination thereof.

[0148] As noted above, in some embodiments, the recombinant polypeptides provided herein do not simply combine to form trimers, but can also aggregate or be aggregated to produce proteins comprising multiple recombinant polypeptides. In some embodiments, the resulting proteins have a macrostructure. In some cases, the macrostructure can confer structural stability to the RSV viral antigen or immunogenic recombinant polypeptide, which in turn provides access to potential antigenic sites that can enhance an immune response.

[0149] In some embodiments, the trimerized recombinant polypeptides aggregate to form a protein comprising a plurality of trimerized recombinant polypeptides. In some embodiments, the plurality of trimerized recombinant polypeptides form a protein having a macrostructure. In some embodiments, the protein comprises rosette-like oligomers comprising F protein peptide trimers as crutch-shaped rods.

[0150] In some embodiments, provided herein are complexes comprising a recombinant polypeptide selected from the group consisting of SEQ ID NOs: 1-16, or fragments, variants, or mutants thereof, in any suitable combination. In some embodiments, provided herein are complexes comprising a trimer of a recombinant polypeptide selected from the group consisting of SEQ ID NOs: 1-16, or fragments, variants, or mutants thereof, wherein the recombinant polypeptides are trimerized via inter-polypeptide disulfide bonds to form the trimer.

[0151] In some embodiments, a protein described herein comprising multiple recombinant polypeptides is an immunogen. In some embodiments, a protein described herein comprising multiple recombinant polypeptides is incorporated into a nanoparticle. For example, in some embodiments, the protein is directly bound to a nanoparticle, e.g., a protein nanoparticle. In some embodiments, the protein is indirectly bound to a nanoparticle. In some embodiments, a protein described herein comprising multiple recombinant polypeptides is incorporated into a virus-like particle (VLP).

[0152] 2. Polynucleotides and Vectors Also provided are polynucleotides (nucleic acid molecules) encoding the RSV antigens or immunogens and recombinant polypeptides provided herein, as well as vectors for genetically modifying cells to express such RSV antigens or immunogens and recombinant polypeptides.

[0153] In some embodiments, provided are polynucleotides encoding the recombinant polypeptides provided herein. In some aspects, the polynucleotides comprise a single nucleic acid sequence, such as a nucleic acid sequence encoding the recombinant polypeptide. In other cases, the polynucleotides comprise a first nucleic acid sequence encoding a recombinant polypeptide comprising a particular RSV viral antigen or immunogen or a second nucleic acid sequence encoding a recombinant polypeptide comprising a different RSV viral antigen or immunogen.

[0154] In some embodiments, the polynucleotide encoding the recombinant polypeptide comprises at least one promoter operably linked to control expression of the recombinant polypeptide, hi some embodiments, the polynucleotide comprises two, three, or more promoters operably linked to control expression of the recombinant polypeptide.

[0155] In some embodiments, for example, when a polynucleotide comprises two or more nucleic acid coding sequences, e.g., sequences encoding recombinant polypeptides comprising different RSV viral antigens or immunogens, at least one promoter is operably linked to control expression of the two or more nucleic acid sequences, hi some embodiments, the polynucleotide comprises two, three, or more promoters operably linked to control expression of the recombinant polypeptides.

[0156] In some embodiments, expression of the recombinant polypeptide is inducible or conditional. Thus, in some aspects, the polynucleotide encoding the recombinant polypeptide comprises a conditional promoter, enhancer, or transactivator. In some such aspects, the conditional promoter, enhancer, or transactivator is an inducible promoter, enhancer, or transactivator or a repressible promoter, enhancer, or transactivator. For example, in some embodiments, an inducible or conditional promoter can be used to restrict expression of the recombinant polypeptide to a specific microenvironment. In some embodiments, expression driven by an inducible or conditional promoter is regulated by exposure to an exogenous agent, such as heat, irradiation, or a drug.

[0157] When a polynucleotide comprises two or more nucleic acid sequences encoding a recombinant polypeptide, the polynucleotide may further comprise a nucleic acid sequence encoding a peptide between one or more of the nucleic acid sequences. In some cases, the nucleic acid located between the nucleic acid sequences encodes a peptide that separates the translation products of the nucleic acid sequences during or after translation. In some embodiments, the peptide comprises an internal ribosome entry site (IRES), a self-cleaving peptide, or a peptide that causes ribosome skipping, such as a T2A peptide.

[0158] In some embodiments, a polynucleotide encoding a recombinant polypeptide is introduced into a composition comprising cultured cells (e.g., host cells), such as by retroviral transduction, gene transfer, or transformation. In some embodiments, this can allow for expression (e.g., production) of the recombinant polypeptide. In some embodiments, the expressed recombinant polypeptide is purified.

[0159] In some embodiments, the polynucleotides (nucleic acid molecules) provided herein encode a RSV viral antigen or immunogen described herein, hi some embodiments, the polynucleotides (nucleic acid molecules) provided herein encode a recombinant polypeptide comprising a RSV viral antigen or immunogen described herein, such as the RSV F peptide protein.

[0160] Also provided are vectors or constructs comprising the nucleic acid molecules described herein. In some embodiments, the vectors or constructs comprise one or more promoters operably linked to the nucleic acid molecules encoding the recombinant polypeptides so as to drive expression thereof. In some embodiments, the promoters are operably linked to one or more nucleic acid molecules, e.g., nucleic acid molecules encoding recombinant polypeptides comprising different RSV viral antigens or immunogens.

[0161] In some embodiments, the vector is a viral vector. In some embodiments, the vector is a retroviral vector. In some embodiments, the retroviral vector is a lentiviral vector. In some embodiments, the retroviral vector is a gamma retroviral vector.

[0162] In some embodiments, a vector or construct comprises a single promoter that drives expression of one or more polynucleotide nucleic acid molecules. In some embodiments, such a promoter can be multicistronic (bicistronic or tricistronic, see, e.g., U.S. Pat. No. 6,060,273). For example, in some embodiments, a transcription unit can be engineered as an IRES (internal ribosome entry site)-containing bicistronic unit, which allows for simultaneous expression of gene products (e.g., encoding different recombinant polypeptides) by messages from a single promoter. In some embodiments, the vectors provided herein are bicistronic, allowing the vector to contain and express two nucleic acid sequences. In some embodiments, the vectors provided herein are tricistronic, allowing the vector to contain and express three nucleic acid sequences.

[0163] In some embodiments, a single promoter directs the expression of RNA containing two or three genes (e.g., encoding a chimeric signaling receptor and encoding a recombinant receptor) in a single open reading frame (ORF), separated from each other by sequences encoding self-cleaving peptides (e.g., 2A sequences) or protease recognition sites (e.g., furin). Thus, the ORF encodes a single polypeptide, which is processed into individual proteins during translation (in the case of 2A) or post-translation. In some cases, peptides such as T2A can cause ribosomes to skip peptide bond synthesis at the C-terminus of the 2A element (ribosomal skipping), separating the end of the 2A sequence from the next downstream peptide (see, e.g., de Felipe. Genetic Vaccines and Ther. 2:13 (2004) and de Felipe et al. Traffic 5:616-626 (2004)). Many 2A elements are known in the art. Examples of 2A sequences that can be used in the methods and nucleic acids disclosed herein include, but are not limited to, 2A sequences from foot and mouth disease virus (F2A), equine rhinitis A virus (E2A), Thosea asigna virus (T2A), and porcine teschovirus-1 (P2A), as described in U.S. Patent Application Publication No. 20070116690.

[0164] In some embodiments, the vector is comprised in a virus. In some embodiments, the virus is a pseudovirus. In some embodiments, the virus is a virus-like particle. In some embodiments, the vector is comprised in a cell. In some embodiments, the vector or the cell containing the vector comprises a recombinant genome.

[0165] III. Immunogenic Compositions and Formulations In some embodiments, provided herein are immunogenic compositions comprising a trimer of a recombinant polypeptide comprising a sequence selected from the group consisting of SEQ ID NOs: 1-16, or a combination of any two or more trimers. In some embodiments, a unit dose of the immunogenic composition may comprise about 10 μg to about 100 μg of RSV F antigen, preferably about 25 μg to about 75 μg of RSV F antigen, preferably about 40 μg to about 60 μg of RSV F antigen, or about 50 μg of RSV F antigen. In some embodiments, the dose comprises 3 μg of RSV F antigen. In other embodiments, the dose comprises 9 μg of RSV F antigen. In further embodiments, the dose comprises 30 μg of RSV F antigen.

[0166] In some cases, it may be desirable to combine the disclosed immunogens with other pharmaceuticals (e.g., vaccines) that induce a protective response against other agents. For example, compositions comprising the recombinant RSV F antigens described herein, e.g., trimers or proteins, can be administered simultaneously or sequentially with other vaccines, such as influenza vaccines or varicella-zoster vaccines, recommended by the Advisory Committee on Immunization Practices (ACIP; cdc.gov / vaccines / acip / index.html) for target age groups (e.g., infants aged approximately 1 to 6 months). Thus, the disclosed immunogens comprising the recombinant RSV F antigens described herein can be administered simultaneously or sequentially with vaccines against, for example, hepatitis B (HepB), diphtheria, tetanus, and pertussis (DTaP), pneumococcal virus (PCV), Haemophilus influenzae type b (Hib), polio, influenza, and rotavirus.

[0167] Multivalent or combination vaccines provide protection against multiple pathogens. In some embodiments, multivalent vaccines can protect against multiple strains of the same pathogen. In some embodiments, multivalent vaccines, such as the combination vaccine Tdap, which protects against strains of tetanus, pertussis, and diphtheria, protect against multiple pathogens. Multivalent vaccines are highly desirable because they minimize the number of immunizations required to confer protection against multiple pathogens or pathogenic strains, reduce administration costs, and increase vaccination rates. This is particularly beneficial, for example, when vaccinating infants or children.

[0168] In some embodiments, for example, vaccines comprising the immunogenic compositions described herein are multivalent vaccines. In some embodiments, the antigenic material for incorporation into the multivalent vaccine compositions of the invention is derived from RSV type A or B, or a combination thereof. Antigens for incorporation into the multivalent vaccine compositions of the invention can be derived from a single strain of RSV, or from multiple strains, e.g., 2-5 strains, to provide a broader spectrum of protection. In one embodiment, antigens for incorporation into the multivalent vaccine compositions of the invention are derived from multiple strains of RSV virus. Other useful antigens include live, attenuated, and inactivated viruses, such as inactivated poliovirus (Jiang et al., J. Biol. Stand., (1986) 14:103-9), attenuated strains of hepatitis A virus (Bradley et al., J. Med. Virol., (1984) 14:373-86), attenuated measles virus (James et al., N. Engl. J. Med., (1995) 332:1262-6), and epitopes of pertussis virus (e.g., ACEL-IMUNErM cell-free DTP, Wyeth-Lederle vaccine, and pediatric vaccines).

[0169] In some aspects, the vaccines provided herein are universal vaccines. In some embodiments, universal vaccines are vaccines that protect against multiple strains of the same virus, such as multiple strains of RSV. The development of an effective universal RSV vaccine would reduce the cost and effort of, for example, using seasonal vaccine formulations, and allow for more robust pandemic preparedness.

[0170] In some embodiments, a universal vaccine is comprised of multiple epitopes from different virus strains. In some embodiments, a universal vaccine is comprised of a single epitope that is conserved across different virus strains. For example, a universal vaccine can be based on a relatively conserved domain of the RSV F protein.

[0171] Immunogenic compositions comprising the disclosed immunogens (e.g., nucleic acid molecules encoding the disclosed recombinant RSV F antigens or promoters of the disclosed recombinant RSV F antigens) and a pharmaceutically acceptable carrier are also provided. In some embodiments, the immunogenic compositions comprise a trimerized recombinant polypeptide provided herein, and optionally a pharmaceutically acceptable carrier. In some embodiments, the immunogenic compositions comprise a protein comprising a plurality of trimerized recombinant polypeptides provided herein, and optionally a pharmaceutically acceptable carrier. In some embodiments, the immunogenic compositions comprise protein nanoparticles provided herein, and optionally a pharmaceutically acceptable carrier. In some embodiments, the immunogenic compositions comprise VLPs provided herein, and optionally a pharmaceutically acceptable carrier. In some embodiments, the immunogenic compositions comprise an isolated nucleic acid provided herein, and optionally a pharmaceutically acceptable carrier. In some embodiments, the immunogenic compositions comprise a vector provided herein, and optionally a pharmaceutically acceptable carrier. In some embodiments, the immunogenic compositions comprise a virus provided herein, and optionally a pharmaceutically acceptable carrier. In some embodiments, the immunogenic composition comprises a pseudovirus provided herein and, optionally, a pharmaceutically acceptable carrier. In some embodiments, the immunogenic composition comprises a cell provided herein and, optionally, a pharmaceutically acceptable carrier. In some embodiments, an immunogenic composition such as those described herein is a vaccine. In some embodiments, the vaccine is a prophylactic vaccine. In some embodiments, the vaccine is a therapeutic vaccine. In some embodiments, the vaccine is a prophylactic and therapeutic vaccine. Such pharmaceutical compositions can be administered to a subject by a variety of administration methods known to those skilled in the art, for example, intramuscular, intradermal, subcutaneous, intravenous, intraarterial, intraarticular, intraperitoneal, intranasal, sublingual, tonsillar, oropharyngeal, or other parenteral mucosal routes. In some embodiments, a pharmaceutical composition comprising one or more disclosed immunogens is an immunogenic composition.Actual methods for preparing administrable compositions will be known or apparent to those skilled in the art and are described in more detail in publications such as Remington's Pharmaceutical Sciences, 19th Ed., Mack Publishing Company, Easton, Pa., 1995.

[0172] Therefore, immunogens, such as the recombinant RSV F antigens described herein, such as trimers, proteins, can be formulated with pharmaceutically acceptable carriers to maintain biological activity and simultaneously enhance stability during storage within acceptable temperature ranges. Potential carriers include, but are not limited to, physiologically unbiased media, phosphate-buffered saline solution, water, emulsions (e.g., oil / water or water / oil emulsions), various types of wetting agents, cryoprotective additives or stabilizers, such as proteins, peptides or hydrolysates (e.g., albumin, gelatin), sugars (e.g., sucrose, lactose, sorbitol), amino acids (e.g., sodium glutamate), or other protective agents. The resulting aqueous solution can be packaged for immediate use or lyophilized. The lyophilized formulation is mixed with a sterile solution prior to administration for single or multiple doses.

[0173] Formulations, particularly liquid formulations, may contain bacteriostatic agents, including but not limited to, benzyl alcohol, phenol, m-cresol, chlorobutanol, methylparaben, and / or propylparaben, at effective concentrations (usually 1% w / v) to prevent or minimize degradation during storage. Bacteriostatic agents may be contraindicated for some patients; therefore, lyophilized formulations may be reconstituted in solutions with or without such ingredients.

[0174] The immunogenic compositions of the present disclosure may contain pharmaceutically acceptable vehicle substances such as pH adjusters and buffers, osmotic pressure adjusters, wetting agents, etc., to approximate physiological conditions, such as sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, and triethanolamine oleate. The immunogenic compositions may optionally contain an adjuvant to enhance the host's immune response. Suitable adjuvants include, for example, toll-like receptor agonists, alum, AlPO4, alhydrogel, lipid-A and its derivatives or variants, oil emulsions, saponins, neutral liposomes, liposomes containing vaccines and cytokines, nonionic block copolymers, and chemokines. Nonionic block polymers containing polyoxyethylene (POE) and polyoxypropylene (POP), such as POE-POP-POE block copolymer, MPL® (3-O-deacylated monophosphoryl lipid A; Corixa, Hamilton, Ind.), and IL-12 (Genetics Institute, Cambridge, Mass.), among many other suitable adjuvants known in the art, can be used as adjuvants (Newman et al., 1998, Critical Reviews in Therapeutic Drug Carrier Systems 15:89-142). These adjuvants have the advantage that they help nonspecifically stimulate the immune system, thus enhancing the immune response to pharmaceuticals. In some embodiments, the immunogenic compositions of the present disclosure can include or be administered together with two or more adjuvants. In some embodiments, the immunogenic compositions of the present disclosure can include or be administered together with two adjuvants. In some embodiments, the immunogenic compositions of the present disclosure may include or be administered in conjunction with multiple adjuvants, e.g., in some cases, vaccines comprising the immunogenic compositions provided herein may include or be administered in combination with multiple adjuvants.

[0175] For vaccine compositions, examples of suitable adjuvants include, for example, aluminum hydroxide, lecithin, Freund's adjuvant, MPL®, and IL-12. In some embodiments, the vaccine compositions or nanoparticle immunogens disclosed herein (e.g., RSV vaccine compositions) can be formulated as controlled-release or sustained-release formulations. This can be achieved with compositions containing slow-release polymers, or via microencapsulated delivery systems or bioadhesive gels. Various pharmaceutical compositions can be prepared according to standard procedures well known in the art.

[0176] In some embodiments, the immunogenic compositions of the present disclosure can include an adjuvant formulation comprising a metabolizable oil (e.g., squalene) and alpha-tocopherol in the form of an oil-in-water emulsion, and polyoxyethylene sorbitan monooleate (Tween 80). In some embodiments, the adjuvant formulation can include about 2% to about 10% squalene, about 2% to about 10% alpha-tocopherol (e.g., D-alpha-tocopherol), and about 0.3% to about 3% polyoxyethylene sorbitan monooleate. In some embodiments, the adjuvant formulation can include about 5% squalene, about 5% tocopherol, and about 0.4% polyoxyethylene sorbitan monooleate. In some embodiments, immunogenic compositions of the present disclosure may include 3 De-O-acylated monophosphoryl lipid A (3D-MPL) and an adjuvant in the form of an oil-in-water emulsion, the adjuvant comprising a metabolizable oil, alpha-tocopherol, and polyoxyethylene sorbitan monooleate. In some embodiments, immunogenic compositions of the present disclosure may include QS21 (extract of Quillaja Saponaria Molina: fraction 21), 3D-MPL, and an oil-in-water emulsion, the oil-in-water emulsion comprising a metabolizable oil, alpha-tocopherol, and polyoxyethylene sorbitan monooleate. In some embodiments, immunogenic compositions of the present disclosure may include QS21, 3D-MPL, and an oil-in-water emulsion, the oil-in-water emulsion having the following composition: a metabolizable oil such as squalene, alpha-tocopherol, and Tween 80. In some embodiments, the immunogenic compositions of the present disclosure may include an adjuvant in the form of a liposomal composition.

[0177] In some embodiments, the immunogenic compositions of the disclosure may include an adjuvant formulation comprising a metabolizable oil (e.g., squalene), polyoxyethylene sorbitan monooleate (Tween 80), and Span 85. In some embodiments, the adjuvant formulation may comprise about 5% (w / v) squalene, about 0.5% (w / v) polyoxyethylene sorbitan monooleate, and about 0.5% (w / v) Span 85.

[0178] In some embodiments, the immunogenic compositions of the present disclosure may include an adjuvant formulation comprising Quillaja saponin, cholesterol, and a phosphorlipid, e.g., in the form of a nanoparticle composition. In some embodiments, the immunogenic compositions of the present disclosure may include a mixture of separately purified fractions of Quillaja saponaria Molina, which are then formulated with cholesterol and a phospholipid.

[0179] In some embodiments, the immunogenic compositions of the present disclosure may comprise an adjuvant selected from the group consisting of MF59®, Matrix-A®, Matrix-C®, Matrix-M®, AS01, AS02, AS03, and AS04.

[0180] In some embodiments, the immunogenic compositions of the present disclosure may include a Toll-like receptor 9 (TLR9) agonist, wherein the TLR9 agonist is an oligonucleotide of 8 to 35 nucleotides in length containing an unmethylated cytidine phosphoguanosine (CpG) motif, and the RSV antigen and oligonucleotide are present in the immunogenic composition in an amount effective to stimulate an immune response to the RSV antigen in a mammalian subject, such as a human subject, in need thereof. TLR9 (CD289) recognizes the unmethylated cytidine phosphoguanosine (CpG) motif found in microbial DNA, which can be mimicked using synthetic CpG-containing oligodeoxynucleotides (CpG-ODNs). CpG-ODNs are known to enhance antibody production and stimulate T helper 1 (Th1) cell responses (Coffman et al., Immunity, 33:492-503, 2010). Optimal oligonucleotide TLR9 agonists often contain a palindromic sequence of the following general formula: 5'-purine-purine-CG-pyrimidine-pyrimidine-3', or 5'-purine-purine-CG-pyrimidine-pyrimidine-CG-3'. U.S. Patent No. 6,589,940, which is incorporated herein by reference in its entirety, is incorporated by reference. In some embodiments, the CpG oligonucleotide is linear. In some embodiments, the CpG oligonucleotide is circular or contains a hairpin loop. The CpG oligonucleotide can be single-stranded or double-stranded. In some embodiments, the CpG oligonucleotide can contain modifications, including, but not limited to, modifications of the 3'OH or 5'OH group, modifications of the nucleotide base, modifications of the sucrose moiety, and modifications of the phosphate group. Modified bases can be incorporated into the palindromic sequence of a CpG oligonucleotide, as long as it maintains the same specificity for its natural complement through Watson-Crick base pairing (e.g., the palindromic portion is still self-complementary). In some embodiments, the CpG oligonucleotide comprises a non-standard base. In some embodiments, the CpG oligonucleotide comprises a modified nucleoside.In some embodiments, the modified nucleoside is selected from the group consisting of 2'-deoxy-7-deazaguanosine, 2'-deoxy-6-thioguanosine, arabinoguanosine, 2'-deoxy-2'-substituted-arabinoguanosine, and 2'-O-substituted-arabinoguanosine. The CpG oligonucleotide may contain modifications of the phosphate group. For example, in addition to phosphodiester linkages, phosphate modifications include, but are not limited to, methylphosphonate, phosphorothioate, phosphoramidate (bridged or unbridged), phosphotriester, and dithiophosphate, and may be used in any combination. Other non-phosphate linkages may also be used. In some embodiments, the oligonucleotide contains only a phosphorothioate backbone. In some embodiments, the oligonucleotide contains only a phosphodiester backbone. In some embodiments, the oligonucleotide contains a combination of phosphate linkages in the phosphate backbone, for example, a combination of phosphodiester and phosphorothioate linkages. Oligonucleotides with phosphorothioate backbones may be more immunogenic than those with phosphodiester backbones and appear to be more resistant to degradation after injection into a host (Braun et al., J Immunol, 141:2084-2089, 1988; and Latimer et al., Mol Immunol, 32:1057-1064, 1995). The CpG oligonucleotides of the present disclosure contain at least one, two, or three internucleotide phosphorothioate ester linkages. In some embodiments, when multiple CpG oligonucleotide molecules are present in a pharmaceutical composition containing at least one excipient, both stereoisomers of the phosphorothioate ester linkage are present in the multiple CpG oligonucleotide molecules. In some embodiments, all internucleotide linkages of the CpG oligonucleotide are phosphorothioate linkages, or in other words, the CpG oligonucleotide has a phosphorothioate backbone.

[0181] Any suitable CpG oligodeoxynucleotide (ODN) or a combination thereof can be used as an adjuvant in the present disclosure. For example, K-type ODN (also called B-type) encodes multiple CpG motifs on a phosphorothioate backbone. K-type ODNs have the following sequence: TCCATGGA CG TTCCTGAG CG The use of phosphorothioate nucleotides, compared to natural phosphodiester nucleotides, increases resistance to nuclease digestion and results in a substantially longer in vivo half-life. K-type ODNs induce pDC differentiation and TNFα production, as well as B cell proliferation and IgM secretion. D-type ODNs (also called A-type) are constructed from a mixed phosphodiester / phosphorothioate backbone, contain a single CpG motif flanked by palindromic sequences, and have poly-G tails (structural motifs that promote the formation of concatemers) at the 3' and 5' ends. D-type ODNs contain the following sequence: GGTGCAT CG It can be based on ATGCAGGGGGG. D-type ODN induces pDC maturation and IFNα secretion, but has no effect on B cells. C-type ODN is similar to K-type in that it is composed entirely of phosphorothioate nucleotides, but similar to D-type in that it contains a palindromic CpG motif. C-type ODN has the following sequence T CG T CG TT CG AA CG A CG This class of ODN can be based on TTGAT. This class of ODN stimulates B cells to secrete IL-6 and pDC to produce IFNα. P-type ODN contains two palindromic sequences, allowing them to form higher-order structures. P-type ODNs contain the following sequence: T CG T CG A CG AT CG G CGCGCG C CG P-type ODN activates B cells and pDCs and induces substantially more IFNα production compared to C-type ODN. In this paragraph, bold letters in ODN sequences indicate self-complementary palindromic structures, and CpG motifs are underlined.

[0182] Exemplary CpG ODNs, such as CpG7909 (5'-TCGTCGTTTTGTCGTTTTGTCGTT-3') and CpG1018 (5'-TGACTGTGAACGTTCGAGATGA-3'), are known and are described in U.S. Patent Nos. 7,255,868, 7,491,706, 7,479,285, 7,745,598, and 7,785. ,610, 8,003,115, 8,133,874, 8,114,418, 8,222,398, 8,333,980, 8,597,665, 8,669,237, 9,028,845, and 10,052,378; U.S. Patent Application Publication No. 2020 / 0002704; and Bode et al., "CpG DNA as a vaccine adjuvant," Expert Rev Vaccines (2011), 10(4):499-511, all of which are incorporated by reference in their entirety for all purposes.

[0183] One or more adjuvants may be used in combination, including, but not limited to, alum (aluminum salts), oil-in-water emulsions, water-in-oil emulsions, liposomes, and microparticles such as poly(lactide-co-glycolide) microparticles (Shah et al., Methods Mol Biol, 1494:1-14, 2017). In some embodiments, the immunogenic composition further comprises an aluminum salt adjuvant to which the RSV antigen is adsorbed. In some embodiments, the aluminum salt adjuvant comprises one or more selected from the group consisting of amorphous aluminum hydroxyphosphate sulfate, aluminum hydroxide, aluminum phosphate, and aluminum potassium sulfate. In some embodiments, the aluminum salt adjuvant comprises one or both of aluminum hydroxide and aluminum phosphate. In some embodiments, the aluminum salt adjuvant comprises aluminum hydroxide. In some embodiments, a unit dose of the immunogenic composition comprises about 0.25 to about 0.50 mg of aluminum. 3+ , or approximately 0.35 mg of Al3+ In some embodiments, the immunogenic composition further comprises an additional adjuvant. Other suitable adjuvants include, but are not limited to, a squalene-in-water emulsion (e.g., MF59 or AS03), a TLR3 agonist (e.g., polyIC or polyICLC), a TLR4 agonist (e.g., a bacterial lipopolysaccharide derivative such as monophosphoryl lipid A (MPL) and / or a saponin such as Quil A or QS-21 as in AS01 or AS02), a TLR5 agonist (bacterial flagellin), and an agonist for TLR7, TLR8, and / or TLR9 (imidazoquinoline derivatives such as imiquimod and resiquimod) (Coffman et al., Immunity, 33:492-503, 2010). In some embodiments, the additional adjuvant comprises MPL and alum (e.g., AS04). For veterinary use and for the production of antibodies in non-human animals, the mitogenic components of Freund's adjuvant (both complete and incomplete) can be used.

[0184] In some embodiments, the immunogenic composition comprises a pharmaceutically acceptable excipient, such as, for example, a solvent, a bulking agent, a buffer, a tonicity adjusting agent, and a preservative (Pramanick et al., Pharma Times, 45:65-77, 2013). In some embodiments, the immunogenic composition may comprise an excipient that functions as one or more of a solvent, a bulking agent, a buffer, and a tonicity adjusting agent (e.g., sodium chloride in saline can function as both an aqueous vehicle and a tonicity adjusting agent).

[0185] In some embodiments, the immunogenic composition comprises an aqueous vehicle as a solvent. Suitable vehicles include, for example, sterile water, saline, phosphate-buffered saline, and Ringer's solution. In some embodiments, the composition is isotonic.

[0186] The immunogenic composition may contain a buffering agent. The buffering agent controls the pH during processing, storage, and optionally reconstitution to prevent degradation of the active agent. Suitable buffers include salts, including, for example, acetate, citrate, phosphate, or sulfate. Other suitable buffers include amino acids, such as arginine, glycine, histidine, and lysine. The buffering agent may further contain hydrochloric acid or sodium hydroxide. In some embodiments, the buffering agent maintains the pH of the composition within the range of 6 to 9. In some embodiments, the pH is greater than 6, 7, or 8 (the lower limit). In some embodiments, the pH is less than 9, 8, or 7 (the upper limit). That is, the pH is in the range of about 6 to 9, where the lower limit is less than the upper limit.

[0187] The immunogenic composition may include a osmolality modifier. Suitable osmolality modifiers include, for example, dextrose, glycerol, sodium chloride, glycerin, and mannitol.

[0188] The immunogenic composition may contain a bulking agent. Bulking agents are particularly useful when the pharmaceutical composition is to be lyophilized prior to administration. In some embodiments, the bulking agent is a protective agent that helps stabilize and prevent degradation of the active agent during freezing or spray drying and / or storage. Suitable bulking agents are sugars (mono-, di-, and polysaccharides), such as sucrose, lactose, trehalose, mannitol, sorbitol, glucose, and raffinose.

[0189] The immunogenic compositions may contain preservatives. Suitable preservatives include, for example, antioxidants and antimicrobial agents. However, in preferred embodiments, the immunogenic compositions are prepared under sterile conditions and are packaged in single-use containers, thus obviating the need for preservatives.

[0190] In some embodiments, the composition can be provided as a sterile composition. Pharmaceutical compositions generally contain an effective amount of the disclosed immunogen and can be prepared by conventional techniques. Typically, the amount of immunogen in each dose of the immunogenic composition is selected to induce an immune response without significant adverse side effects. In some embodiments, the composition can be provided as a unit dosage form for use in inducing an immune response in a subject. The unit dosage form contains a single preselected dose suitable for administration to a subject, or two or more appropriately labeled or measured preselected unit dosages, and / or a metering mechanism for administering a unit dose or multiples thereof. In other embodiments, the composition further comprises an adjuvant.

[0191] IV. Methods for Inducing an Immune Response In some embodiments, provided herein are methods of generating an immune response to a surface antigen of RSV in a subject, the method comprising administering to the subject an effective amount of a complex comprising a recombinant polypeptide selected from the group consisting of SEQ ID NOs: 1-16. In some embodiments, provided herein are methods of generating an immune response to a surface antigen of RSV in a subject, the surface antigen comprising an F protein or an antigenic fragment thereof, the method comprising administering to the subject an effective amount of a complex comprising a recombinant polypeptide selected from the group consisting of SEQ ID NOs: 1-16. In some embodiments, provided herein are methods of generating an immune response to a surface antigen of RSV in a subject, the surface antigen comprising a sequence selected from the group consisting of SEQ ID NOs: 17-47, the method comprising administering to the subject an effective amount of a complex comprising a recombinant polypeptide selected from the group consisting of SEQ ID NOs: 1-16. In some embodiments, provided herein are methods for generating an immune response to a surface antigen of RSV in a subject, wherein the surface antigen comprises an RSV F protein or an antigenic fragment thereof, and optionally the surface antigen comprises one or more of the sequences set forth in SEQ ID NOs: 17-47 or antigenic fragments thereof, the method comprising administering to the subject an effective amount of a complex comprising a recombinant polypeptide comprising a sequence set forth in any one of SEQ ID NOs: 1-16.

[0192] In some embodiments, provided herein are methods for generating an immune response to a surface antigen of RSV in a subject, wherein the surface antigen comprises an F protein or an antigenic fragment thereof, the method comprising administering to the subject an effective amount of a complex comprising a recombinant polypeptide comprising a sequence selected from the group consisting of SEQ ID NOs: 1-16, or a combination of any two or more of the complexes.

[0193] The disclosed immunogens (e.g., nucleic acid molecules (e.g., RNA molecules) or vectors encoding recombinant RSV F antigens described herein, such as trimers, proteins, disclosed recombinant RSV F antigens, protein nanoparticles or virus-like particles containing the disclosed recombinant RSV F antigens) can be administered to a subject to induce an immune response against the corresponding RSV F antigen in the subject. In a specific example, the subject is a human. The immune response can be a protective immune response, e.g., a response that suppresses subsequent infection with the corresponding RSV. Induction of an immune response can also be used to treat or suppress infections and diseases associated with the corresponding RSV.

[0194] Subjects who are infected with RSV or who are at risk of developing an infection, for example, due to exposure or potential exposure to RSV, can be selected for treatment. After administration of the disclosed immunogens, subjects can be monitored for infection, symptoms associated with RSV, or both.

[0195] Typical subjects for treatment with the therapeutic agents and methods of the present disclosure include humans, as well as non-human primates and other animals. To identify subjects for prevention or treatment with the methods of the present disclosure, recognized screening methods are used to determine risk factors associated with the target or suspected disease or condition, or to identify the subject's current status of the disease or condition. These screening methods include, for example, routine examinations to identify risk factors that may be associated with environmental, familial, occupational, and other target or suspected diseases or conditions, as well as diagnostic methods such as various ELISAs and other immunoassays for detecting and / or characterizing RSV infection. These and other routine methods allow clinicians to select patients who require treatment using the methods and pharmaceutical compositions of the present disclosure. According to these methods and principles, the compositions can be administered according to the teachings of the present specification or other conventional methods as an independent prevention or treatment program, or as a follow-up, adjunct, or coordinated therapy for other treatments.

[0196] The administration of the disclosed immunogens, such as RSV F antigens, e.g., trimers, proteins, can be for prophylactic or therapeutic purposes. When administered prophylactically, the disclosed therapeutic agents are administered before any symptoms, e.g., before infection. Prophylactic administration of the disclosed therapeutic agents serves to prevent or ameliorate any subsequent infection. When administered therapeutically, the disclosed therapeutic agents are administered at or after the onset of disease or infection symptoms, e.g., after the onset of symptoms of RSV infection corresponding to the RSV F antigen, or after diagnosis of RSV infection. Thus, the therapeutic agent can be administered before expected exposure to RSV, after exposure or suspected exposure to the virus, or after the actual onset of infection to reduce the severity, duration, or extent of the expected infection and / or associated disease symptoms.

[0197] The immunogens and immunogenic compositions described herein are administered to a subject, preferably a human, in an effective amount to induce or enhance an immune response to the RSV F antigen. The actual dosage of the disclosed immunogens will vary depending on factors such as the disease being treated and the specific condition of the subject (e.g., the subject's age, size, fitness, severity of symptoms, susceptibility factors, etc.), the time and route of administration, other drugs or treatments being administered concomitantly, and the specific pharmacology of the composition that elicits the desired activity or biological response of the subject. The administration regimen can be adjusted to obtain an optimal prophylactic or therapeutic response.

[0198] Pharmaceutical compositions containing one or more of the disclosed immunogens can be used in coordinated (or prime-boost) vaccination protocols or combination formulations. In certain embodiments, the novel combination immunogenic compositions and coordinated immunization protocols each use separate immunogens or formulations to induce an antiviral immune response, such as an immune response to the RSV F antigen. Separate immunogenic compositions that induce an antiviral immune response can be combined into a multivalent immunogenic composition administered to a subject in a single immunization step, or they can be administered separately (in a monovalent immunogenic composition) in a coordinated (or prime-boost) immunization protocol.

[0199] There may be several boosts, and each boost may be a different disclosed immunogen. In some examples, a boost may be another boost or the same immunogen as the prime. The prime and boost may be administered as a single dose or multiple doses, e.g., two, three, four, five, six, or more doses administered over a period of days, weeks, or months. Multiple boosts may also be administered, from one to five (e.g., 1, 2, 3, 4, or 5 boosts), or more. Different dosages may be used in a series of sequential immunizations. For example, a relatively large dose may be administered in the primary immunization, followed by a relatively smaller dose of boost.

[0200] In some embodiments, the boost can be administered about 2, about 3-8 weeks, or about 4 weeks after priming, or about several months after priming. In some embodiments, the boost can be administered about 5, about 6, about 7, about 8, about 10, about 12, about 18, or about 24 months after priming, or at approximately the same time after priming. Periodic additional boosts can also be used at appropriate times to strengthen the subject's "immunological memory." The appropriateness of selected vaccination parameters, e.g., formulation, dosage, therapy, etc., can be determined by collecting aliquots of serum from the subject and assaying antibody titers over the course of the immunization program. In addition, the subject's clinical condition can be monitored for the desired effect, e.g., prevention of infection or amelioration of disease (e.g., reduction in viral load). If such monitoring indicates suboptimal vaccination, the subject can be boosted with additional doses of the immunogenic composition, or vaccination parameters can be altered in a manner predicted to enhance the immune response.

[0201] In some embodiments, a prime-boost method can include a DNA primer and protein boost vaccination protocol for a subject. The method can include two or more administrations of the nucleic acid molecule or protein.

[0202] For protein therapeutics, typically each human dose contains 1 to 1000 μg of protein, e.g., about 1 μg to about 100 μg, e.g., about 1 μg to about 50 μg, e.g., about 1 μg, about 2 μg, about 5 μg, about 10 μg, about 15 μg, about 20 μg, about 25 μg, about 30 μg, about 40 μg, or about 50 μg of protein.

[0203] The amount utilized in the immunogenic composition is selected based on the target population (e.g., infants or the elderly). Optimal amounts for a particular composition can be ascertained by standard testing, including observation of antibody titers and other responses in subjects. It is understood that a therapeutically effective amount of the disclosed immunogen, such as the disclosed recombinant RSV F antigen, e.g., trimer, protein, viral vector, or nucleic acid molecule in the immunogenic composition, can include an amount that is ineffective in eliciting an immune response upon administration of a single dose, but is effective upon administration of multiple doses, for example, in a prime-boost protocol.

[0204] Upon administration of the disclosed immunogens of the present disclosure, the subject's immune system typically responds to the immunogenic composition by producing antibodies specific for the RSV F protein peptide contained in the immunogen, indicating that an immunologically effective amount has been delivered to the subject.

[0205] In some embodiments, the subject's antibody response is determined during the assessment of an effective dosage / immunization protocol. In most cases, it is sufficient to assess the antibody titer in serum or plasma obtained from the subject. The decision on whether to administer a booster vaccination and / or change the amount of therapeutic agent administered to an individual can be made at least in part based on the antibody titer level. The antibody titer level can be based, for example, on an immunobinding assay that measures the concentration of antibodies in serum that bind to an antigen containing a recombinant RSV F antigen, such as a trimer protein.

[0206] RSV infection does not need to be completely eliminated, reduced, or prevented for the method to be effective. For example, eliciting an immune response against RSV with one or more disclosed immunogens can reduce or suppress RSV infection by a desired amount, for example, by at least 10%, at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 100% (elimination or prevention of detectable infected cells) compared to RSV infection in the absence of the immunogen. In a further example, RSV replication can be reduced or suppressed by the disclosed method. RSV replication does not need to be completely eliminated for the method to be effective. For example, an immune response elicited using one or more disclosed immunogens can reduce corresponding RSV replication by a desired amount, e.g., by at least 10%, at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 100% (eliminating or preventing detectable replication of RSV) compared to RSV replication in the absence of the immunogen.

[0207] In some embodiments, the disclosed immunogen is administered to the subject simultaneously with administration of the adjuvant, hi other embodiments, the disclosed immunogen is administered to the subject after administration of the adjuvant within a time sufficient to induce an immune response.

[0208] One approach to administering nucleic acids is direct immunization using plasmid DNA, such as with mammalian expression plasmids. Immunization with nucleic acid constructs is well known in the art and is taught, for example, in U.S. Pat. No. 5,643,578, which describes a method for immunizing vertebrates by introducing DNA encoding a desired antigen, and U.S. Pat. Nos. 5,593,972 and 5,817,637, which describe operably linking a nucleic acid sequence encoding an antigen to a regulatory sequence that enables expression. U.S. Pat. No. 5,880,103 describes several methods for delivering nucleic acids encoding immunogenic peptides or other antigens to organisms. Methods include liposomal delivery of nucleic acids (or synthetic peptides themselves) and immunostimulatory constructs, or ISCOMS®, negatively charged cage-like structures 30-40 nm in size that spontaneously form by mixing cholesterol and Quil A®. Protective immunity has been generated in various experimental models of infectious diseases, including toxoplasmosis and Epstein-Barr virus-induced tumors, using ISCOMS® as antigen delivery vehicles (Mowat and Donachie, Immunol. Today 12:383, 1991). Doses as low as 1 μg of antigen encapsulated in ISCOMS® have been shown to generate class I-mediated CTL responses (Takahashi et al., Nature 344:873, 1990).

[0209] In some embodiments, the disclosed immunogens are expressed in a subject using a plasmid DNA vaccine. For example, nucleic acid molecules encoding the disclosed immunogens can be administered to a subject to induce an immune response to the RSV F antigen. In some embodiments, the nucleic acid molecules can be incorporated into a plasmid vector, such as the pVRC8400 vector, for DNA immunization (see Barouch et al., J. Virol. 79, 8828-8834, 2005, which is incorporated herein by reference).

[0210] In another approach for using nucleic acids for immunization, the disclosed recombinant RSV F antigen, e.g., trimer, protein, can be expressed by an attenuated viral host or vector or a bacterial vector. Recombinant vaccinia virus, adeno-associated virus (AAV), herpesvirus, retrovirus, cytomegalovirus, or other viral vectors can be used to express peptides or proteins, thereby eliciting CTL responses. For example, vaccinia vectors and methods useful for immunization protocols are described in U.S. Patent No. 4,722,848. BCG (bacillus Calmette-Guerin) provides another vector for peptide expression (see Stover, Nature 351:456-460, 1991).

[0211] In one embodiment, a nucleic acid encoding the disclosed recombinant RSV F antigen is directly introduced into cells. For example, the nucleic acid can be loaded onto gold microspheres by standard methods and introduced into the skin using a device such as Bio-Rad's HELIOS® gene gun. The nucleic acid can be "naked," consisting of a plasmid under the control of a strong promoter. Typically, the DNA is injected into muscle, although it can also be injected directly into other sites. The dosage for injection is usually about 0.5 μg / kg to about 50 mg / kg, typically about 0.005 mg / kg to about 5 mg / kg (see, e.g., U.S. Patent No. 5,589,466).

[0212] For example, nucleic acids can be loaded onto gold microspheres using standard methods and introduced into the skin using a device such as Bio-Rad's HELIOS® gene gun. The nucleic acid can be "naked," consisting of a plasmid under the control of a strong promoter. Typically, DNA is injected into the muscle, but it can also be injected directly into other sites. The dosage for injection is usually about 0.5 μg / kg to about 50 mg / kg, typically about 0.005 mg / kg to about 5 mg / kg (see, e.g., U.S. Pat. No. 5,589,466).

[0213] In another embodiment, a nucleic acid encoding the disclosed recombinant RSV F antigen can be delivered directly to cells using an mRNA-based immunization protocol. In some embodiments, mRNA-based nucleic acid vaccines can provide a powerful alternative to previously mentioned approaches. mRNA vaccines eliminate safety concerns regarding DNA integration into the host genome and are delivered directly into the host cytoplasm. Furthermore, simple cell-free in vitro synthesis of RNA avoids the manufacturing complexities associated with viral vectors. Two exemplary forms of RNA-based vaccination that can be used to deliver nucleic acids encoding the disclosed recombinant RSV F antigens include conventional, non-amplified mRNA immunization (see, e.g., Petsch et al., "Protective efficacy of in vitro synthesized, specific mRNA vaccines against influenza A virus infection," Nature biotechnology, 30(12):1210-6, 2012) and self-amplified mRNA immunization (see, e.g., Geall et al., "Nonviral delivery of self-amplifying RNA vaccines," PNAS, 109(36):14604-14609, 2012; Magini et al., "Self-Amplifying mRNA Vaccines Expressing Multiple Conserved Influenza Antigens Confer Protection against Homologous and Heterosubtypic Viral Challenge," PLoS One, 11(8):e0161193, 2016; and Brito et al., "Self-amplifying mRNA vaccines," Adv Genet., 89:179-233, 2015).

[0214] In some embodiments, administration of a therapeutically effective amount of one or more of the disclosed immunogens to a subject induces a neutralizing immune response in the subject. To assess neutralizing activity, serum can be collected from the subject at appropriate time points after immunization, frozen, and stored for neutralization testing. Methods for assaying neutralizing activity are known to those skilled in the art and are further described herein, including, but not limited to, plaque reduction neutralization (PRNT) assays, microneutralization assays, flow cytometry-based assays, and single-cycle infection assays. In some embodiments, serum neutralizing activity can be assayed using a panel of RSV pseudoviruses.

[0215] In some embodiments, administration of a therapeutically effective amount of one or more of the disclosed immunogens to a subject induces a neutralizing immune response in the subject. To assess neutralizing activity, serum can be collected from the subject at appropriate time points after immunization, frozen, and stored for neutralization testing. Methods for assaying neutralizing activity are known to those skilled in the art and are further described herein, including, but not limited to, plaque reduction neutralization (PRNT) assays, microneutralization assays, flow cytometry-based assays, and single-cycle infection assays. In some embodiments, serum neutralizing activity can be assayed using a panel of RSV pseudoviruses.

[0216] In some embodiments, the neutralizing immune response induced by the immunogens disclosed herein generates neutralizing antibodies against RSV. In some embodiments, the neutralizing antibodies herein bind to a cellular receptor or coreceptor for RSV or a component thereof. Nucleolin is an entry coreceptor for RSV and also mediates the internalization of influenza, parainfluenza viruses, some enteroviruses, and the bacteria that cause tularemia. Binding of the pre-fusion RSV-F glycoprotein to the insulin-like growth factor 1 receptor (IGF1R) can also activate protein kinase C zeta (PKCζ), localizing nucleolin from the nucleus to the plasma membrane and binding to RSV-F on virions. In some embodiments, the viral receptor or coreceptor is a paramyxovirus receptor or coreceptor, preferably a pneumovirus receptor or coreceptor, more preferably a human RSV receptor or coreceptor. For example, CCR1, CCR2, CCR3, CCR4, CCR5, and / or CCR8 receptors can be involved in human RSV infection. RhoA is another example of a host cell RSV receptor or coreceptor. In some embodiments, the neutralizing antibodies herein modulate, reduce, antagonize, alleviate, block, inhibit, suppress, and / or interfere with at least one RSV activity or binding, such as RSV release, RSV receptor signaling, membrane RSV cleavage, RSV activity, RSV production and / or synthesis, or RSV receptor activity or binding, in vitro, in situ, and / or in vivo. In some embodiments, the immunogens disclosed herein induce neutralizing antibodies against RSV that modulate, reduce, antagonize, alleviate, block, inhibit, suppress, and / or interfere with RSV binding to a RSV receptor or coreceptor, such as nucleolin, IGF1R, CCR1, CCR2, CCR3, CCR4, CCR5, CCR8, and / or RhoA.

[0217] V. Product or Kit Also provided are articles of manufacture or kits containing the provided recombinant polypeptides, proteins, and immunogenic compositions. The articles of manufacture may include a container and a label or package insert on or attached to the container. Suitable containers include, for example, bottles, vials, syringes, test tubes, IV solution bags, etc. The containers may be formed from a variety of materials, such as glass or plastic. In some embodiments, the containers have a sterile access port. Exemplary containers include intravenous solution bags and vials, including those with stoppers pierceable by injection needles. The articles of manufacture or kits may further include a package insert indicating that the composition can be used to treat a particular condition, such as a condition described herein (e.g., RSV infection). Alternatively, or in addition, the articles of manufacture or kits may further include a separate or the same container containing a pharmaceutically acceptable buffer. Other materials, such as other buffers, diluents, filters, needles, and / or syringes, may further be included.

[0218] The label or package insert can indicate that the composition is used to treat RSV infection in individuals.The label or package insert on or attached to the container can indicate how to reconstitute and / or use the formulation.The label or package insert can further indicate that the formulation is useful or intended for subcutaneous, intravenous, or other administration methods for treating RSV infection in individuals.

[0219] In some embodiments, the container holds a composition, alone or in combination with another composition, that is effective in treating, preventing, and / or diagnosing a condition. The article of manufacture or kit may include (a) a first container having contained therein an immunogenic composition or protein or recombinant polypeptide composition thereof (i.e., a first agent); and (b) a second container having contained therein a composition comprising an adjuvant or an additional agent, such as a therapeutic agent (i.e., a second agent), wherein the article of manufacture or kit further includes instructions on a label or package insert for treating a subject with an effective amount of the second agent.

[0220] Terminology Unless otherwise specified, all technical, notational, and other technical and scientific terms or terminology used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter belongs. In some instances, terms having commonly understood meanings are defined herein for clarity and / or ready reference, and the inclusion of such definitions herein should not necessarily be construed as representing a substantial difference from what is commonly understood in the art.

[0221] The terms "polypeptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues and are not limited to a minimum length. The receptor and other polypeptides provided, e.g., polypeptides containing linkers or peptides, can contain amino acid residues, including natural and / or unnatural amino acid residues. The term also includes post-expression modifications of the polypeptide, e.g., glycosylation, sialylation, acetylation, and phosphorylation. In some embodiments, a polypeptide can contain modifications to the native or native sequence, so long as the protein maintains the desired activity. These modifications can be deliberate, such as by site-directed mutagenesis, or accidental, such as by host mutations that generate proteins or errors due to PCR amplification.

[0222] As used herein, a "subject" is a mammal, such as a human, or other animal, typically a human. In some embodiments, the subject, e.g., a patient to whom an agent, cell, cell population, or composition is administered, is a mammal, typically a primate, such as a human. In some embodiments, the primate is a monkey or ape. The subject may be male or female and of any suitable age, including infants, juveniles, adults, and geriatric subjects. In some embodiments, the subject is a non-primate mammal, such as a rodent.

[0223] As used herein, "treatment" (and grammatical variations thereof, such as "treat" or "treating") means a complete or partial amelioration or reduction of a disease or condition or disorder, or a symptom, adverse effect or consequence, or associated phenotype. Desirable effects of treatment include, but are not limited to, prevention of disease onset or recurrence, alleviation of symptoms, reduction of any direct or indirect pathological consequence of disease, prevention of metastasis, slowing of disease progression, amelioration or palliation of the condition, and remission or improved prognosis. The term does not imply a complete cure of a disease, or the complete elimination of any symptom or effect, as opposed to all symptoms or consequences.

[0224] As used herein, "delaying the onset of disease" means to postpone, prevent, slow, decelerate, stabilize, inhibit, and / or delay the onset of a disease (such as cancer). This delay can be for varying lengths of time depending on the disease being treated and / or the individual's medical history. In some embodiments, a sufficient or significant delay effectively encompasses prevention, in that the individual does not develop the disease. For example, late-stage cancer, such as the development of metastases, can be delayed.

[0225] As used herein, "preventing" includes providing protection against the occurrence or recurrence of a disease in a subject who is susceptible to the disease but has not yet been diagnosed with the disease. In some embodiments, the provided cells and compositions are used to delay the onset of the disease or slow the progression of the disease.

[0226] As used herein, "inhibiting" a function or activity means reducing the function or activity as compared to otherwise identical conditions or parameters of interest, or as compared to another condition. For example, a cell that inhibits tumor growth reduces the rate of tumor growth as compared to the rate of tumor growth in the absence of the cell.

[0227] An "effective amount" of an agent, e.g., a pharmaceutical formulation, cell, or composition, means an amount effective, in the context of administration, as a dosage / amount, for a period of time necessary, to achieve a desired result, such as a therapeutic or prophylactic result.

[0228] A "therapeutically effective amount" of an agent, e.g., pharmaceutical formulation, cell, means an amount effective, in the context of administration, as a therapeutic dose, for a period of time necessary to achieve the desired therapeutic result for the treatment of a disease, condition, or disorder, and / or the pharmacokinetic or pharmacodynamic effect of the treatment. A therapeutically effective amount may vary depending on factors such as the disease state, age, sex, and weight of the subject to be administered, and the population of cells administered. In some embodiments, the provided methods include administering cells and / or compositions in an effective amount, e.g., a therapeutically effective amount.

[0229] A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, but not necessarily, the prophylactically effective amount will be less than the therapeutically effective amount, since prophylactic administration is used in subjects before or at an early stage of disease. In the case of lower tumor burdens, in some embodiments, the prophylactically effective amount will be higher than the therapeutically effective amount.

[0230] As used herein, the term "about" refers to a normal range of error for each value, which is readily apparent to one of ordinary skill in the art. As used herein, the reference to "about" preceding a value or parameter includes (and describes) embodiments directed to that value or parameter itself.

[0231] As used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, "a" or "an" means "at least one" or "one or more."

[0232] Throughout this disclosure, various aspects of the claimed subject matter are presented in range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the claimed subject matter. Accordingly, the description of a range should be considered to include all possible subranges specifically disclosed, as well as individual numerical values ​​within that range. For example, when a range of values ​​is provided, it is understood that each intervening value between the upper and lower limits of that range and any other stated or intervening value in that stated range is encompassed within the claimed subject matter. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the claimed subject matter, subject to the existence of any specifically excluded range within the stated range. When a stated range includes one or both of its limits, ranges excluding either or both of those included limits are also encompassed within the claimed subject matter. This applies regardless of the broadness of the range.

[0233] As used herein, a composition means any mixture of two or more products, substances, or compounds, including cells, which may be a solution, suspension, liquid, powder, paste, aqueous solution, non-aqueous solution, or any combination thereof.

[0234] As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors as self-replicating nucleic acid structures as well as vectors that have integrated into the genome of a host cell into which they have been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0235] Exemplary Embodiments Embodiment 1. A protein comprising a plurality of recombinant polypeptides, each recombinant polypeptide comprising a respiratory syncytial virus (RSV) F protein peptide or a fragment or epitope thereof linked to a C-terminal propeptide of collagen, wherein the C-terminal propeptides of the recombinant polypeptides form an inter-polypeptide disulfide bond.

[0236] Embodiment 2. The protein of embodiment 1, wherein the RSV is subtype A or subtype B.

[0237] Embodiment 3. The protein of embodiment 1 or 2, wherein the epitope is a linear epitope or a conformational epitope.

[0238] Embodiment 4. The protein of any of embodiments 1-3, wherein the F protein peptide comprises an F1 subunit peptide, an F2 subunit peptide, or any combination thereof, and the protein comprises three recombinant polypeptides.

[0239] Embodiment 5. The protein of any of embodiments 1-4, wherein the F protein peptide comprises a signal peptide, a heptad repeat C (HRC) peptide, a pep27 peptide, a fusion peptide (FP), a heptad repeat A (HRA) peptide, a domain I peptide, a domain II peptide, or a heptad repeat B (HRB) peptide, or any combination thereof.

[0240] Embodiment 6. The protein of any one of embodiments 1 to 5, wherein the F protein peptide comprises the F1 subunit of the F protein but not the F2 subunit, or vice versa.

[0241] Embodiment 7. The protein of any one of embodiments 1 to 6, wherein the F protein peptide comprises the F1 and F2 subunits of an F protein, optionally lacking pep27, and optionally wherein the F1 and F2 subunits are linked by a disulfide bond or an artificially introduced linker.

[0242] Embodiment 8. The protein of any of embodiments 1-7, wherein the F protein peptide does not comprise a transmembrane (TM) domain peptide and / or a cytoplasmic (CP) domain peptide.

[0243] Embodiment 9. The protein of any of embodiments 1-8, wherein the F protein peptide comprises a protease cleavage site, and the protease is optionally furin, trypsin, factor Xa, or cathepsin L.

[0244] Embodiment 10. The protein of any of embodiments 1-8, wherein the F protein peptide does not comprise a protease cleavage site, and the protease is optionally furin, trypsin, factor Xa, or cathepsin L.

[0245] Embodiment 11. The protein of any of embodiments 1-10, wherein the F protein peptide is soluble or does not directly bind to a lipid bilayer, e.g., a membrane or viral envelope.

[0246] Embodiment 12. The protein of any of embodiments 1 to 11, wherein the F protein peptide is the same or different between recombinant polypeptides of the protein.

[0247] Embodiment 13. The protein of any of embodiments 1-12, wherein the F protein peptide is fused directly to the C-terminal propeptide or is attached to the C-terminal propeptide via a linker, such as a linker comprising a glycine-XY repeat, where X and Y are independently any amino acid, optionally proline or hydroxyproline.

[0248] Embodiment 14. The protein of any of embodiments 1 to 13, which is soluble or does not directly bind to a lipid bilayer, e.g., a membrane or viral envelope.

[0249] Embodiment 15. The protein of any one of embodiments 1 to 14, wherein the protein is capable of forming rosette-like oligomers comprising F protein peptide trimers.

[0250] Embodiment 16. The protein of any of embodiments 1 to 15, wherein the protein is capable of binding to a cell surface adhesion factor or receptor of a subject, and optionally the subject is a mammal such as a primate, e.g., a human.

[0251] Embodiment 17. The protein of any one of embodiments 1 to 16, wherein the C-terminal propeptide is human collagen.

[0252] Embodiment 18. The protein of any of embodiments 1-17, wherein the C-terminal propeptide comprises the C-terminal polypeptide of proα1(I), proα1(II), proα1(III), proα1(V), proα1(XI), proα2(I), proα2(V), proα2(XI), or proα3(XI), or a fragment thereof.

[0253] Embodiment 19. The protein of any of embodiments 1 to 18, wherein the C-terminal propeptides are the same or different between the recombinant polypeptides.

[0254] Embodiment 20. The protein of any of embodiments 1 to 19, wherein the C-terminal propeptide comprises SEQ ID NO: 48, or an amino acid sequence at least 90% identical thereto, capable of forming an inter-polypeptide disulfide bond to trimerize the recombinant polypeptide.

[0255] Embodiment 21. The protein of any of embodiments 1-19, wherein the C-terminal propeptide comprises SEQ ID NO: 49, or an amino acid sequence at least 90% identical thereto, capable of forming an inter-polypeptide disulfide bond to trimerize the recombinant polypeptide.

[0256] Embodiment 22. The protein of any of embodiments 1 to 19, wherein the C-terminal propeptide comprises SEQ ID NO: 50, or an amino acid sequence at least 90% identical thereto, capable of forming an inter-polypeptide disulfide bond to trimerize the recombinant polypeptide.

[0257] Embodiment 23. The protein of any of embodiments 1 to 19, wherein the C-terminal propeptide comprises SEQ ID NO: 51, or an amino acid sequence at least 90% identical thereto, capable of forming an inter-polypeptide disulfide bond to trimerize the recombinant polypeptide.

[0258] Embodiment 24. The protein of any of embodiments 1 to 19, wherein the C-terminal propeptide comprises SEQ ID NO: 52, or an amino acid sequence at least 90% identical thereto, capable of forming an inter-polypeptide disulfide bond to trimerize the recombinant polypeptide.

[0259] Embodiment 25. The protein of any of embodiments 1 to 19, wherein the C-terminal propeptide comprises SEQ ID NO: 53, or an amino acid sequence at least 90% identical thereto, capable of forming an inter-polypeptide disulfide bond to trimerize the recombinant polypeptide.

[0260] Embodiment 26. The protein of any of embodiments 1 to 19, wherein the C-terminal propeptide comprises SEQ ID NO: 54, or an amino acid sequence at least 90% identical thereto, capable of forming an inter-polypeptide disulfide bond to trimerize the recombinant polypeptide.

[0261] Embodiment 27. The protein of any of embodiments 1 to 19, wherein the C-terminal propeptide comprises any of SEQ ID NOs: 55-59 or an amino acid sequence at least 90% identical thereto, which is capable of forming an inter-polypeptide disulfide bond to trimerize the recombinant polypeptide.

[0262] Embodiment 28. The protein of any of embodiments 1 to 19, wherein the C-terminal propeptide comprises SEQ ID NO: 60, or an amino acid sequence at least 90% identical thereto, capable of forming an inter-polypeptide disulfide bond to trimerize the recombinant polypeptide.

[0263] Embodiment 29. The protein of any of embodiments 1 to 19, wherein the C-terminal propeptide comprises any of SEQ ID NOs: 61-63 or an amino acid sequence at least 90% identical thereto, which is capable of forming an inter-polypeptide disulfide bond to trimerize the recombinant polypeptide.

[0264] Embodiment 30. The protein of any of embodiments 1-29, wherein the C-terminal propeptide comprises an amino acid sequence comprising a glycine-XY repeat attached to the N-terminus of any of SEQ ID NOs: 48-63, or an amino acid sequence at least 90% identical thereto, which is capable of forming an inter-polypeptide disulfide bond to trimerize the recombinant polypeptide, where X and Y are independently any amino acid, optionally proline or hydroxyproline.

[0265] Embodiment 31. In some embodiments, the F protein peptide in each recombinant polypeptide is in a pre-fusion conformation or a post-fusion conformation, and optionally, the protein comprises a rosette-like oligomer comprising F protein peptide trimers as crutch-shaped rods.

[0266] Embodiment 32. The protein of any one of embodiments 1 to 31, wherein the F protein peptide in each recombinant polypeptide comprises any one of SEQ ID NOs: 17 to 47, or an amino acid sequence at least 80% identical thereto.

[0267] Embodiment 33. The protein of any one of embodiments 1 to 31, wherein the recombinant polypeptide comprises an amino acid sequence of any one of SEQ ID NOs: 1 to 16, or at least 80% identical thereto.

[0268] Embodiment 34. An immunogen comprising the protein according to any one of embodiments 1 to 33.

[0269] Embodiment 35. A protein nanoparticle comprising the protein according to any one of embodiments 1 to 33 directly or indirectly bound to the nanoparticle.

[0270] Embodiment 36. A virus-like particle (VLP) comprising a protein according to any one of embodiments 1 to 33.

[0271] Embodiment 37. An isolated nucleic acid encoding one, two, three or more recombinant polypeptides of a protein according to any one of embodiments 1 to 33.

[0272] Embodiment 38. The isolated nucleic acid of embodiment 37, wherein a polypeptide encoding the F protein peptide is fused in frame to a polypeptide encoding the C-terminal propeptide of collagen.

[0273] Embodiment 39. The isolated nucleic acid of embodiment 37 or 38, which is operably linked to a promoter.

[0274] Embodiment 40. The isolated nucleic acid of any one of embodiments 37 to 39, which is a DNA molecule.

[0275] Embodiment 41. The isolated nucleic acid of any of embodiments 37 to 39, which is an RNA molecule, optionally an mRNA molecule, such as a nucleoside-modified mRNA, a non-amplified mRNA, a self-amplified mRNA, or a trans-amplified mRNA.

[0276] Embodiment 42. A vector comprising the isolated nucleic acid of any one of embodiments 37 to 41.

[0277] Embodiment 43. The vector of embodiment 42, which is a viral vector.

[0278] Embodiment 44. A virus, pseudovirus, or cell comprising the vector of embodiment 42 or 43, optionally wherein the virus or cell has a recombinant genome.

[0279] Embodiment 45. An immunogenic composition comprising a protein, immunogen, protein nanoparticle, VLP, isolated nucleic acid, vector, virus, pseudovirus, or cell according to any one of embodiments 1 to 44, and a pharmaceutically acceptable carrier.

[0280] Embodiment 46. A vaccine comprising the immunogenic composition of embodiment 45 and optionally an adjuvant, which is optionally a subunit vaccine, and optionally a prophylactic and / or therapeutic vaccine.

[0281] Embodiment 47. The vaccine of embodiment 46, wherein the vaccine comprises multiple different adjuvants.

[0282] Embodiment 48. A method for producing a protein, comprising expressing in a host cell an isolated nucleic acid or vector described in any one of embodiments 37 to 43 to produce in the host cell a protein described in any one of embodiments 1 to 33; and purifying the protein.

[0283] Embodiment 49. A protein produced by the method of embodiment 48.

[0284] Embodiment 50. A method for generating an immune response in a subject against a RSV F protein peptide or a fragment or epitope thereof, comprising administering to a subject an effective amount of a protein, immunogen, protein nanoparticle, VLP, isolated nucleic acid, vector, virus, pseudovirus, cell, immunogenic composition, or vaccine described in any one of embodiments 1 to 47 and 49, to generate an immune response.

[0285] Embodiment 51. The method of embodiment 50 for treating or preventing infection with RSV.

[0286] Embodiment 52. The method of embodiment 50 or 51, wherein the generation of an immune response inhibits or reduces replication of RSV in the subject.

[0287] Embodiment 53. The method of any of embodiments 50-52, wherein the immune response comprises a cell-mediated response and / or a humoral response, and optionally includes the production of one or more neutralizing antibodies, such as polyclonal or monoclonal antibodies.

[0288] Embodiment 54. The method of any one of embodiments 50 to 53, wherein the immune response is directed against a RSV F protein peptide or a fragment or epitope thereof, but not against a C-terminal propeptide.

[0289] Embodiment 55. The method of any of embodiments 50-54, wherein the administration does not result in antibody-dependent enhancement (ADE) in the subject due to one or more prior exposures to RSV.

[0290] Embodiment 56. The method of any of embodiments 50-55, wherein the administration does not result in antibody-dependent enhancement (ADE) in the subject upon one or more subsequent exposures to RSV.

[0291] Embodiment 57. The method of any one of embodiments 50 to 56, further comprising a priming step and / or a boosting step.

[0292] Embodiment 58. The method of any of embodiments 50-57, wherein the administering step is carried out via topical, transdermal, subcutaneous, intradermal, oral, intranasal (e.g., intranasal spray), intratracheal, sublingual, buccal, rectal, vaginal, inhalation, intravenous (e.g., intravenous injection), intraarterial, intramuscular (e.g., intramuscular injection), intracardiac, intraosseous, intraperitoneal, transmucosal delivery, intravitreal, subretinal, intra-articular, periarticular, local, or epicutaneous administration.

[0293] Embodiment 59. The method of any of embodiments 50-58, wherein the effective amount is administered in a single dose or in a series of doses separated by one or more intervals.

[0294] Embodiment 60. The method of any one of embodiments 50-59, wherein the effective amount is administered without an adjuvant.

[0295] Embodiment 61. The method of any one of embodiments 50 to 59, wherein the effective amount is administered together with an adjuvant.

[0296] Embodiment 62. A method comprising administering to a subject an effective amount of a protein according to any one of embodiments 1 to 33, and producing neutralizing antibodies or neutralizing antisera against RSV in the subject.

[0297] Embodiment 63. The method of embodiment 62, wherein the subject is a mammal, optionally a human or non-human primate.

[0298] Embodiment 64 The method of embodiment 62 or 63, further comprising isolating neutralizing antibodies or neutralizing antisera from the subject.

[0299] Embodiment 65. The method of embodiment 64, further comprising administering an effective amount of an isolated neutralizing antibody or neutralizing antiserum to a human subject via passive immunization to prevent or treat infection with RSV.

[0300] Embodiment 66. The method of any one of embodiments 62 to 65, wherein the neutralizing antibody or neutralizing antiserum against RSV comprises a polyclonal antibody against a RSV F protein peptide or a fragment or epitope thereof, and optionally, the neutralizing antibody or neutralizing antiserum does not contain or is substantially free of antibodies against the C-terminal propeptide of collagen.

[0301] Embodiment 67. The method of any one of embodiments 62 to 65, wherein the neutralizing antibody comprises a monoclonal antibody against a RSV F protein peptide or a fragment or epitope thereof, and optionally, the neutralizing antibody does not contain or is substantially free of antibodies against the C-terminal propeptide of collagen.

[0302] Embodiment 68. The protein, immunogen, protein nanoparticle, VLP, isolated nucleic acid, vector, virus, pseudovirus, cell, immunogenic composition, or vaccine of any one of embodiments 1 to 47 and 49 for use in eliciting an immune response to RSV in a subject provided herein and / or for treating or preventing infection with RSV.

[0303] Embodiment 69. Use of a protein, immunogen, protein nanoparticle, VLP, isolated nucleic acid, vector, virus, pseudovirus, cell, immunogenic composition, or vaccine described in any one of embodiments 1 to 47 and 49 for inducing an immune response to RSV in a subject and / or for treating or preventing infection with RSV.

[0304] Embodiment 70. Use of a protein, immunogen, protein nanoparticle, VLP, isolated nucleic acid, vector, virus, pseudovirus, cell, immunogenic composition, or vaccine according to any one of embodiments 1 to 47 and 49 for the manufacture of a medicament or prophylactic for inducing an immune response against RSV in a subject and / or for the treatment or prevention of infection with RSV.

[0305] Embodiment 71. A method for analyzing a sample, comprising contacting the sample with a protein according to any one of embodiments 1 to 33, and detecting binding between the protein and an analyte capable of specifically binding to a RSV F protein peptide or a fragment or epitope thereof.

[0306] Embodiment 72. The method of embodiment 71, wherein the analyte is an antibody, receptor, or cell that recognizes an F protein peptide or a fragment or epitope thereof.

[0307] Embodiment 73. The method of embodiment 71 or 72, wherein binding indicates the presence of the analyte in the sample and / or RSV infection of the subject from which the sample is derived.

[0308] Embodiment 74. A kit comprising a protein according to any one of embodiments 1 to 33 and a substrate, pad or vial containing or immobilizing the protein, and optionally an ELISA or lateral flow assay kit.

[0309] The following examples are included for illustrative purposes only and are not intended to limit the scope of the present invention.

[0310] Example 1: Production of recombinant polypeptides containing RSV F protein peptides A secreted recombinant polypeptide containing an RSV F protein peptide was generated as a candidate vaccine.

[0311] The RSV F glycoprotein construct was derived from the RSV A2 strain (accession number AAC55970). The sequence encoding residues 1-520 of the F protein peptide was codon-optimized, synthesized, and subcloned into a mammalian expression vector encoding the human C-propeptide of α1 collagen at the Hind III and Bgl II sites. Figure 1A shows a schematic diagram of an exemplary recombinant polypeptide.

[0312] The recombinant plasmid was transfected into GH-CHO (dfhr-) cells and selected without hypoxanthine thymine (HT) (Invitrogen). For high-titer expression of the fusion protein in serum-free culture, CD007-4 TM1 medium (Jianshun Biosciences) was used for stepwise gene amplification with increasing concentrations of MTX (Sigma). Exemplary recombinant polypeptides were first purified via affinity binding to Endo180 using a salt gradient elution and further purified on a Superdex 200 gel filtration column (GE Healthcare). The purity of exemplary recombinant polypeptides containing the RSV F peptide was determined by size exclusion chromatography (SEC-HPLC) according to the manufacturer's instructions (Sepax Technologies).

[0313] The product titer of disulfide-linked fusion peptides (e.g., trimers) was found to be up to approximately 0.15 g / L in serum-free fed-batch cultures (Figure 1B). Culture supernatants containing trimerized recombinant polypeptides were first purified via affinity binding to the Fc-tagged collagen receptor uPARAP / Endo180 (a member of the mannose receptor family) (Thomas et al., (2005) J. Biol. Chem. 280, 22596-22605). This was pre-captured on a Protein A chromatography column, followed by gel filtration chromatography. SEC-HPLC analysis indicated that the purity of exemplary recombinant polypeptide trimers was approximately 95% (Figure 1C).

[0314] Purified exemplary trimerized recombinant polypeptides (0.1 μg) were separated by 8% SDS-PAGE under non-reducing or reducing conditions and transferred to a PVDF membrane. After blocking with 5% non-fat milk in PBS, the membrane was incubated with mouse anti-RSV F monoclonal antibody (Millipore) or palivizumab (AbbVie) and rabbit anti-C-propeptide of type I procollagen (CICP) polyclonal antibody (Millipore). 2 μg of purified exemplary recombinant polypeptide trimers were subjected to Coomassie blue staining. Figure 2A shows that the fusion peptide is expressed as a covalently linked trimeric protein.

[0315] The structural features and integrity of this purified recombinant polypeptide were further confirmed by Western blot analysis using antibodies specific for F and the trimerization peptide (Fig. 2A).

[0316] Purified recombinant polypeptide trimers were analyzed by negative staining electron microscopy. Purified recombinant polypeptides were diluted to 50 μg / mL and applied to a carbon-coated 400 CU mesh grid glow-discharged at 12 mA for 20 seconds for 5 seconds. The grid was negatively stained with 1% (w / v) uranyl formate for 20 seconds. Samples were collected on an FEI Tecnai Spirit electron microscope operating at 120 keV, and micrographs were acquired at 180,000x magnification. Figure 2B shows the recombinant polypeptide trimers aggregated in the protein, with macrostructures primarily in the form of rosette-like oligomers, similar to observations made with full-length F protein (Calder et al., (2000) Virology 271, 122-131; Smith et al., (2012) PloS One 7, e50852). The molecules in the rosette were crutch-shaped rods with their more flared ends protruding away from the center, consistent with the reported post-fusion conformation of F (Swanson et al., (2011) Prot. Natl. Acad. Sci. USA 108, 9619-9624).

[0317] The affinity binding of the monoclonal antibody palivizumab to an exemplary recombinant polypeptide trimer was measured using biolayer interferometry (BLI) on a fortebio OCTET QKe system (Pall). 5 μg / mL of palivizumab was directly immobilized on a Protein A sensor, then balanced in PBS and placed in wells containing 2-fold diluted fusion peptides (starting at 20 μg / mL). Dissociation was performed by immersion in PBS, and data were processed by subtracting the buffer reference using Data Analyze software for a 1:1 binding model.

[0318] The binding affinity of a purified exemplary recombinant polypeptide of palivizumab is less than 1 picomolar K D (FIG. 2C) showing that antigenic site II is exposed on the exemplary fusion peptide.

[0319] Example 2: Functional characterization of recombinant polypeptides containing RSV F protein peptides To evaluate the immunogenicity and protective efficacy of the exemplary recombinant polypeptide purified as described in Example 1, randomly grouped BALB / c mice were immunized twice by intramuscular injection on days 1 and 21 with one of three doses (1, 6, and 30 μg) of the exemplary fusion peptide with or without alum adjuvant (Imject alum adjuvant (Thermo Scientific)). An additional group immunized with PBS served as a control. Serum was collected on day 49, followed by intranasal (in) administration of 1x10 6 pfu RSV A2 strain (Fig. 3A). Animals were observed daily and euthanized on day 54 for lung tissue collection.

[0320] Sera were evaluated by enzyme-linked immunosorbent assay (ELISA). Briefly, 96-well plates were coated with 2 μg / mL of purified exemplary fusion peptide (in PBS) overnight at 4°C and blocked with 1 mg / mL BSA. The plates were washed with PBST and then incubated with serial 2-fold dilutions of serum (1:64 to 1:262,144) at room temperature for 2 hours. Bound antibodies were detected with HRP-conjugated goat anti-mouse IgG (SouthernBiotech) at room temperature for 1 hour. The enzyme reaction was developed with TMB (Thermo) and stopped by adding 2 M HCl, and the absorbance at 450 nm was recorded. Sera from PBS-immunized mice were used as a negative control at the same dilution, and the antibody titer was defined as the serum dilution that resulted in a ratio of 2.0 between the OD of the RSV F trimer and the OD of PBS.

[0321] Serum analysis revealed that all groups immunized with the exemplary recombinant polypeptides had high levels of RSV F protein peptide-specific antibodies, which were dose- and adjuvant-dependent (Fig. 3B). The neutralizing activity of anti-F antibodies was measured by microneutralization assay.

[0322] The RSV microneutralization assay was performed using HeLa cells and the RSV A2 strain. Serum was heat-inactivated at 56°C for 30 minutes and serially diluted in serum-free DMEM in 96-well culture plates (50 μL / well). An equal volume of virus (1,000 pfu / mL prepared in serum-free DMEM) was added to the plate, and the serum / virus mixture was incubated for 1 hour at 37°C. Approximately 5 x 10 4HeLa cells were added to the plate and incubated at 37°C until the positive control (virus only) wells showed 100% CPE. The plate was washed with PBST and fixed with 80% pre-chilled acetone in PBS for 10 minutes. 100 ng / mL palivizumab was added to the wells, blocked with 1 mg / mL BSA for 1 hour, and then incubated at room temperature for 2 hours. After three washes, the enzyme reaction was developed by adding HRP-conjugated goat anti-human IgG (SouthernBiotech), and the OD at 450 nm was recorded. The dilution resulting in 50% inhibition of CPE formation was determined as the neutralizing antibody titer.

[0323] Microneutralization assays demonstrated that exemplary recombinant polypeptide-induced anti-F antibodies possessed potent RSV neutralizing activity, and co-injection of alum adjuvant induced higher neutralizing antibody titers than the same dose of exemplary recombinant polypeptide. These results were consistent with the anti-F antibody results (Figure 3C).

[0324] The protective efficacy of exemplary recombinant polypeptides was assessed by measuring viral replication in the lungs 5 ​​days after viral challenge. Mice were sacrificed 5 days after intranasal RSV challenge, and the left lungs were harvested, weighed, and homogenized in 1 mL of serum-free DMEM. The homogenate was clarified by centrifugation at 1,000 x g for 10 minutes at 4°C, and viral titers in lung samples were determined by plaque assay as previously described.

[0325] The results showed that mice immunized with the exemplary recombinant polypeptide were completely protected from RSV replication, with no detectable virus, whereas the PBS-immunized control group exhibited high levels of viral load (Figure 3D).

[0326] Because antigenic site II is exposed on the exemplary recombinant polypeptides (see Example 1 and Figure 2C), a palivizumab competitive ELISA was performed to determine whether antibodies elicited by the exemplary recombinant polypeptides were directed against this site.

[0327] 5x10 6Palivizumab competitive ELISA was performed using 96-well ELISA plates coated with pfu / mL heat-inactivated RSV (HI-RSV in 50 mM carbonate-bicarbonate buffer, pH 9.2) and incubated overnight at 4°C. Uncoated surfaces were blocked with 1 mg / mL BSA. Two-fold dilutions (1:32 to 1:4,096) of serum mixtures containing 100 ng / mL palivizumab were added to the wells and incubated for 2 hours at room temperature. Bound palivizumab was detected using HRP-conjugated goat anti-human IgG (SouthernBiotech) and TMB substrate. Wells containing immunized mouse serum served as a noncompetitive positive control, and percent inhibition was calculated as ((OD of PBS - OD of RSV F trimer) / OD of PBS) x 100%. Competitive binding titers were expressed as the dilution producing 50% inhibition.

[0328] Inhibition of palivizumab binding to heat-inactivated RSV (HI-RSV) particles was observed in all serum samples collected from mice immunized with RSV F trimers, with or without alum adjuvant (Figure 3E). These results indicated that neutralizing antibodies elicited by exemplary fusion peptide immunization could effectively prevent RSV replication in the lungs of mice administered at least via the targeted antigenic site II of the virus.

[0329] FI-RSV vaccination unexpectedly increased disease severity (Kim et al., (1969) Am. J. Epidemiol. 89, 422-434; Chin et al., (1969) Am. J. Epidemiol. 89, 449-463), so safety monitoring is a top priority in RSV candidate vaccine development (Murata, Y. (2009) Clin. Lab. Med. 29, 725-739). Histological examination of lung tissue from immunized mice 5 days after challenge was performed to assess the safety of exemplary recombinant polypeptides.

[0330] For histopathological evaluation, the collected lung tissues were fixed in 10% neutral buffered formalin, embedded in paraffin, sectioned at 5 μm, and stained with H&E. Photographs were taken at 200x magnification under a Nikon microscope.

[0331] H&E staining revealed that the PBS-immunized control group exhibited some degree of alveolitis, peribronchiolitis, and perivasculitis, accompanied by significant inflammatory cell infiltration (Figure 4). In contrast, animals immunized with the exemplary recombinant polypeptide at any dose, with or without alum adjuvant, experienced limited immune cell infiltration and no obvious pathological changes (Figure 4). This result supports the use of the exemplary recombinant polypeptide immunization to protect against vaccine-associated disease enhancement following RSV infection.

[0332] The present invention is not intended to be limited in scope to the particular disclosed embodiments, which are provided, for example, to illustrate various aspects of the present invention. Various modifications to the described compositions and methods will become apparent from the descriptions and teachings herein. Such modifications can be made without departing from the true scope and spirit of the present disclosure, and are intended to be within the scope of the present disclosure.

[0333] [Table 3]

[0334] [Table 4]

[0335] [Table 5]

[0336] [Table 6]

[0337] Table 7

[0338] Table 8

[0339] Table 9

[0340] Table 10

[0341] Table 11

[0342] Table 12

[0343] Table 13

Claims

1. A recombinant subunit vaccine used to prevent infection with respiratory syncytial virus (RSV) in a mammal, the recombinant subunit vaccine comprising a soluble RSV viral surface antigen linked by in-frame fusion of the C-terminal portion of procollagen to the N-terminus to form a disulfide-bond linked trimeric fusion protein, the RSV viral surface antigen comprising an F protein consisting of an F1 peptide and an F2 peptide.

2. 2. The recombinant subunit vaccine of claim 1, wherein the RSV is subtype A or subtype B.

3. 3. The recombinant subunit vaccine of claim 1 or 2, wherein the RSV viral surface antigen comprises a mutant F protein, and the mutant F protein comprises one or more mutations selected from the group consisting of P102A, R109A, R136A, E161P, E218A, S215P, I379A, and M447V.

4. The recombinant subunit vaccine of any one of claims 1 to 3, wherein the fusion protein comprises the sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13 or 15.

5. The recombinant subunit vaccine of any one of claims 1 to 4, wherein the fusion protein comprises the sequence shown in SEQ ID NO:

37.

6. The recombinant subunit vaccine of any one of claims 1 to 5, wherein the fusion protein comprises the sequence shown in SEQ ID NO:

40.

7. 7. The recombinant subunit vaccine of claim 1, wherein the fusion protein comprises a first sequence set forth in any of SEQ ID NOs: 17-32 linked to a second sequence set forth in any of SEQ ID NOs: 48-63, and the C-terminus of the first sequence is linked directly or indirectly to the N-terminus of the second sequence.

8. The recombinant subunit vaccine of any one of claims 1 to 7, wherein the recombinant subunit vaccine is administered via intramuscular injection.

9. The recombinant subunit vaccine of any one of claims 1 to 8, wherein the recombinant subunit vaccine is administered via a nasal spray.

10. The recombinant subunit vaccine of any of claims 1 to 9, wherein the recombinant subunit vaccine is administered in a single dose or in a series of doses separated by intervals of several weeks or months.

11. The recombinant subunit vaccine of any one of claims 1 to 10, wherein the recombinant subunit vaccine is administered without an adjuvant.

12. The recombinant subunit vaccine of any one of claims 1 to 10, wherein the recombinant subunit vaccine is administered together with an adjuvant.

13. The recombinant subunit vaccine of any one of claims 1 to 10, wherein the recombinant subunit vaccine is administered together with two or more adjuvants.

14. A method for detecting antibodies to respiratory syncytial virus (RSV) from mammalian serum, comprising the step of contacting the serum with a soluble RSV surface antigen linked by in-frame fusion of the C-terminal portion of procollagen to the N-terminus to form a disulfide-bond linked trimeric fusion protein, wherein the RSV surface antigen comprises an F protein consisting of an F1 peptide and an F2 peptide.

15. 15. The method of claim 14, wherein the soluble RSV surface antigen is an F protein.

16. A recombinant subunit vaccine for use in a method comprising immunizing a mammal and purifying the neutralizing antibodies produced, and treating a patient infected with RSV via passive immunization using the neutralizing antibodies, wherein the recombinant subunit vaccine comprises a soluble surface antigen derived from respiratory syncytial virus (RSV), linked by in-frame fusion of the C-terminal portion of procollagen to the N-terminus to form a disulfide-bond linked trimeric fusion protein, the RSV-derived surface antigen comprising an F protein consisting of an F1 peptide and an F2 peptide.

17. 17. The recombinant subunit vaccine of claim 16, wherein the neutralizing antibody comprises a polyclonal antibody.

18. The recombinant subunit vaccine of claim 16, wherein the neutralizing antibody is a monoclonal antibody.

19. 17. The recombinant subunit vaccine of claim 16, wherein the neutralizing antibody is a monoclonal antibody against the F protein or peptide.

20. 17. The recombinant subunit vaccine of claim 16, wherein the neutralizing antibody is a monoclonal antibody against the F1 peptide.

21. 17. The recombinant subunit vaccine of claim 16, wherein the neutralizing antibody is a monoclonal antibody against the F2 peptide.

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