RSV vaccine composition, method, and use thereof

Abstract

An immunogenic composition comprising a recombinant peptide and a protein, wherein the recombinant peptide and protein comprises a respiratory syncytial virus (RSV) viral antigen and an immunogen, such as an RSV F protein peptide. The immunogenic composition comprises a secreted fusion protein comprising a soluble RSV viral antigen, wherein the soluble RSV viral antigen is linked to the C-terminal portion of collagen via in-frame fusion to form a trimer fusion protein linked by a disulfide bond. The immunogenic composition can be used to induce an immune response, for example, to treat or prevent RSV infection. The immunogenic composition can be used in a vaccine composition, for example, as part of a prophylactic and / or therapeutic vaccine. Methods for producing the recombinant peptide and protein, methods for prevention, treatment and / or diagnosis, and related kits are further provided.

Description

[Technical Field]

[0001] In some aspects, this disclosure relates to immunogenic compositions comprising recombinant peptides and proteins, wherein the recombinant peptides and proteins comprise a viral antigen and immunogen of respiratory syncytial virus (RSV), such as RSV F protein peptide, and the immunogenic compositions are intended for the treatment and / or prevention of RSV infection. [Background technology]

[0002] Respiratory syncytial virus (RSV) causes respiratory infections in adults and children, and is a leading cause of lower respiratory tract infections and hospitalizations in infancy and childhood. Despite the high incidence of RSV infection, treatment options such as prophylactic agents, therapeutic agents, and vaccines are limited or unavailable. Improved methods for treating RSV are needed. This specification provides compositions, methods, uses, and products that meet these and other needs. [Overview of the project]

[0003] In one embodiment, this specification provides 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 the C-terminal propeptide of collagen, wherein the C-terminal propeptide of the recombinant polypeptide forms an interpolypeptide disulfide bond. In some embodiments, the RSV belongs to subtype A or subtype B. In some embodiments, the epitope is a linear epitope or a conformational epitope.

[0004] In some embodiments, this specification discloses recombinant subunit vaccines comprising an extracellular domain of the RSV F protein or a fragment thereof (e.g., lacking a transmembrane and cytoplasmic domain), wherein the extracellular domain is in-frame fused with a collagen C propeptide capable of forming a homotrimer linked by a disulfide bond. The produced recombinant subunit vaccine, e.g., the F trimer, can be expressed from transfected cells and purified, and is expected to have a native-like conformation in trimer form. This solves the misfolding problem that often occurs when viral antigens are expressed as recombinant peptides or proteins in a soluble form lacking a transmembrane and / or cytoplasmic domain. Such misfolded viral antigens often fail to accurately retain the conformation of the native viral antigen and fail to induce neutralizing antibodies.

[0005] 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 hepta-repeat sequence C (HRC) peptide, a pep27 peptide, a fusion peptide (FP), a hepta-repeat sequence A (HRA) peptide, a domain I peptide, a domain II peptide, or a hepta-repeat sequence B (HRB) peptide, or any combination thereof. In some embodiments, the F protein peptide comprises the F1 subunit of the F protein but not the F2 subunit, or vice versa. In some embodiments, the F protein peptide comprises the F1 and F2 subunits of the F protein, optionally lacking pep27, and optionally linking the F1 and F2 subunits via a disulfide bond or an artificially introduced linker. In some embodiments, the F protein peptide does not contain a transmembrane (TM) domain peptide and / or a cytoplasmic (CP) domain peptide. In some embodiments, the F protein peptide includes a protease cleavage site, where the protease is optionally furin protease, trypsin, factor Xa, thrombin, or cathepsin L. In some embodiments, the F protein peptide does not include a protease cleavage site, where the protease is optionally furin protease, trypsin, factor Xa, thrombin, or cathepsin L.

[0006] In some embodiments, the F protein peptide is soluble or does not directly bind to a lipid bilayer, such as a membrane or viral envelope. In some embodiments, the F protein peptide is the same as or different from the recombinant polypeptide of the protein. In some embodiments, the F protein peptide is directly fused to the C-terminal propeptide or linked to the C-terminal propeptide via a linker, such as a linker comprising a glycine-XY repeat sequence, where X and Y are independently any amino acids, and optionally proline or hydroxyproline.

[0007] In some embodiments, the protein is soluble or does not directly bind to a lipid bilayer, such as a membrane or viral envelope. In some embodiments, the protein can form a rosette-like oligomer containing an F protein peptide trimer. In some embodiments, the protein can bind to cell surface adhesion factors or receptors of a subject, and optionally, the subject is a mammal, such as a primate, such as a human.

[0008] In some embodiments, the C-terminal propeptide belongs to human collagen. In some embodiments, the C-terminal propeptide comprises a C-terminal polypeptide or fragment thereof of pro α1(I), pro α1(II), pro α1(III), pro α1(V), pro α1(XI), pro α2(I), pro α2(V), pro α2(XI), or pro α3(XI). In some embodiments, the C-terminal propeptide is the same or different among the recombinant polypeptides. In some embodiments, the C-terminal propeptide comprises an amino acid sequence that can form an interpolypeptide disulfide bond and trimerize the recombinant polypeptide, having at least 90% identity with any one of SEQ ID NO: 48-63.

[0009] In some embodiments, the F protein peptide in each recombinant polypeptide exhibits either a pre-fusion conformation or a post-fusion conformation, and optionally, the protein comprises a rosette-like oligomer, the rosette-like oligomer comprising a cane-shaped rod-like F protein peptide trimer. In any one of the above embodiments, the F protein peptide in each recombinant polypeptide may comprise any one of SEQ ID NO: 17-47 or an amino acid sequence having at least 80% identity thereto.

[0010] In any one of the above embodiments, the recombinant polypeptide may include one of SEQ ID NO: 1-16 and 64-71 or an amino acid sequence having at least 80% identity thereto. In any one of the above embodiments, the recombinant polypeptide may include one of SEQ ID NO: 17-47 and 72-79 or an amino acid sequence having at least 80% identity thereto, which is directly or indirectly linked to one of SEQ ID NO: 48-63 or an amino acid sequence having at least 90% identity thereto.

[0011] This specification further provides immunogens containing proteins according to this specification. This specification provides protein nanoparticles containing proteins according to this specification that are directly or indirectly linked to nanoparticles. This specification provides virus-like particles (VLPs) containing proteins according to this specification.

[0012] This specification also provides isolated nucleic acids, which encode one, two, three or more recombinant polypeptides of proteins according to this specification. In some embodiments, a polypeptide encoding an F protein peptide is fused in frame with a polypeptide encoding a C-terminal propeptide of collagen. In some embodiments, the isolated nucleic acids according to this specification are operably linked to a promoter.

[0013] In some embodiments, the nucleic acid isolated according to this specification is a DNA molecule. In some embodiments, the nucleic acid isolated according to this specification is an RNA molecule, and selectively, an mRNA molecule, such as nucleoside-modified mRNA, unamplified mRNA, self-amplified mRNA, or trans-amplified mRNA.

[0014] This specification also provides vectors comprising nucleic acids isolated according to this specification. In some embodiments, the vector is a viral vector.

[0015] In some embodiments, this specification provides viruses, pseudoviruses, or cells comprising vectors according to this specification, wherein the viruses or cells selectively have a recombinant genome. In some embodiments, this specification provides immunogenic compositions comprising proteins, immunogens, protein nanoparticles, VLPs, isolated nucleic acids, vectors, viruses, pseudoviruses, or cells according to this specification, and pharmaceutically acceptable carrier agents.

[0016] This specification also provides vaccines comprising immunogenic compositions and optional adjuvants according to this specification, wherein the vaccine is optionally a subunit vaccine. In some embodiments, the vaccine is a prophylactic and / or therapeutic vaccine.

[0017] In some embodiments, this specification provides a method for producing a protein, the method comprising expressing an isolated nucleic acid or vector according to this specification in a host cell to produce a protein according to this specification, and purifying the protein. This specification provides proteins produced by the methods according to this specification.

[0018] This specification provides a method for inducing an immune response in a subject to the F protein peptide or fragment or epitope thereof of RSV, the method comprising administering an effective amount of the specified protein, immunogen, protein nanoparticles, VLP, isolated nucleic acid, vector, virus, pseudovirus, cell, immunogenic composition, or vaccine to the subject to induce the immune response. In some embodiments, the method according to this specification is for treating or preventing RSV infection. In some embodiments, the immune response induces suppression or reduction of 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 against the F protein peptide or fragment or epitope thereof of RSV, but not against the C-terminal propeptide. In some embodiments, administration to the subject does not result in antibody-dependent enhancement (ADE) due to the subject's prior exposure to one or more RSVs. In some embodiments, the administration does not result in antibody-dependent enhancement (ADE) when the subject is subsequently exposed to one or more RSVs. In some embodiments, the method further includes a priming step and / or a boosting step. In some embodiments, the administration step is carried out by topical, transdermal, subcutaneous, intradermal, oral, intranasal (e.g., intranasal spray), intratracheal, sublingual, buccal, rectal, vaginal, inhalation, intravenous (e.g., intravenous injection), intra-arterial, intramuscular (e.g., intramuscular injection), intracardiac, intraosseous, intraperitoneal, transmucosal, intravitreous, subretinal, intra-articular, peri-articular, topical, or superficial administration. In some embodiments, the effective dose is administered as a single dose or in a schedule of multiple doses with multiple intervals. In some embodiments, the effective dose is administered without an adjuvant. In some embodiments, the effective dose is administered with an adjuvant.

[0019] This specification provides a method comprising administering an effective amount of the protein specified herein to a subject to produce a neutralizing antibody or neutralizing antiserum against RSV in the subject. In some embodiments, the subject is a mammal, and optionally, a human or a non-human primate. In some embodiments, the method further comprises isolating the neutralizing antibody or neutralizing antiserum from the subject. In some embodiments, the method comprises administering an effective amount of the isolated neutralizing antibody or neutralizing antiserum to a human subject by passive immunization to prevent or treat RSV infection. In some embodiments, the neutralizing antibody or neutralizing antiserum against RSV comprises a polyclonal antibody against the RSV F protein peptide or a fragment or epitope thereof, and optionally, the neutralizing antibody or neutralizing antiserum does not contain or substantially contains an antibody against the collagen C-terminal propeptide. In some embodiments, the neutralizing antibody comprises a monoclonal antibody against the RSV F protein peptide or a fragment or epitope thereof, and optionally, the neutralizing antibody does not contain or substantially contains an antibody against the collagen C-terminal propeptide.

[0020] In some embodiments, the proteins, immunogens, protein nanoparticles, VLPs, isolated nucleic acids, vectors, viruses, pseudoviruses, cells, immunogenic compositions, or vaccines described herein are intended to induce an immune response to RSV in a subject and / or to treat or prevent RSV infection.

[0021] In some embodiments, the present specification provides the use of a protein, immunogen, protein nanoparticle, VLP, isolated nucleic acid, vector, virus, pseudovirus, cell, immunogenic composition or vaccine according to the present specification for inducing an immune response against RSV in a subject and / or for treating or preventing RSV infection. In some embodiments, the present specification provides the use of a protein, immunogen, protein nanoparticle, VLP, isolated nucleic acid, vector, virus, pseudovirus, cell, immunogenic composition or vaccine according to the present specification, which is for manufacturing a drug or prophylactic agent, and the drug or prophylactic agent is for inducing an immune response against RSV virus in a subject and / or for treating or preventing RSV infection.

[0022] The present specification further provides a method for analyzing a sample, the method including contacting the sample with a protein according to the present specification and detecting the binding of the protein to an analyte that can specifically bind to the F protein peptide of RSV 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, the binding indicates the presence of the analyte in the sample and / or RSV infection in the subject from whom the sample is derived.

[0023] The present specification provides a kit, the kit including a protein according to the present specification and a substrate, pad or vial for containing or immobilizing the protein, and optionally, the kit is an ELISA or lateral flow assay kit. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] [Figure 1] Schematic diagram showing an exemplary fusion peptide containing an extracellular F domain fused to a trimerization peptide. [Figure 2]This figure shows the OD values ​​in the supernatant of 293T cells on day 3. On day 3, the amounts of total protein and pre-fusion F protein in the supernatant were measured by ELISA. [Figure 3] This figure shows the OD values ​​in the CHO cell supernatant on day 3. On day 3, the amounts of total protein and pre-fusion F protein in the supernatant were measured by ELISA. [Figure 4] This figure shows the OD values ​​in the CHO cell supernatant on day 7. On day 7, the amounts of total protein and pre-fusion F protein in the supernatant were measured by ELISA. [Figure 5] This figure shows the expression levels of peptides analyzed by 8% SDS-PAGE using exemplary fusion peptide expression from serum-free fed-batch cell cultures. SCB-N25C: Cell-free culture media from day 1 to day 13 were separated under non-reducing and reducing conditions, and stained with Coomassie brilliant blue, with a sample volume of 26 μl. SCB-N25: Cell-free culture media from day 1 to day 13 were separated under non-reducing and reducing conditions, and stained with Coomassie brilliant blue, with a sample volume of 26 μl. [Figure 6] This figure shows the purity evaluation of exemplary purified fusion peptides by SEC-HPLC, with the main peak area of ​​the exemplary SCB-N25 fusion protein being 92.8% and the main peak area of ​​the SCB-N25C protein being 81.7%. [Figure 7]Figures illustrating affinity kinetic studies. Figures A and B show biolayer interferometry studies of binding of palivizumab to an exemplary fusion peptide containing RSV F protein peptide. First, 5 μg / mL of palivizumab was immobilized on a protein A sensor, and then the sensor was immersed in exemplary fusion peptides of different concentrations to measure binding kinetics. By subtracting a buffer reference value, the resulting curves were fitted to a 1:1 binding model to obtain K association and K dissociation, the values ​​of which are shown in the table below. Figures C and D show binding studies between antibody D25 and an exemplary fusion peptide containing RSV F protein peptide. Corresponding KD, Kon, and Kdis from antibody D25 binding experiments are indicated by the labels below. SCB-N25C showed high affinity for both palivizumab and antibody D25. [Figure 8]This figure shows the results of immunization tests using an exemplary fusion peptide containing the RSV F protein peptide. Figure 8A is a schematic diagram of the experimental method, in which mice were immunized on day 0 and day 21, and serum was collected on day 0 and day 35, respectively. Figure 8B shows the (D35)-RSV A2 strain microneutralizing antibody titers in serological experiments against the purified exemplary fusion peptide, which contains the RSV F protein peptide and the adjuvant Alum, Alum+CpG 1018, or CAS-1. The fusion protein with Alum+CpG 1018 as an adjuvant showed higher potency than the corresponding fusion proteins with Alum or CAS-1 as adjuvants. SCB-N25C showed higher potency than the other antigens. Figure 8C shows the competitive IgG titers of D25 and palivizumab, which provide 50% inhibition of binding of D25 and palivizumab to heat-inactivated RSV (HI-RSV) particles, as measured by serum sample dilutions. Values ​​are expressed as log2 and mean ± SEM. All antigens that bound to Alum showed lower antibody (DCA: D25 competitive antibody and PCA: palivizumab competitive antibody) responses than the other two adjuvants. When used in combination with Alum + CpG1018 adjuvant, both DS-CAV1-Trimer and N20 showed high DCA efficacy and PCA titer, with no significant difference between them. N25 showed the lowest. None of the four candidate antigens used in combination with CAS-1 adjuvant showed effective DCA titer. N20 and N25 showed high PCA titer, while DS-CAV1-Trimer showed the lowest. Figure 8D shows the results of the Elispot trial. The N25C antigen, when used in combination with the Alum adjuvant, can induce a strong Th1 cell immune response. The antigens, when used in combination with the Alum+CpG1018 adjuvant, all induced a low Th2 cell immune response. The antigens, when used in combination with the CAS-1 adjuvant, all induced high Th1 and Th2 cell immune responses. CAS-1 contains squalene, α-tocopherol, and Zween-80. [Modes for carrying out the invention]

[0025] In some embodiments, compositions and methods of use of recombinant soluble surface antigens derived from RNA viruses that exhibit a covalently linked trimer morphology have been disclosed. In some embodiments, the produced fusion protein is secreted as a homotrimer linked by disulfide bonds, which is structurally more stable while retaining the conformation of native-like trimer viral antigens, and can therefore be used as a more effective vaccine against these dangerous pathogens.

[0026] In some embodiments, this specification discloses methods for preventing viral infection using a viral antigen trimer as a vaccine or as part of a polyvalent vaccine, the methods of which use or do not use adjuvants, or use one or more adjuvants, and which are administered optionally by intramuscular injection or nasal administration.

[0027] In some embodiments, this specification discloses a method for diagnosing viral infection by detecting an antibody that recognizes a viral antigen, such as an IgM or IgG neutralizing antibody, using a viral antigen trimer as the antigen.

[0028] In some embodiments, this specification discloses methods for producing polyclonal or monoclonal antibodies, such as neutralizing mAbs for treating RSV infection in infants, that can be used for passive immunization, using viral antigen trimers as antigens.

[0029] In some embodiments, the Specified herein discloses viral antigen trimers as a vaccine or as part of a polyvalent vaccine, wherein the vaccine comprises a plurality of trimer subunit vaccines, the plurality of trimer subunit vaccines comprising viral antigens of the same viral protein or comprising viral antigens of one or more viruses or two or more different proteins of one or more strains of the same virus.

[0030] In some embodiments, this specification has disclosed a monovalent vaccine comprising the viral antigen trimer disclosed herein. In some embodiments, this specification has disclosed a bivalent vaccine comprising the viral antigen trimer disclosed herein. In some embodiments, this specification has disclosed a trivalent vaccine comprising the viral antigen trimer disclosed herein. In some embodiments, this specification has disclosed a tetravalent vaccine comprising the viral antigen trimer disclosed herein.

[0031] In some embodiments, this specification has disclosed a monovalent vaccine comprising the F trimer disclosed herein. In some embodiments, this specification has disclosed a bivalent vaccine comprising the F trimer disclosed herein. In some embodiments, this specification has disclosed a bivalent vaccine 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 virus species or strains / subtypes, or from two or more different F proteins of one or more virus species or one or more strains / subtypes of the same virus species. In some embodiments, this specification has disclosed a trivalent vaccine comprising the F trimer disclosed herein. In some embodiments, this specification has disclosed a trivalent vaccine 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 virus species or strains / subtypes, or from two, three, or more different F proteins of one or more virus species or strains / subtypes of the same virus species. In some embodiments, the Specified herein includes a quadrivalent vaccine comprising the F trimers disclosed herein. In some embodiments, the Specified herein has disclosed a quadrivalent vaccine 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 virus species or strains / subtypes, or from two, three, four, or more different F proteins of one or more virus species or strains / subtypes of the same virus species.

[0032] This specification provides immunogenic compositions, methods, and uses, for example, for the prophylactic and therapeutic treatment of RSV infection, comprising fusion peptides and proteins of RSV virus antigen or immunogen. Respiratory syncytial virus (RSV) is considered the leading cause of acute lower respiratory tract infections (ALRTIs) in infants and young children, causing 7,000 to 20,000 child deaths worldwide annually. RSV infection is the second leading cause of infant mortality in developing countries. Furthermore, RSV can cause serious illness in the elderly and immunocompromised individuals. Effective prophylactic humanized mAb palivizumab (SYNAGIS) (登録商標) ) can only be used as a passive immunization measure for infants at high risk of RSV infection.

[0033] Despite decades of research, the development of an RSV vaccine has not been successful for various reasons. For example, problems with the production, stability, and efficacy of RSV candidate vaccines are difficult to overcome, and safety has become a major concern, especially since formalin-inactivated RSV (FI-RSV) vaccines have been recognized as mediating vaccine-induced disease enhancement (VED).

[0034] The proteins comprising RSV virus antigen and immunogen according to this specification, including recombinant polypeptides and fusion proteins, can be used to effectively and safely treat RSV infection (e.g., therapeutically and prophylactically). For example, the proteins comprising RSV virus antigen and immunogen according to this specification can treat RSV infection without considering VED and / or antibody-dependent enhancement (ADE). Furthermore, the proteins comprising RSV virus antigen and immunogen according to this specification are easily produced and exhibit stability under high-stress conditions, such as high temperature, extreme pH, and high and low osmotic pressure. Therefore, the proteins and immunogenic compositions according to this specification avoid and satisfy the production, stability, safety, and efficacy problems that have hindered the development of RSV vaccines.

[0035] In some embodiments, the RSV viral antigens and immunogens according to this specification include RSV glycoprotein (F), which is also referred herein to as RSV F protein peptide or peptide. RSV F protein peptide is a homotrimeric type I transmembrane protein that mediates the entry of the membrane and the virus into host cells. RSV F protein peptide is synthesized as an F0 proprotein precursor and converted to the mature forms F1 and F2, which are cleaved by furin proteases at two sites and then linked via disulfide bonds. 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.

[0036] In some embodiments, a protein containing the RSV virus antigen or immunogen, such as the RSV F protein peptide, can induce an immune response, such as an immune response to the RSV F peptide protein. In some embodiments, the immune response suppresses or reduces the replication of RSV in a subject, such as 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 suppress or reduce the replication of RSV in a subject, such as a patient. In some embodiments, administration of the protein to a subject (e.g., in the form of an immunogenic composition) does not result in antibody-dependent enhancement (ADE) due to the subject's prior exposure to RSV. In some embodiments, a protein containing the RSV virus antigen and immunogen, such as the RSV F protein peptide, is used as a vaccine.

[0037] In some embodiments, the RSV virus antigen and an immunogen, such as the 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 virus antigen or immunogen is linked can associate with the protein or peptide of the fusion protein or recombinant polypeptide, for example, and can be linked covalently or noncovalently. Therefore, in some cases, the protein or peptide to which the RSV virus antigen or immunogen is linked is a polymerizing domain.

[0038] In some embodiments, the RSV virus antigen and an immunogen, such as the RSV F protein peptide, are linked to the C-terminus of a collagen propeptide, such as the collagen propeptide, to form a fusion peptide or recombinant polypeptide. Thus, in some embodiments, the protein according to this specification comprises a recombinant polypeptide, the recombinant polypeptide containing the RSV virus antigen and immunogen, such as the RSV F protein peptide or a fragment or epitope thereof, linked to the C-terminal propeptide of collagen. In some embodiments, the collagen propeptide is derived from human α1-collagen C propeptide and can self-trimerize.

[0039] In some embodiments, linking the RSV viral antigen and immunogen, such as RSV F protein peptide, to the C-terminus of a collagen propeptide, such as collagen propeptide, contributes to the protein's ability to generate an immune response. For example, by producing recombinant proteins, the tertiary and quaternary structures of the RSV F protein peptide can be preserved, which may be important for the stability of the native conformation of the RSV F protein peptide and, consequently, for the accessibility of the antigen site on the surface of proteins (e.g., neutralizing antibodies) that can induce an immune response. Furthermore, linking the RSV F protein peptide to a self-trimerizable protein or peptide enables aggregation of recombinant proteins, thereby mimicking the native homotrimeric structure of the RSV F protein peptide on the viral envelope.

[0040] In some embodiments, a self-trimerizing recombinant polypeptide is obtained by linking an RSV F protein peptide to the C-terminal propeptide of collagen. In some embodiments, the protein according to this specification comprises a plurality of self-trimerizing RSV F protein peptides and a propeptide of a collagen recombinant polypeptide, and optionally, the plurality of recombinant proteins form a structure, for example, a rosette (see, for example, Figure 2B PCT / CN202l / 099286, the PCT patent application which is incorporated herein by reference in whole for all purposes). In some embodiments, the trimer properties of the recombinant protein contribute to the stability of the protein. In some embodiments, the macrostructure (e.g., rosette) of the plurality of self-trimerizing recombinant proteins contributes to the stability of the protein. In some embodiments, the trimer properties of the recombinant protein and the macrostructure (e.g., rosette) of the plurality of self-trimerizing recombinant proteins contribute to the stability of the protein. In some embodiments, the trimer properties of the recombinant protein contribute to the ability of the protein to elicit an immune response. In some embodiments, the macrostructure (e.g., rosette-like) of multiple self-trimerized recombinant proteins contributes to the protein's ability to generate an immune response. In some embodiments, the trimer properties of recombinant proteins and the macrostructure of multiple self-trimerized recombinant proteins contribute to the protein's ability to generate an immune response.

[0041] This specification further provides immunogenic compositions containing the protein specified herein, methods for producing the protein specified herein, methods for treating subjects with the protein and composition specified herein, and kits.

[0042] All publications, including patent documents, scientific papers, and databases, referenced in this application are incorporated by reference as a whole for any purpose, to the same extent that each individual publication is incorporated by reference. If any definition contained herein contradicts or is inconsistent with any definition contained herein in a patent, application, published application, or other publication incorporated herein by reference, the definition contained herein shall prevail over the definition incorporated herein by reference.

[0043] The section headings used in this specification are for organizational purposes only and should not be construed as limiting the subject matter.

[0044] I. Viral Antigens and Immunogens Respiratory syncytial virus (RSV) is the most common cause of acute lower respiratory tract infections in infants and young children, and a major disease burden in the elderly. Despite the RSV virus being characterized more than half a century ago, vaccines against RSV remain limited, and vaccine-mediated disease enhancement in children administered formalin-inactivated RSV in the 1960s has hindered development efforts. Challenges related to antigen production, purity, stability, and efficacy of RSV candidate vaccines have also been obstacles to development.

[0045] In some embodiments, the proteins according to this specification include RSV virus antigens and / or immunogens. In some embodiments, RSV virus antigens and / or immunogens can promote or stimulate cell-mediated and / or humoral responses. In some embodiments, the response (e.g., cell-mediated or humoral response) includes the production of antibodies, such as neutralizing antibodies. In some embodiments, neutralizing antibodies (NAbs) against the viral antigens and / or immunogens provided adaptive immune protection against RSV exposure by inhibiting infection of susceptible cells. In some embodiments, the effectiveness of a vaccine against multiple viruses is due to and / or related to its NAb-inducing ability. In some embodiments, the RSV virus antigen or immunogen is an RSV F protein peptide disclosed herein.

[0046] The RSV F protein peptide is the envelope glycoprotein of respiratory syncytial virus (RSV). The RSV F protein peptide is translated as a single precursor polypeptide (designated F0). The RSV F protein mediates viral entry into cells and intercellular fusion, is a target of neutralizing antibodies, and is highly conserved between RSV A and B strains. F0 is cleaved into three fragments by cellular furin proteases at Arg109 and Arg136. The short F2 polypeptide is covalently linked to the long F1 polypeptide via two disulfide bonds at its N-terminus, the latter having an 18-amino acid fusion domain at the N-terminus and a hydrophobic transmembrane region near the C-terminus, releasing an intermediate 27-amino acid fragment. The neutralizing monoclonal antibodies palivizumab and motavizumab have been shown to bind to the RSV F antigen site II (Asn258-Val278) and provide protection against RSV disease of the lower and upper respiratory tracts in high-risk infants and full-term infants. The structure of the RSV F epitope polypeptide that binds to these neutralizing antibodies is larger than that of the linear peptide; palivizumab binds to RSV F with nanomolar affinity, while motavizumab binds to RSV F with picomolar affinity. Modeling predicts that complete binding of palivizumab and motavizumab requires one or two amino acids from the RSV F protomer, respectively. Therefore, preserving the tertiary and quaternary structures of RSV F may be crucial in the development of RSV F vaccines that retain the native conformation of this important neutralizing region.

[0047] In some embodiments, the F0 precursor polypeptide has a length of 574 amino acids, as shown in SEQ ID NO:31. [ka]

[0048] In some embodiments, the F0 precursor polypeptide has a length of 574 amino acids, as shown in SEQ ID NO:32.

change

[0049] In some embodiments, the RSV F protein peptides herein include proline or alanine at residue 102. In some embodiments, the RSV F protein peptides herein include substitutions, deletions, and / or insertions at and / or near residue 102 in SEQ ID NO: 31 or 32. In some embodiments, the RSV F protein peptides herein include a substitution of A from residue 102 in SEQ ID NO: 31. In some embodiments, the RSV F protein peptides herein include glutamic acid or alanine at residue 218. In some embodiments, the RSV F protein peptides herein include substitutions, deletions, and / or insertions at and / or near residue 218 in SEQ ID NO: 31 or 32. In some embodiments, the RSV F protein peptides herein include a substitution of glutamic acid from alanine at residue 218 in SEQ ID NO: 32. In some embodiments, the RSV F protein peptides herein include a substitution of T from I at residue 523 in SEQ ID NO: 32. In some embodiments, the RSV F protein peptides herein include valine or isoleucine at residue 379. In some embodiments, the RSV F protein peptides herein include substitutions, deletions, and / or insertions at and / or near residue 379 of SEQ ID NO: 31 or 32. In some embodiments, the RSV F protein peptides herein include valine or methionine at residue 447. In some embodiments, the RSV F protein peptides herein include substitutions, deletions, and / or insertions at and / or near residue 447 of SEQ ID NO: 31 or 32. In some embodiments, the RSV F protein peptides herein include substitutions, deletions, and / or insertions at and / or near one or more of the following: 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 peptides herein include substitutions, deletions, and / or insertions in and / or near one or more residues 102, 218, 379, and 447 of SEQ ID NO: 31 or 32. In some embodiments, the RSV F protein peptides herein include substitutions, deletions, and / or insertions in and / or near one or more other residues of SEQ ID NO: 31 or 32.

[0050] In some embodiments, the RSV F protein peptides 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 herein include glutamine or asparagine at residues 108 and / or 109. In some embodiments, the RSV F protein peptides herein include glutamine at residues 108 and 109. In some embodiments, the RSV F protein peptides herein include asparagine at residues 108 and 109. In some embodiments, the RSV F protein peptides 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 peptides herein contain 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 peptides herein contain 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 peptides herein contain glutamine at residues 133, 135, and 136 of SEQ ID NO: 31 or 32.

[0051] In some embodiments, the RSV F protein peptides herein include a substitution of alanine to glutamic acid at residue 218 of SEQ ID NO:32, and substitutions at one or more of residues 106, 107, 108, 109, 133, 135, and 136, wherein the substituted amino acids are freely selected from cysteine, alanine, threonine, tyrosine, glycine, or serine or any combination thereof.

[0052] In some embodiments, the RSV F protein peptides herein include a substitution of alanine to glutamic acid at residue 218 of SEQ ID NO:32, as well as substitutions in one or more of residues 106, 107, 108, 109, 133, 135, and 136. In some embodiments, the RSV F protein peptides herein include a substitution of alanine to glutamic acid at residue 218 of SEQ ID NO:32, as well as substitutions in one or more of residues 106, 108, 109, 133, 135, and 136. In some embodiments, the RSV F protein peptides herein include a substitution of alanine to glutamic acid at residue 218 of SEQ ID NO:32, as well as substitutions in one or more of residues 106, 108, 109, 133, 135, and 136. In some embodiments, the RSV F protein peptides herein include a substitution of alanine to glutamic acid at residue 218 of SEQ ID NO:32, as well as substitutions of cysteine ​​at all of residues 106, 108, 109, 133, 135, and 136. The RSV F protein peptides herein include a substitution of alanine to glutamic acid at residue 218 of SEQ ID NO:32, as well as substitutions of cysteine ​​at all of residues 108, 109, 133, 135, and 136. The RSV F protein peptides herein include a substitution of alanine to glutamic acid at residue 218 of SEQ ID NO:32, as well as substitutions of cysteine ​​at all of residues 109, 133, 135, and 136. The RSV F protein peptide described herein includes a substitution of alanine to glutamic acid at residue 218 of SEQ ID NO:32, as well as substitution of all residues 133, 135, and 136 with cysteine. The RSV F protein peptide described herein includes a substitution of alanine to glutamic acid at residue 218 of SEQ ID NO:32, as well as substitution of all residues 135 and 136 with cysteine.The RSV F protein peptide described herein includes a substitution of alanine to glutamic acid at residue 218 of SEQ ID NO:32, as well as a substitution of cysteine ​​at residue 136. The RSV F protein peptide described herein includes a substitution of alanine to glutamic acid at residue 218 of SEQ ID NO:32, as well as a substitution of cysteine ​​at residue 135. The RSV F protein peptide described herein includes a substitution of alanine to glutamic acid at residue 218 of SEQ ID NO:32, as well as substitution of cysteine ​​at all of residues 106, 133, 135, and 136. The RSV F protein peptide described herein includes a substitution of alanine to glutamic acid at residue 218 of SEQ ID NO:32, as well as substitution of cysteine ​​at all of residues 106, 135, and 136. The RSV F protein peptide described herein includes a substitution of alanine to glutamic acid at residue 218 of SEQ ID NO:32, as well as substitution of all residues 106, 133, and 136 with cysteine. The RSV F protein peptide described herein includes a substitution of alanine to glutamic acid at residue 218 of SEQ ID NO:32, as well as substitution of all residues 106, 133, and 135 with cysteine. The RSV F protein peptide described herein includes a substitution of alanine to glutamic acid at residue 218 of SEQ ID NO:32, as well as substitution of all residues 106 and 136 with cysteine. The RSV F protein peptide described herein includes a substitution of alanine to glutamic acid at residue 218 of SEQ ID NO:32, as well as substitution of all residues 106 and 135 with cysteine. The RSV F protein peptide described herein includes a substitution of alanine to glutamic acid at residue 218 of SEQ ID NO:32, as well as substitution of all residues 108 and 136 with cysteine. The RSV F protein peptide described herein includes a substitution of alanine to glutamic acid at residue 218 of SEQ ID NO:32, as well as substitution of all residues 108 and 135 with cysteine.The RSV F protein peptide described herein includes a substitution of alanine to glutamic acid at residue 218 of SEQ ID NO:32, as well as substitution of all residues 106 and 133 with cysteine. The RSV F protein peptide described herein includes a substitution of alanine to glutamic acid at residue 218 of SEQ ID NO:32, as well as substitution of all residues 106 and 133 with cysteine. The RSV F protein peptide described herein includes a substitution of alanine to glutamic acid at residue 218 of SEQ ID NO:32, as well as substitution of all residues 108 and 133 with cysteine. The RSV F protein peptide described herein includes a substitution of alanine to glutamic acid at residue 218 of SEQ ID NO:32, as well as substitution of all residues 108 and 133 with cysteine.

[0053] In some embodiments, the RSV F protein peptides 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 herein include alanine or proline at 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 herein include alanine at residue 109 of SEQ ID NO: 31 or 32. In some embodiments, the RSV F protein peptides herein include alanine at residue 136 of SEQ ID NO: 31 or 32. In some embodiments, the RSV F protein peptides herein include alanine at residues 109 and 136 of SEQ ID NO: 31 or 32. In some embodiments, the RSV F protein peptides herein include proline at residue 161 of SEQ ID NO: 31 or 32. In some embodiments, the RSV F protein peptide according to this specification contains proline at residue 215 of SEQ ID NO: 31 or 32. In some embodiments, the RSV F protein peptide according to this specification contains proline at residues 161 and 215 of SEQ ID NO: 31 or 32. In some embodiments, the RSV F protein peptide according to this specification contains alanine at residues 109 and 136 and proline at residues 161 and 215 of SEQ ID NO: 31 or 32.

[0054] In some embodiments, the RSV F protein peptides herein include substitutions, deletions, and / or insertions in and / or near one or more residues 131-154 of SEQ ID NO:31 or 32. In some embodiments, the RSV F protein peptides herein include two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, or more deletions from residues 131-154 of SEQ ID NO:31 or 32. In some embodiments, the RSV F protein peptides herein include deletions in one or more residues 137-154 of SEQ ID NO:31 or 32. In some embodiments, the RSV F protein peptides herein include deletions in one or more residues 137-146 of SEQ ID NO:31 or 32. In some embodiments, the RSV F protein peptides described herein include glutamine at residues 133, 135, and 136 of SEQ ID NO: 31 or 32 and deletions at residues 137-146.

[0055] In some embodiments, the RSV F protein peptide according to this specification comprises amino acids 1-25 of the F0 precursor, which is the signal peptide MELLILKANAITTILTAVTFCFASG (SEQ ID NO: 33). In some embodiments, the precursor polypeptide F0 formed a precursor trimer. In some embodiments, the RSV F protein peptide according to this specification is hydrolytically cleaved by one or more cellular proteases, for example, at a conservative furin protease consensus cleavage site, thereby producing the Pep 27 polypeptide (also called p27), the F1 polypeptide, and the F2 polypeptide. In some embodiments, the Pep 27 polypeptide (e.g., amino acids 110-136 of the F0 precursor) is removed and, in some embodiments, does not become 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 F0 precursor. In some embodiments, the F1 polypeptide (hereafter referred to as "F1" or "F1 subunit peptide") comprises amino acid residues 137–574 of the F0 precursor and may also include an extracellular domain (e.g., residues 137–524), a transmembrane domain (e.g., residues 525–550), and a cytoplasmic domain (e.g., residues 551–574).

[0056] In some embodiments, the RSV F protein peptides herein comprise F1 and F2 polypeptides linked via a disulfide bond to form a heterodimer, referred to as the RSV F "protomer." In some embodiments, the RSV F protein peptides herein comprise three protomers that form an RSV F trimer, and are therefore homotrimers of the three protomers. In some embodiments, the RSV F protein peptides herein are mature RSV F trimers. In some embodiments, the RSV F protein peptides herein are membrane-bound. In some embodiments, the RSV F protein peptides herein are not membrane-bound. In some embodiments, the RSV F protein peptides herein are soluble and lack a transmembrane region and a cytoplasmic region or fragments thereof. For example, conversion to a soluble form can be achieved by shortening the RSV F protein at amino acids 513 (by removing amino acids from 514 onwards), 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 to the cell membrane. 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 docks with the cell membrane, the RSV F protein trimer undergoes a series of conformational changes and transitions to a highly stable post-fusion ("post-F") conformation.

[0057] In some embodiments, the RSV virus antigen or immunogen is a signal peptide (SP) (e.g., amino acids 1-22 of SEQ ID NO: 31 or 32) or a fragment thereof and / or mutant sequence, a heptar repeat C (HRC) (e.g., F2, which may be amino acids 23-109 of SEQ ID NO: 31 or 32) or a fragment thereof and / or mutant sequence, a furin protease cleavage site (FCS) (e.g., at the boundary between amino acids 109 / 110 of SEQ ID NO: 31 or 32) or a fragment thereof and / or mutant sequence, a 27-mer fragment (pep27) (e.g., amino acids 110-136 of SEQ ID NO: 31 or 32) or a fragment thereof and / or mutant sequence, a putative fusion peptide (FP) (e.g., amino acids 137-155 of SEQ ID NO: 31 or 32) or a fragment thereof and / or mutant sequence, a heptar repeat A (HRA) (e.g., SEQ ID This includes any suitable combination of amino acids 156-214 of SEQ ID NO:31 or 32 or fragments thereof and / or mutant sequences, domains I and II (e.g., amino acids 215-476 of SEQ ID NO:31 or 32) or fragments thereof and / or mutant sequences, heptarette repeat B (HRB) (e.g., amino acids 477-524 of SEQ ID NO:31 or 32) or fragments thereof and / or mutant sequences, transmembrane (TM) domains (e.g., amino acids 525-550 of SEQ ID NO:31 or 32) or fragments thereof and / or mutant sequences, and / or cytoplasmic (CP) domains (e.g., amino acids 551-574 of SEQ ID NO:31 or 32) or fragments thereof and / or mutant sequences.

[0058] In some embodiments, the RSV virus antigen or immunogen is an RSV F protein peptide of the RSV A subtype. In some embodiments, the RSV virus antigen or immunogen is an RSV F protein peptide of the RSV A2 subtype. In some embodiments, the RSV virus antigen or immunogen is an RSV F protein peptide of the RSV B subtype. In some cases, the RSV F protein peptide is conserved among RSV subtypes.

[0059] In some cases, the RSV virus antigen or immunogen is a fragment of the RSV F protein peptide. In some embodiments, the RSV virus antigen or immunogen is an epitope of the 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 the RSV F protein peptide or its fragment exist as the RSV virus antigen or immunogen.

[0060] In some cases, for example, if the RSV virus antigen or immunogen is a fragment of the RSV F protein peptide, only a single subunit of the RSV F protein peptide may be present.

[0061] In some embodiments, the RSV virus antigen or immunogen is or comprises an F1 subunit peptide. In some embodiments, the F1 subunit peptide is or comprises the 137-574 amino acid sequence of the wild-type F protein. In some embodiments, the RSV virus antigen or immunogen is or comprises an F2 subunit peptide. In some embodiments, the RSV virus antigen or immunogen comprises an RSV F protein peptide containing a signal peptide, a hepta repeat C (HRC) peptide, a pep27 peptide, a fusion peptide (FP), a hepta repeat A (HRA) peptide, a domain I peptide, a domain II peptide, or a hepta repeat B (HRB) peptide, or any combination thereof. In some embodiments, the RSV virus antigen or immunogen comprises an RSV F protein peptide containing a signal peptide. In some embodiments, the RSV virus antigen or immunogen comprises an RSV F protein peptide containing a pep27 peptide. In some embodiments, the RSV virus antigen or immunogen comprises an RSV F protein peptide containing a fusion peptide (FP) (also called a fusion domain (FD)). In some embodiments, the RSV virus antigen or immunogen comprises an RSV F protein peptide containing a signal peptide, a pep27 peptide, and a fusion peptide (FP).

[0062] In some embodiments, the RSV virus antigen or immunogen comprises an RSV F protein peptide containing the F1 and F2 subunits of the F protein. In some embodiments, the RSV virus antigen or immunogen comprises an RSV F protein peptide containing the F1 and F2 subunit peptides of the F protein but not containing the pep 27 peptide. In some embodiments, the RSV virus antigen or immunogen comprises an RSV F protein peptide containing the F1 subunit peptide, F2 subunit peptide, and pep 27 peptide of the F protein. In some embodiments, the RSV virus antigen or immunogen comprises an RSV F protein peptide containing the F1 subunit peptide, F2 subunit peptide, pep 27 peptide, and FP of the F protein.

[0063] In some cases, for example, if the viral antigen or immunogen contains both the F1 and F2 subunit peptides of the RSV F protein peptide, the F1 and F2 subunits are linked. In some embodiments, the F1 and F2 subunits are linked via a disulfide bond. In some embodiments, the F1 and F2 subunits are linked via an artificially introduced linker. In some embodiments, the F1 and F2 subunits are linked via the pep27 peptide. For example, in some embodiments, the N-terminus-C-terminus orientation is or includes F2-pep27-F1. In some embodiments, the N-terminus-C-terminus 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.

[0064] In some cases, the RSV viral antigen or immunogen is an RSV F protein peptide that does not contain a transmembrane (TM) domain peptide. In some cases, the RSV F protein does not contain a cytoplasmic (CP) domain peptide. In some cases, the RSV F protein does not contain a TM domain peptide or a CP domain peptide.

[0065] In some embodiments, the RSV virus antigen or immunogen comprises an RSV F protein peptide containing a protease cleavage site. In some embodiments, the protease cleavage site is specific to the cleavage of protease furin protease. In some embodiments, the protease cleavage site is specific to the cleavage of protease trypsin. In some embodiments, the protease cleavage site is specific to the cleavage of protease factor Xa. In some embodiments, the protease cleavage site is specific to the cleavage of protease cathepsin L.

[0066] In some cases, the RSV virus antigen or immunogen contains an RSV F protein peptide that does not contain a protease cleavage site. In some cases, the RSV virus antigen or immunogen contains an RSV F protein peptide that does not contain a protease cleavage site specific to the cleavage of furin protease. In some cases, the RSV virus antigen or immunogen contains an RSV F protein peptide that does not contain a protease cleavage site specific to the cleavage of trypsin. In some cases, the RSV virus antigen or immunogen contains an RSV F protein peptide that does not contain a protease cleavage site specific to the cleavage of protease factor Xa. In some cases, the RSV virus antigen or immunogen contains an RSV F protein peptide that does not contain a protease cleavage site specific to the cleavage of cathepsin L.

[0067] In some embodiments, the RSV virus 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.

[0068] In some embodiments, the RSV F protein peptide is produced from an optimized nucleic acid sequence. In some embodiments, the RSV F protein peptide is produced from a nucleic acid sequence that is not codon-optimized.

[0069] In some embodiments, the RSV F protein peptide may include any F protein sequence known in the art, for example, the sequence disclosed in U.S. Patent No. 10,017,543, which is incorporated herein by reference in whole for all purposes.

[0070] In some embodiments, the RSV virus antigen or immunogen is or comprises an RSV F protein peptide having an amino acid sequence of 1 to 520 of SEQ ID NO: 31 or 32. In some embodiments, the RSV virus antigen or immunogen is or comprises an RSV F protein peptide having an amino acid sequence of 26 to 520 of SEQ ID NO: 31 or 32.

[0071] In some embodiments, the RSV virus antigen or immunogen is or comprises the sequence of F2, the sequence of pep27, and the sequence of F1 (e.g., F2-pep27-F1). In some embodiments, the RSV virus antigen or immunogen comprises exposure to a fusion peptide and has a post-fusion conformation. In some embodiments, the RSV virus antigen or immunogen comprises a furin protease cleavage site mutation. In some embodiments, the RSV virus antigen or immunogen comprises a furin protease site I mutation (e.g., R109A) and / or a furin protease site II mutation (e.g., R136A), and in some of these examples, the RSV virus antigen or immunogen has a post-fusion conformation, while in other examples, the RSV virus antigen or immunogen has a pre-fusion conformation. In some embodiments, the RSV virus antigen or immunogen has furin protease site I mutations and furin protease site II mutations (e.g., R109A / R136A), and in some of these examples, the RSV virus antigen or immunogen contains full-length F0, is not exposed to the fusion peptide, and has a pre-fusion conformation. In some embodiments, the RSV virus antigen or immunogen contains one or more mutations, the mutations prevent the formation of long helices and / or stabilize the α4-α5 hinge loop. In some embodiments, the RSV virus antigen or immunogen contains one or more mutations, the mutations retain the pre-fusion conformation. In some embodiments, the RSV virus antigen or immunogen contains one or more mutations, the mutations improved expression. In some embodiments, substitutions at positions 161, 182 and 215 (e.g., by proline) resulted in higher expression levels, and E161P and S215P also improved protein stability. In some embodiments, the RSV virus antigen or immunogen comprises E161P and / or S215P and has a pre-fusion conformation.In some embodiments, the RSV virus antigen or immunogen comprises R109A, R136A, E161P and / or S215P and has a pre-fusion conformation.

[0072] In some embodiments, the viral antigen or immunogen comprises the sequence shown in SEQ ID NO:17. In some embodiments, the viral antigen or immunogen comprises an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence of SEQ ID NO:17, which comprises a sequence that includes substitutions, deletions, and / or insertions at one or more amino acid positions.

[0073] In some embodiments, the viral antigen or immunogen comprises the sequence shown in SEQ ID NO:18. In some embodiments, the viral antigen or immunogen comprises an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence of SEQ ID NO:18, which comprises a sequence that includes substitutions, deletions, and / or insertions at one or more amino acid positions.

[0074] In some embodiments, the viral antigen or immunogen comprises the sequence shown in SEQ ID NO:19. In some embodiments, the viral antigen or immunogen comprises an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence of SEQ ID NO:19, which comprises a sequence that includes substitutions, deletions, and / or insertions at one or more amino acid positions.

[0075] In some embodiments, the viral antigen or immunogen comprises the sequence shown in SEQ ID NO:20. In some embodiments, the viral antigen or immunogen comprises an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence of SEQ ID NO:20, which comprises a sequence that includes substitutions, deletions, and / or insertions at one or more amino acid positions.

[0076] In some embodiments, the viral antigen or immunogen comprises the sequence shown in SEQ ID NO:21. In some embodiments, the viral antigen or immunogen comprises an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence of SEQ ID NO:21, which comprises a sequence that includes substitutions, deletions, and / or insertions at one or more amino acid positions.

[0077] In some embodiments, the viral antigen or immunogen comprises the sequence shown in SEQ ID NO:22. In some embodiments, the viral antigen or immunogen comprises an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence of SEQ ID NO:22, which comprises a sequence that includes substitutions, deletions, and / or insertions at one or more amino acid positions.

[0078] In some embodiments, the viral antigen or immunogen comprises the sequence shown in SEQ ID NO:23. In some embodiments, the viral antigen or immunogen comprises an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence of SEQ ID NO:23, which comprises a sequence that includes substitutions, deletions, and / or insertions at one or more amino acid positions.

[0079] In some embodiments, the viral antigen or immunogen comprises the sequence shown in SEQ ID NO:24. In some embodiments, the viral antigen or immunogen comprises an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence of SEQ ID NO:24, which comprises a sequence that includes substitutions, deletions, and / or insertions at one or more amino acid positions.

[0080] In some embodiments, the viral antigen or immunogen comprises the sequence shown in SEQ ID NO:25. In some embodiments, the viral antigen or immunogen comprises an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence of SEQ ID NO:25, which comprises a sequence that includes substitutions, deletions, and / or insertions at one or more amino acid positions.

[0081] In some embodiments, the viral antigen or immunogen comprises the sequence shown in SEQ ID NO:26. In some embodiments, the viral antigen or immunogen comprises an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence of SEQ ID NO:26, which comprises a sequence that includes substitutions, deletions, and / or insertions at one or more amino acid positions.

[0082] In some embodiments, the viral antigen or immunogen comprises the sequence shown in SEQ ID NO:27. In some embodiments, the viral antigen or immunogen comprises an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence of SEQ ID NO:27, which comprises a sequence that includes substitutions, deletions, and / or insertions at one or more amino acid positions.

[0083] In some embodiments, the viral antigen or immunogen comprises the sequence shown in SEQ ID NO:28. In some embodiments, the viral antigen or immunogen comprises an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence of SEQ ID NO:28, which comprises a sequence that includes substitutions, deletions, and / or insertions at one or more amino acid positions.

[0084] In some embodiments, the viral antigen or immunogen comprises the sequence shown in SEQ ID NO:29. In some embodiments, the viral antigen or immunogen comprises an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence of SEQ ID NO:29, which comprises a sequence that includes substitutions, deletions, and / or insertions at one or more amino acid positions.

[0085] In some embodiments, the viral antigen or immunogen comprises the sequence shown in SEQ ID NO:30. In some embodiments, the viral antigen or immunogen comprises an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence of SEQ ID NO:30, which comprises a sequence that includes substitutions, deletions, and / or insertions at one or more amino acid positions.

[0086] In some embodiments, the viral antigen or immunogen comprises the sequence shown in SEQ ID NO:31. In some embodiments, the viral antigen or immunogen comprises an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence of SEQ ID NO:31, which comprises a sequence that includes substitutions, deletions, and / or insertions at one or more amino acid positions.

[0087] In some embodiments, the viral antigen or immunogen comprises the sequence shown in SEQ ID NO:32. In some embodiments, the viral antigen or immunogen comprises an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence of SEQ ID NO:32, which comprises a sequence that includes substitutions, deletions, and / or insertions at one or more amino acid positions.

[0088] In some embodiments, the viral antigen or immunogen comprises the sequence shown in SEQ ID NO:32. In some embodiments, the viral antigen or immunogen comprises an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence of SEQ ID NO:32, which comprises a sequence that includes substitutions, deletions, and / or insertions at one or more amino acid positions. In some embodiments, the RSV F protein peptides herein include a substitution from A to E at residue 218 of SEQ ID NO:32, as well as substitutions at one or more of residues 106, 107, 108, 109, 133, 135, and 136. In some embodiments, the RSV F protein peptides herein include a substitution from A to E at residue 218 of SEQ ID NO:32, as well as substitutions in one or more of residues 106, 108, 109, 133, 135, and 136. In some embodiments, the RSV F protein peptides herein include a substitution from alanine to glutamic acid at residue 218 of SEQ ID NO:32, as well as substitutions to cysteine ​​in one or more of residues 106, 108, 109, 133, 135, and 136. In some embodiments, the RSV F protein peptides herein include a substitution from alanine to glutamic acid at residue 218 of SEQ ID NO:32, as well as substitutions to cysteine ​​in all of residues 106, 108, 109, 133, 135, and 136. The RSV F protein peptide described herein includes a substitution of alanine to glutamic acid at residue 218 of SEQ ID NO:32, as well as substitution of all residues 108, 109, 133, 135, and 136 with cysteine. The RSV F protein peptide described herein includes a substitution of alanine to glutamic acid at residue 218 of SEQ ID NO:32, as well as substitution of all residues 109, 133, 135, and 136 with cysteine.The RSV F protein peptide described herein includes a substitution of alanine to glutamic acid at residue 218 of SEQ ID NO:32, as well as substitution of all residues 133, 135, and 136 with cysteine. The RSV F protein peptide described herein includes a substitution of alanine to glutamic acid at residue 218 of SEQ ID NO:32, as well as substitution of all residues 135 and 136 with cysteine. The RSV F protein peptide described herein includes a substitution of alanine to glutamic acid at residue 218 of SEQ ID NO:32, as well as substitution of cysteine ​​at residue 136. The RSV F protein peptide described herein includes a substitution of alanine to glutamic acid at residue 218 of SEQ ID NO:32, as well as substitution of cysteine ​​at residue 135. The RSV F protein peptide described herein includes a substitution of alanine to glutamic acid at residue 218 of SEQ ID NO:32, as well as substitution of all residues 106, 133, 135, and 136 with cysteine. The RSV F protein peptide described herein includes a substitution of alanine to glutamic acid at residue 218 of SEQ ID NO:32, as well as substitution of all residues 106, 135, and 136 with cysteine. The RSV F protein peptide described herein includes a substitution of alanine to glutamic acid at residue 218 of SEQ ID NO:32, as well as substitution of all residues 106, 133, and 136 with cysteine. The RSV F protein peptide described herein includes a substitution of alanine to glutamic acid at residue 218 of SEQ ID NO:32, as well as substitution of all residues 106, 133, and 135 with cysteine. The RSV F protein peptide described herein includes a substitution of alanine to glutamic acid at residue 218 of SEQ ID NO:32, as well as substitution of all residues 106 and 136 with cysteine. The RSV F protein peptide described herein includes a substitution of alanine to glutamic acid at residue 218 of SEQ ID NO:32, as well as substitution of all residues 106 and 135 with cysteine.The RSV F protein peptide described herein includes a substitution of alanine to glutamic acid at residue 218 of SEQ ID NO:32, as well as substitution of all residues 108 and 136 with cysteine. The RSV F protein peptide described herein includes a substitution of alanine to glutamic acid at residue 218 of SEQ ID NO:32, as well as substitution of all residues 108 and 135 with cysteine. The RSV F protein peptide described herein includes a substitution of alanine to glutamic acid at residue 218 of SEQ ID NO:32, as well as substitution of all residues 106 and 133 with cysteine. The RSV F protein peptide described herein includes a substitution of alanine to glutamic acid at residue 218 of SEQ ID NO:32, as well as substitution of all residues 106 and 133 with cysteine. The RSV F protein peptide described herein includes a substitution of alanine to glutamic acid at residue 218 of SEQ ID NO:32, as well as substitution of all residues 108 and 133 with cysteine.

[0089] In some embodiments, the viral antigen or immunogen comprises the sequence shown in SEQ ID NO:33. In some embodiments, the viral antigen or immunogen comprises an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence of SEQ ID NO:33, which comprises a sequence that includes substitutions, deletions, and / or insertions at one or more amino acid positions.

[0090] In some embodiments, the viral antigen or immunogen comprises the sequence shown in SEQ ID NO:34. In some embodiments, the viral antigen or immunogen comprises an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence of SEQ ID NO:34, which comprises a sequence that includes substitutions, deletions, and / or insertions at one or more amino acid positions.

[0091] In some embodiments, the viral antigen or immunogen comprises the sequence shown in SEQ ID NO:35. In some embodiments, the viral antigen or immunogen comprises an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence of SEQ ID NO:35, which comprises a sequence that includes substitutions, deletions, and / or insertions at one or more amino acid positions.

[0092] In some embodiments, the viral antigen or immunogen comprises the sequence shown in SEQ ID NO:36. In some embodiments, the viral antigen or immunogen comprises an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence of SEQ ID NO:36, which comprises a sequence that includes substitutions, deletions, and / or insertions at one or more amino acid positions.

[0093] In some embodiments, the viral antigen or immunogen comprises the sequence shown in SEQ ID NO:37. In some embodiments, the viral antigen or immunogen comprises an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence of SEQ ID NO:37, which comprises a sequence that includes substitutions, deletions, and / or insertions at one or more amino acid positions.

[0094] In some embodiments, the viral antigen or immunogen comprises the sequence shown in SEQ ID NO:38. In some embodiments, the viral antigen or immunogen comprises an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence of SEQ ID NO:38, which comprises a sequence that includes substitutions, deletions, and / or insertions at one or more amino acid positions.

[0095] In some embodiments, the viral antigen or immunogen comprises the sequence shown in SEQ ID NO:39. In some embodiments, the viral antigen or immunogen comprises an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence of SEQ ID NO:39, which comprises a sequence that includes substitutions, deletions, and / or insertions at one or more amino acid positions.

[0096] In some embodiments, the viral antigen or immunogen comprises the sequence shown in SEQ ID NO:40. In some embodiments, the viral antigen or immunogen comprises an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence of SEQ ID NO:40, which comprises a sequence that includes substitutions, deletions, and / or insertions at one or more amino acid positions.

[0097] In some embodiments, the viral antigen or immunogen comprises the sequence shown in SEQ ID NO:41. In some embodiments, the viral antigen or immunogen comprises an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence of SEQ ID NO:41, which comprises a sequence that includes substitutions, deletions, and / or insertions at one or more amino acid positions.

[0098] In some embodiments, the viral antigen or immunogen comprises the sequence shown in SEQ ID NO:42. In some embodiments, the viral antigen or immunogen comprises an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence of SEQ ID NO:42, which comprises a sequence that includes substitutions, deletions, and / or insertions at one or more amino acid positions.

[0099] In some embodiments, the viral antigen or immunogen comprises the sequence shown in SEQ ID NO:43. In some embodiments, the viral antigen or immunogen comprises an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence of SEQ ID NO:43, which comprises a sequence that includes substitutions, deletions, and / or insertions at one or more amino acid positions.

[0100] In some embodiments, the viral antigen or immunogen comprises the sequence shown in SEQ ID NO:44. In some embodiments, the viral antigen or immunogen comprises an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence of SEQ ID NO:44, which comprises a sequence that includes substitutions, deletions, and / or insertions at one or more amino acid positions.

[0101] In some embodiments, the viral antigen or immunogen comprises the sequence shown in SEQ ID NO:45. In some embodiments, the viral antigen or immunogen comprises an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence of SEQ ID NO:45, which comprises a sequence that includes substitutions, deletions, and / or insertions at one or more amino acid positions.

[0102] In some embodiments, the viral antigen or immunogen comprises the sequence shown in SEQ ID NO:46. In some embodiments, the viral antigen or immunogen comprises an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence of SEQ ID NO:46, which comprises a sequence that includes substitutions, deletions, and / or insertions at one or more amino acid positions.

[0103] In some embodiments, the viral antigen or immunogen comprises the sequence shown in SEQ ID NO:47. In some embodiments, the viral antigen or immunogen comprises an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence of SEQ ID NO:47, which comprises a sequence that includes substitutions, deletions, and / or insertions at one or more amino acid positions.

[0104] In some embodiments, the viral antigen or immunogen comprises a sequence freely selected from SEQ ID NO: 72-79. In some embodiments, the viral antigen or immunogen comprises an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with a sequence freely selected from SEQ ID NO: 64-79, which comprises a sequence that includes substitutions, deletions, and / or insertions at one or more amino acid positions.

[0105] In some embodiments, the viral antigen or immunogen comprises a sequence freely selected from SEQ ID NO:76. In some embodiments, the viral antigen or immunogen comprises an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence of SEQ ID NO:76, which comprises a sequence that includes substitutions, deletions, and / or insertions at one or more amino acid positions.

[0106] In some embodiments, the viral antigens or immunogens of this specification may include RSV glycoprotein (G) or its fragments, variants or mutants, RSV small hydrophobic protein (SH) or its fragments, variants or mutants, RSV fusion protein (F) or its fragments, variants or mutants, RSV matrix protein (M) or its fragments, variants or mutants, RSV nucleoprotein (N) or its fragments, variants or mutants, RSV phosphoprotein (P) or its fragments, variants or mutants, RSV "giant" protein (L) or its fragments, variants or mutants, M2-1 protein or its fragments, variants or mutants, RSV M2-2 protein or its fragments, variants or mutants, RSV NS-1 protein or its fragments, variants or mutants, or RSV Ns-2 protein or its fragments, variants or mutants, or any combination thereof.

[0107] In some embodiments, the viral antigen or immunogen is produced from a codon-optimized nucleic acid sequence. In some embodiments, the viral antigen or immunogen is produced from a non-codon-optimized nucleic acid sequence.

[0108] In some embodiments, the RSV viral antigen or immunogen referred to herein may comprise a recombinant polypeptide or fusion polypeptide containing the viral antigen or immunogen. The term viral antigen or immunogen may be used to refer to a protein containing the RSV viral antigen or immunogen. In specific cases, the RSV viral antigen or immunogen is the RSV protein peptide as defined herein.

[0109] II. Recombinant Peptides and Proteins The RSV virus antigen and immunogen, e.g., RSV F protein peptide (see Section I), as described herein, are assumed to be able to bind to other proteins or peptides, for example, by ligation, to form recombinant polypeptides containing a fusion peptide. In some embodiments, a single recombinant polypeptide (e.g., monomer) as described herein may associate to form a polymer of recombinant polypeptide, e.g., a trimer. In some embodiments, the association of a single recombinant polypeptide monomer occurs via covalent interactions. In some embodiments, the association of a single recombinant polypeptide monomer occurs via non-covalent interactions. In some embodiments, the interaction (e.g., covalent or non-covalent) is influenced by the protein or peptide ligated to the RSV virus antigen or immunogen (e.g., RSV F protein peptide). In some embodiments, e.g., if the RSV virus antigen or immunogen is the RSV F protein peptide described herein, the protein or peptide it ligates to may be selected such that the native homotrimeric structure of the glycoprotein is preserved. This may be advantageous for inducing a potent and effective immunogenic response to the RSV F protein peptide. For example, maintaining and / or preserving the native conformation of the RSV virus antigen or immunogen (e.g., RSV F protein peptide) can improve or enable proximity to antigenic sites that may produce an immune response. In some cases, recombinant polypeptides comprising the RSV F protein peptide described herein (e.g., Section I) are referred to herein, by alternative, as recombinant RSV F antigen, recombinant RSV F immunogen, or recombinant RSV F protein.

[0110] In some cases, the recombinant polypeptide or its polymerized recombinant polypeptide may aggregate or be able to aggregate, thereby forming a protein containing multiple RSV virus antigens and / or immunogen recombinant polypeptides. The formation of such a protein may be advantageous for producing a potent and effective immunogenic response to RSV virus antigens and / or immunogens. For example, the formation of a protein of multiple recombinant polypeptides and the resulting formation of multiple RSV virus antigens (e.g., RSV F protein peptides) preserves the tertiary and / or quaternary structure of the viral antigen, thereby enabling an enhanced immune response to the native structure. In some cases, aggregation may confer structural stability to the RSV virus antigen or immunogen, and thus allow it to approach potential antigenic sites that can promote an immune response.

[0111] 1. Fusion peptides and recombinant polypeptides In some embodiments, the RSV virus antigen or immunogen can be linked to a trimerizing domain at its C-terminus (C-terminal linkage), thereby promoting monomeric trimerization. In some embodiments, trimerization stabilized the proximal membrane condition of the RSV virus antigen or immunogen (e.g., RSV F protein peptide) exhibiting trimer conformation.

[0112] Non-limiting examples of exogenous multimerization domains that promote stable trimerization of soluble recombinant proteins include the GCN4 leucine zipper (Harbury et al., 1993 Science 262:1401-1407), trimerization motifs from lung surfactant protein (Hoppe et al., 1994 FEBS Lett 344:191-195), collagen (McAlinden et al., 2003 J Biol Chem 278:42200-42207), and phage T4 fibrinfoldon (Miroshnikov et al., 1998 Protein Eng 11:329-414), any of which can be linked to the recombinant RSV viral antigen or immunogen described herein (e.g., via linkage to the C-terminus of the RSV F peptide), thereby promoting trimerization of the recombinant viral antigen or immunogen. See also U.S. Patents 7,268,116, 7,666,837, 7,691,815, 10,618,949, 10,906,944 and 10,960,070, and U.S. 2020 / 0009244, which are incorporated herein by reference in their entirety for all purposes.

[0113] In some embodiments, one or more peptide linkers (e.g., gly-ser linkers, e.g., 10-amino acid glycine-serine peptide linkers) can be used to link a recombinant viral antigen or immunogen to a multimerizing domain. The trimer may contain any stabilizing mutation (or combination thereof) described herein, provided that the recombinant viral antigen or immunogen trimer retains desired properties (e.g., pre-fusion conformation).

[0114] To make therapeutically feasible, the trimerized protein moiety desirable for the design of a biologic should meet the following criteria: Ideally, it should be part of a native secretory protein, e.g., immunoglobulin protein Fc; abundant in circulation (non-toxic); of human origin (non-immunogenic); relatively stable (long half-life); and capable of efficient self-trimerization (reinforced by covalent disulfide bonds between chains); so that the trimerized RSV virus antigen or immunogen is structurally stable.

[0115] Collagen is a family of fibrous proteins and a major component of the extracellular matrix. It is the most abundant protein in mammals, accounting for nearly 25% of the total protein in the body. Collagen plays a major structural role in the formation of bones, tendons, skin, cornea, cartilage, blood vessels, and teeth. Fibrous collagen types I, II, III, IV, V, and XI are all synthesized as larger trimer precursors called procollagen, where a central uninterrupted triple helix domain consisting of hundreds of "GXY" repeat sequences (or glycine repeat sequences) is flanked by a non-collagenary domain (NC), N-propeptide, and C-propeptide. Both the C-terminus and N-terminus are treated by protein hydrolysis during procollagen secretion, an event that causes the mature protein to assemble into collagen fibers, thereby forming an insoluble cellular matrix. BMP-1 is a protease that recognizes specific peptide sequences of procollagen near the boundary between the glycine repeat sequence and the collagen C-prodomain and is involved in the removal of the propeptide. The uncoated type I collagen trimer C-propeptide is found in the normal adult human serum at concentrations ranging from 50 to 300 ng / mL, with children having higher levels, which indicates active bone formation. In individuals with familially elevated serum type I collagen C-propeptide concentrations, the levels can reach approximately 1 to 6 μg / mL without apparent abnormalities, indicating that the C-propeptide is not toxic. Structural studies of the collagen trimer C-propeptide suggest a trefoil structure in which all three subunits converge at a junctional region near its N-terminus, connecting to the rest of the procollagen molecule. This geometric shape, with the fusion target proteins protruding in one direction, is similar to the geometric shape of the Fc dimer.

[0116] Type I, IV, V, and XI collagens are primarily assembled into heterotrimeric forms consisting of two α-1 chains and one α-2 chain (Type I, IV, and V) or three highly homologous chains in different sequences (Type XI). Type II and III collagens are both homotrimers of the α-1 chain. In the case of the most abundant collagen form, Type I collagen, a stable α(I) homotrimer is also formed and exists in variable levels in various tissues. Most of these collagen C-propeptide chains self-assemble into homotrimers when overexpressed alone in cells. The N-propeptide domain is synthesized first, but the assembly of molecules into trimer collagen begins with the mutual orientation and association of C-propeptides. The C-propeptide complex is thought to be stabilized by the formation of interchain disulfide bonds, but the necessity of disulfide bond formation for proper chain orientation is not clear. It repeats the triple helix of glycine and propagates from the C-terminus to the N-terminus like a zipper, associating with each other. This finding demonstrated the production of non-natural collagen matrices by exchanging C-propeptides of different collagen chains using recombinant DNA technology. Non-collagen proteins, such as cytokines and growth factors, were also fused to the N-terminus of procollagen or mature collagen, thereby forming a novel collagen matrix intended to allow for the sustained release of non-collagen proteins from the cell matrix. However, in both cases, the C-propeptide needs to be cleaved before the recombinant collagen fibrils can assemble into the insoluble cell matrix.

[0117] While other protein trimer domains, such as yeast GCN4, phage T4 fibrin, and the aspartate transcarbamoylase from Escherichia coli, have been described as enabling heterologous protein trimerization, none of these trimer proteins are native human proteins or naturally secreted proteins. Therefore, all trimer fusion proteins must be synthesized intracellularly, which not only can lead to misfolding of naturally secreted proteins (e.g., soluble receptors) but also makes the purification of such fusion proteins from thousands of other intracellular proteins difficult. Furthermore, a fatal drawback of using such non-human protein trimer domains (e.g., from yeast, aphage, and bacteria) in the design of trimer biopharmaceuticals lies in their hypothetical immunogenicity in the human body, which would render such fusion proteins ineffective shortly after injection into the human body.

[0118] Therefore, the use of collagen in recombinant polypeptides as described herein has many advantages, including the following: (1) Collagen is the most abundant protein secreted in the mammalian body, accounting for nearly 25% of all proteins in the body; (2) The primary form of collagen arises spontaneously as a trimer helix, with its globular C-propeptide involved in the initiation of trimerization; (3) Trimeric C-propeptides of collagen, hydrolyzed from mature collagen, are naturally found in mammalian blood at submicrogram / milliliter levels and are known to be non-toxic to the body; (4) The linear triple-helical region of collagen can be included as a linker at a predicted interval of 2.9 A per residue, or excluded from part of the fusion protein, so that the distance between the protein being trimerized and the C-propeptide of collagen can be precisely adjusted to achieve optimal biological activity; (5) The BMP1 recognition site that cleaves the C-propeptide from procollagen can be mutated or deleted to prevent the breakdown of the trimer fusion protein; (6) The C-propeptide domain self-trimerizes via disulfide bonds, providing a universal affinity tag that can be used to purify any secreted fusion protein produced. In some embodiments, the C propeptide of collagen, to which the RSV virus antigen and immunogen (e.g., RSV F protein peptide) are linked, can be recombinantly recombined to produce a covalently linked soluble homotrimeric fusion protein.

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

[0120] In some embodiments, the C-terminal propeptide belongs to human collagen. In some embodiments, the C-terminal propeptide comprises the C-terminal polypeptide or fragment thereof of pro α1(I), pro α1(II), pro α1(III), pro α1(V), pro α1(XI), pro α2(I), pro α2(V), pro α2(XI), or pro α3(XI). In some embodiments, the C-terminal propeptide is or comprises the C-terminal polypeptide of pro α1(I).

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

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

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

[0124] 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), where aspartic acid (D) is replaced with asparagine (N) at the BMP-1 site, for example, RAD is mutated to RAN. 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), where alanine (A) is replaced with asparagine (N) at the BMP-1 site, for example, RAD is mutated to RND. In some embodiments, the C-terminal propeptide herein may comprise a mutated BMP-1 site, for example, RSAN may be included instead of DDAN. In some embodiments, the C-terminal propeptide of this specification may include a BMP-1 site, and for example, a sequence containing 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 polypeptide disclosed herein.

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

[0126] In some embodiments, the C-terminal propeptide may include a sequence containing a glycine-XY repeat sequence, where X and Y are independently any amino acid, or an amino acid sequence having at least 85%, 90%, 92%, 95%, or 97% identity with it, capable of forming an interpolypeptide disulfide bond and trimerizing the recombinant polypeptide. In some embodiments, X and Y are independently proline or hydroxyproline.

[0127] Here, in some cases, the RSV F peptide protein (e.g., RSV viral antigen or immunogen, see Section I, e.g.) is linked to a C-terminal propeptide to form a recombinant polypeptide, which produces a homotrimer of the RSV F protein peptide by forming a trimer. In some embodiments, the trimerized recombinant polypeptide contains a cane-shaped rod-shaped F protein peptide trimer. In some embodiments, the RSV F protein peptide of the trimerized recombinant polypeptide exhibits a pre-fusion conformation. In some embodiments, the RSV F protein peptide of the trimerized recombinant polypeptide exhibits a post-fusion conformation. In some embodiments, the conformational state allows access to different antigenic sites on the F protein peptide. In some embodiments, the antigenic site is an epitope, e.g., a linear epitope or a conformational epitope. One advantage of having the trimerized recombinant polypeptide is that it can enhance the immune response to a variety of potential and different antigenic sites.

[0128] In some embodiments, the trimerized recombinant polypeptide comprises a single recombinant polypeptide containing the same viral antigen or immunogen. In some embodiments, the trimerized recombinant polypeptide comprises a single recombinant polypeptide each containing a different viral antigen or immunogen from the other recombinant polypeptides. In some embodiments, the trimerized recombinant polypeptide comprises a single recombinant polypeptide, where one of the single recombinant polypeptides contains a different viral antigen or immunogen from the other recombinant polypeptides. In some embodiments, the trimerized recombinant polypeptide comprises a single recombinant polypeptide, where two of the single recombinant polypeptides contain the same viral antigen or immunogen, and the viral antigen or immunogen is different from the viral antigen or immunogen contained in the remaining recombinant polypeptide.

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

[0130] In some embodiments, the recombinant polypeptide or the fusion protein comprises a first sequence shown in one of SEQ ID NOs: 17-47 and 72-79, which is ligated to a second sequence shown in one of SEQ ID NOs: 48-63, where the C-terminus of the first sequence is directly ligated to the N-terminus of the second sequence.

[0131] In some embodiments, the recombinant polypeptide or the fusion protein comprises a first sequence represented by SEQ ID NO: 17-47 and 72-79, which is linked to a second sequence represented by SEQ ID NO: 48-63, where the C-terminus of the first sequence is indirectly linked to the N-terminus of the second sequence, for example, via a linker. In some embodiments, the linker comprises a sequence containing a glycine-XY repeat sequence.

[0132] In some embodiments, the recombinant polypeptide is or comprises the sequence shown in SEQ ID NO:1. In some embodiments, the recombinant polypeptide is or comprises an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence of SEQ ID NO:1, wherein it has one or more amino acid positions, for example, The sequence includes substitutions, deletions, and / or insertions of 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, the variant comprising one, two, three, four, five or more mutations or any combination thereof selected from P102A, R109A, R136A, E161P, E218A, S215P, I379A and M447V.

[0133] In some embodiments, the recombinant polypeptide is or comprises the sequence shown in SEQ ID NO:2. In some embodiments, the recombinant polypeptide is or comprises an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence of SEQ ID NO:2, which has one or more amino acid positions, for example, The sequence includes substitutions, deletions, and / or insertions of 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:2, the variant comprising one, two, three, four, five or more mutations or any combination thereof selected from P102A, R109A, R136A, E161P, E218A, S215P, I379A and M447V.

[0134] In some embodiments, the recombinant polypeptide is or comprises the sequence shown in SEQ ID NO:3. In some embodiments, the recombinant polypeptide is or comprises an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence of SEQ ID NO:3, and it has one or more amino acid positions, for example, The sequence includes substitutions, deletions, and / or insertions of 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:3, the variant comprising one, two, three, four, five or more mutations or any combination thereof selected from P102A, R109A, R136A, E161P, E218A, S215P, I379A and M447V.

[0135] In some embodiments, the recombinant polypeptide is or comprises the sequence shown in SEQ ID NO:4. In some embodiments, the recombinant polypeptide is or comprises an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence of SEQ ID NO:4, and it has one or more amino acid positions, for example, The sequence includes substitutions, deletions, and / or insertions of 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, the variant comprising one, two, three, four, five or more mutations or any combination thereof selected from P102A, R109A, R136A, E161P, E218A, S215P, I379A and M447V.

[0136] In some embodiments, the recombinant polypeptide is or comprises the sequence shown in SEQ ID NO: 5. In some embodiments, the recombinant polypeptide is or comprises an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence of SEQ ID NO: 5, and it has one or more amino acid positions, for example, The sequence includes substitutions, deletions, and / or insertions of 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, the variant comprising one, two, three, four, five or more mutations or any combination thereof selected from P102A, R109A, R136A, E161P, E218A, S215P, I379A and M447V.

[0137] In some embodiments, the recombinant polypeptide is or comprises the sequence shown in SEQ ID NO:6. In some embodiments, the recombinant polypeptide is or comprises an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence of SEQ ID NO:6, which has one or more amino acid positions, for example, The sequence includes substitutions, deletions, and / or insertions of 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:6, the variant comprising one, two, three, four, five or more mutations or any combination thereof selected from P102A, R109A, R136A, E161P, E218A, S215P, I379A and M447V.

[0138] In some embodiments, the recombinant polypeptide is or comprises the sequence shown in SEQ ID NO:7. In some embodiments, the recombinant polypeptide is or comprises an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence of SEQ ID NO:7, and it has one or more amino acid positions, for example, The sequence includes substitutions, deletions, and / or insertions of 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:7, the variant comprising one, two, three, four, five or more mutations or any combination thereof selected from P102A, R109A, R136A, E161P, E218A, S215P, I379A and M447V.

[0139] In some embodiments, the recombinant polypeptide is or comprises the sequence shown in SEQ ID NO:8. In some embodiments, the recombinant polypeptide is or comprises an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence of SEQ ID NO:8, and it has one or more amino acid positions, for example, The sequence includes substitutions, deletions, and / or insertions of 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:8, the variant comprising one, two, three, four, five or more mutations or any combination thereof selected from P102A, R109A, R136A, E161P, E218A, S215P, I379A and M447V.

[0140] In some embodiments, the recombinant polypeptide is or comprises the sequence shown in SEQ ID NO:9. In some embodiments, the recombinant polypeptide is or comprises an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence of SEQ ID NO:9, and it has one or more amino acid positions, for example, The sequence includes substitutions, deletions, and / or insertions of 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:9, the variant comprising one, two, three, four, five or more mutations or any combination thereof selected from P102A, R109A, R136A, E161P, E218A, S215P, I379A and M447V.

[0141] In some embodiments, the recombinant polypeptide is or comprises the sequence shown in SEQ ID NO:10. In some embodiments, the recombinant polypeptide is or comprises an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence of SEQ ID NO:10, which includes one or more amino acid positions, for example The sequence includes substitutions, deletions, and / or insertions of 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 position 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:10, the variant comprising one, two, three, four, five or more mutations or any combination thereof selected from P102A, R109A, R136A, E161P, E218A, S215P, I379A and M447V.

[0142] In some embodiments, the recombinant polypeptide is or comprises the sequence shown in SEQ ID NO:11. In some embodiments, the recombinant polypeptide is or comprises an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence of SEQ ID NO:11, wherein it has one or more amino acid positions, for example The sequence includes substitutions, deletions, and / or insertions of 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 position 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:11, the variant comprising one, two, three, four, five or more mutations or any combination thereof selected from P102A, R109A, R136A, E161P, E218A, S215P, I379A and M447V.

[0143] In some embodiments, the recombinant polypeptide is or comprises the sequence shown in SEQ ID NO:12. In some embodiments, the recombinant polypeptide is or comprises an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence of SEQ ID NO:12, which includes one or more amino acid positions, for example The sequence includes substitutions, deletions, and / or insertions of 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 position 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:12, the variant comprising one, two, three, four, five or more mutations or any combination thereof selected from P102A, R109A, R136A, E161P, E218A, S215P, I379A and M447V.

[0144] In some embodiments, the recombinant polypeptide is or comprises the sequence shown in SEQ ID NO:13. In some embodiments, the recombinant polypeptide is or comprises an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence of SEQ ID NO:13, which includes one or more amino acid positions, for example The sequence includes substitutions, deletions, and / or insertions of 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 position 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:13, the variant comprising one, two, three, four, five or more mutations or any combination thereof selected from P102A, R109A, R136A, E161P, E218A, S215P, I379A and M447V.

[0145] In some embodiments, the recombinant polypeptide is or comprises the sequence shown in SEQ ID NO:14. In some embodiments, the recombinant polypeptide is or comprises an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence of SEQ ID NO:14, which includes one or more amino acid positions, for example The sequence includes substitutions, deletions, and / or insertions of 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 position 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:14, the variant comprising one, two, three, four, five or more mutations or any combination thereof selected from P102A, R109A, R136A, E161P, E218A, S215P, I379A and M447V.

[0146] In some embodiments, the recombinant polypeptide is or comprises the sequence shown in SEQ ID NO:15. In some embodiments, the recombinant polypeptide is or comprises an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence of SEQ ID NO:15, which includes one or more amino acid positions, for example The sequence includes substitutions, deletions, and / or insertions of 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 position 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:15, the variant comprising one, two, three, four, five or more mutations or any combination thereof selected from P102A, R109A, R136A, E161P, E218A, S215P, I379A and M447V.

[0147] In some embodiments, the recombinant polypeptide is or comprises the sequence shown in SEQ ID NO:16. In some embodiments, the recombinant polypeptide is or comprises an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence of SEQ ID NO:16, which includes one or more amino acid positions, for example The sequence includes substitutions, deletions, and / or insertions of 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 position 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:16, the variant comprising one, two, three, four, five or more mutations or any combination thereof selected from P102A, R109A, R136A, E161P, E218A, S215P, I379A and M447V.

[0148] In some embodiments, the recombinant polypeptide is freely selected from or includes sequences shown in SEQ ID NO: 64-71. In some embodiments, the recombinant polypeptide is an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with a sequence freely selected from SEQ ID NO: 64-71.

[0149] In some embodiments, the recombinant polypeptide is or contains the sequence shown in SEQ ID NO:64. In some embodiments, the recombinant polypeptide is or comprises an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence of SEQ ID NO: 64, and it comprises one or more amino acid positions, for example, 102, 107, 109, 131, 132, 134, 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 (SEQ ID NO: 64). The recombinant polypeptide comprises a sequence that includes substitutions, deletions and / or insertions to amino acid positions (for NO:31 or 32) or any combination thereof. In some embodiments, the recombinant polypeptide is or comprises a variant of SEQ ID NO:64, the variant comprising one, two, three, four, five or more mutations or any combination thereof selected from P102A, R109A, R136A, E161P, E218A, S215P, I379A and M447V.

[0150] As described above, in some embodiments, the recombinant polypeptides according to this specification can not only associate to form trimers, but can also aggregate or be aggregated to produce proteins containing multiple recombinant polypeptides. In some embodiments, the formed proteins have macrostructures. In some cases, the macrostructures can confer structural stability to the RSV virus antigen or immunogenic recombinant polypeptide, and thus be able to access potential antigenic sites that can promote an immune response.

[0151] In some embodiments, the trimerized recombinant polypeptide aggregates to form a protein containing multiple trimerized recombinant polypeptides. In some embodiments, the multiple trimerized recombinant polypeptides form a protein having a macrostructure. In some embodiments, the protein contains a rosette-like oligomer containing a cane-shaped rod-shaped F protein peptide trimer.

[0152] In some embodiments, this specification provides a complex comprising any suitable combination of recombinant polypeptides or fragments, variants, or mutants selected from SEQ ID NO: 1-16 and 64-71. In some embodiments, this specification provides a complex comprising a trimer of recombinant polypeptides or fragments, variants, or mutants selected from SEQ ID NO: 1-16 and 64-71, wherein the recombinant polypeptide is trimerized via interpolypeptide disulfide bonds to form the trimer.

[0153] In some embodiments, the protein comprising multiple recombinant polypeptides described herein is an immunogen. In some embodiments, the protein comprising multiple recombinant polypeptides described herein is contained in nanoparticles. For example, in some embodiments, the protein is directly linked to nanoparticles, such as protein nanoparticles. In some embodiments, the protein is indirectly linked to nanoparticles. In some embodiments, the protein comprising multiple recombinant polypeptides described herein is contained in virus-like particles (VLPs).

[0154] 2. Polynucleotides and vectors The Specified Provisions also provide polynucleotides (nucleic acid molecules) encoding RSV antigens or immunogens and recombinant polypeptides, as well as vectors for genetically engineering cells to express such RSV antigens or immunogens and recombinant polypeptides.

[0155] In some embodiments, polynucleotides encoding recombinant polypeptides according to this specification have been provided. In some embodiments, the polynucleotide comprises a single nucleic acid sequence, for example, a nucleic acid sequence encoding a recombinant polypeptide. In other cases, the polynucleotide comprises a first nucleic acid sequence encoding a recombinant polypeptide containing a specific RSV virus antigen or immunogen, and a second nucleic acid sequence encoding a recombinant polypeptide containing a different RSV virus antigen or immunogen.

[0156] In some embodiments, the polynucleotide encoding the recombinant polypeptide contains at least one promoter, which is operably linked to control the expression of the recombinant polypeptide. In some embodiments, the polynucleotide contains two, three or more promoters, which are operably linked to control the expression of the recombinant polypeptide.

[0157] In some embodiments, for example, if the polynucleotide includes two or more nucleic acid coding sequences, such as sequences coding recombinant polypeptides containing different RSV virus antigens or immunogens, at least one promoter is operably linked to control the expression of the two or more nucleic acid sequences. In some embodiments, the polynucleotide contains two, three or more promoters, and the promoters are operably linked to control the expression of the recombinant polypeptide.

[0158] In some embodiments, the expression of the recombinant polypeptide is inducible or conditional. Therefore, in some embodiments, the polynucleotide encoding the recombinant polypeptide contains a conditional promoter, enhancer, or transactivator. In some such embodiments, the conditional promoter, enhancer, or transactivator is an inducible promoter, enhancer, or transactivator, or an inhibitory promoter, enhancer, or transactivator. For example, in some embodiments, an inducible or conditional promoter can be used to restrict the expression of the recombinant polypeptide to a specific microenvironment. In some embodiments, the expression driven by an inducible or conditional promoter is regulated by exposure to an exogenous factor such as heat, radiation, or drugs.

[0159] If the polynucleotide comprises one or more nucleic acid sequences encoding recombinant polypeptides, the polynucleotide may further contain nucleic acid sequences encoding peptides between one or more nucleic acid sequences. In some cases, the nucleic acid-encoded peptides located between nucleic acid sequences separate the translation products of the nucleic acid sequences during or after translation. In some embodiments, the peptides contain internal ribosome entry sites (IRESs), self-cleaving peptides, or peptides that induce ribosome skipping, such as T2A peptides.

[0160] In some embodiments, polynucleotides encoding recombinant polypeptides are introduced into a composition containing cultured cells (e.g., host cells) by, for example, retroviral transduction, transfection, or transformation. In some embodiments, this may enable the expression (e.g., production) of the recombinant polypeptide. In some embodiments, the expressed recombinant polypeptide is purified.

[0161] In some embodiments, the polynucleotides (nucleic acid molecules) according to this specification encode the RSV virus antigen or immunogen described herein. In some embodiments, the polynucleotides (nucleic acid molecules) according to this specification encode a recombinant polypeptide containing the RSV virus antigen or immunogen, for example, the RSV F peptide protein described herein.

[0162] Vectors or constructs containing nucleic acid molecules described herein are also provided. In some embodiments, the vector or construct contains one or more promoters, the promoters operably linked to nucleic acid molecules encoding recombinant polypeptides to drive their expression. In some embodiments, the promoters operably linked to one or more nucleic acid molecules, for example, nucleic acid molecules encoding recombinant polypeptides containing different RSV virus antigens or immunogens.

[0163] In some embodiments, the vector is a viral vector. In some embodiments, the viral 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.

[0164] In some embodiments, the vector or construct comprises a single promoter that drives the expression of one or more nucleic acid molecules of polynucleotides. In some embodiments, such a promoter may be multicistronic (biscistronic or tricistronic, see, for example, U.S. Patent No. 6,060,273). For example, in some embodiments, the transcription unit may be operated as a biscistronic unit containing an IRES (Internal Ribosome Entry Site), thereby enabling co-expression of gene products (e.g., encoding different recombinant polypeptides) by signaling from a single promoter. In some embodiments, the vector according to this specification is biscistronic, thereby enabling the vector to contain and express two nucleic acid sequences. In some embodiments, the vector according to this specification is tricistronic, thereby enabling the vector to contain and express three nucleic acid sequences.

[0165] In some embodiments, a single promoter directs the expression of RNA, and the RNA contains two or three genes (e.g., genes encoding chimeric signaling receptors and recombinant receptors) within a single open reading frame (ORF), and the genes are separated from each other by sequences encoding self-cleaving peptides (e.g., 2A sequences) or protease recognition sites (e.g., furin proteases). 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, the peptide, e.g., T2A, causes ribosomes to skip the synthesis of the peptide bond at the C-terminus of the 2A element (ribosome skipping), resulting in separation between the end of the 2A sequence and 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 derived from foot-and-mouth disease virus (F2A), equine rhinitis A virus (E2A), Zosea asigna virus (T2A), and porcine tescovirus-1 (P2A), as described in U.S. Patent Publication No. 20070116690.

[0166] In some embodiments, the vector is contained within 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 contained within a cell. In some embodiments, the virus or cell containing the vector contains a recombinant genome.

[0167] III. Immunogenic Compositions and Preparations In some embodiments, this specification provides immunogenic compositions comprising a trimer of a recombinant polypeptide or any two or more combinations of the trimers, wherein the recombinant polypeptide comprises a sequence selected from SEQ ID NO: 1-16 and 64-71. In some embodiments, a unit dose of the immunogenic composition may contain about 10 μg to about 100 μg of the RSV F antigen, preferably about 25 μg to about 75 μg of the RSV F antigen, preferably about 40 μg to about 60 μg of the RSV F antigen or about 50 μg of the RSV F antigen. In some embodiments, the dose contains 3 μg of the RSV F antigen. In other embodiments, the dose contains 9 μg of the RSV F antigen. In other embodiments, the dose contains 30 μg of the RSV F antigen.

[0168] In some cases, it may be desirable to combine the disclosed immunogen with other pharmaceuticals (e.g., vaccines) that induce a protective response against other factors. For example, compositions comprising the recombinant RSV F antigen (e.g., trimer or protein) described herein can be administered simultaneously (usually separately) or sequentially with other vaccines recommended by the Advisory Committee on Immunization Practices (ACIP; cdc.gov / vaccines / acip / index.html) for the target age group (e.g., infants approximately 1 to 6 months old), such as the influenza vaccine or the varicella-zoster vaccine. Therefore, the disclosed immunogens comprising the recombinant RSV F antigen described herein can be administered simultaneously or sequentially with vaccines against, for example, hepatitis B (HepB), diphtheria, tetanus and pertussis (DTaP), pneumococcal disease (PCV), Haemophilus influenzae type b (Hib), polio, influenza, and rotavirus.

[0169] Polyvalent or combination vaccines provide protection against multiple pathogens. In some cases, a polyvalent vaccine can provide protection against multiple strains or strains of the same pathogen. In other cases, a polyvalent vaccine provides protection against multiple pathogens; for example, the combination vaccine Tdap provides protection against strains of tetanus, pertussis, and diphtheria. Polyvalent vaccines are ideal for minimizing the number of immunizations required to provide protection against multiple pathogens or pathogenic strains, reducing administration costs, and improving reach. This is particularly useful, for example, when vaccinating neonates and children.

[0170] In some embodiments, for example, a vaccine comprising the immunogenic composition described herein is a polyvalent vaccine. In some embodiments, the antigenic substance to be incorporated into the polyvalent vaccine composition of the present invention is derived from type A or type B RSV or a combination thereof. The antigen to be incorporated into the polyvalent vaccine composition of the present invention may be derived from one or more strains of RSV, for example, 2 to 5 strains, in order to provide a broader range of protection. In one embodiment, the antigen to be incorporated into the polyvalent vaccine composition of the present invention is derived from multiple strains of the RSV virus. Other useful antigens include live viruses, attenuated viruses, 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 pertussis virus epitopes (e.g., ACEL-IMUNErM cell-free DTP, Wyeth-Lederle vaccine, and pediatric medicines).

[0171] In some embodiments, the vaccines described herein are universal vaccines. In some embodiments, a universal vaccine is a vaccine that provides protection against multiple strains of the same virus, for example, multiple RSV strains. The development of an effective universal RSV vaccine would, for example, reduce the cost and effort of formulating seasonal vaccines and enable a more robust pandemic response.

[0172] In some embodiments, a universal vaccine is a vaccine containing multiple epitopes derived from different virus strains. In some embodiments, a universal vaccine contains a single conserved epitope in different virus strains. For example, a universal vaccine may be based on a relatively conserved domain of the RSV F protein.

[0173] Furthermore, immunogenic compositions are provided, comprising a disclosed immunogen (e.g., a nucleic acid molecule encoding a disclosed recombinant RSV F antigen or a protocol of a disclosed recombinant RSV F antigen) and a pharmaceutically acceptable carrier. In some embodiments, the immunogenic composition comprises a trimerized recombinant polypeptide according to this specification and an optional pharmaceutically acceptable carrier. In some embodiments, the immunogenic composition comprises a protein containing a plurality of trimerized recombinant polypeptides according to this specification and an optional pharmaceutically acceptable carrier. In some embodiments, the immunogenic composition comprises protein nanoparticles according to this specification and an optional pharmaceutically acceptable carrier. In some embodiments, the immunogenic composition comprises a VLP according to this specification and an optional pharmaceutically acceptable carrier. In some embodiments, the immunogenic composition comprises an isolated nucleic acid according to this specification and an optional pharmaceutically acceptable carrier. In some embodiments, the immunogenic composition comprises a vector according to this specification and an optional pharmaceutically acceptable carrier. In some embodiments, the immunogenic composition comprises a virus according to this specification and an optionally pharmaceutically acceptable carrier. In some embodiments, the immunogenic composition comprises a pseudovirus according to this specification and an optionally pharmaceutically acceptable carrier. In some embodiments, the immunogenic composition comprises cells according to this specification and an optionally pharmaceutically acceptable carrier. In some embodiments, the immunogenic composition, for example, the immunogenic composition 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 both a prophylactic and a therapeutic vaccine. Such pharmaceutical compositions may be administered to a subject by various modes of administration known to those skilled in the art, for example, intramuscular, intradermal, subcutaneous, intravenous, intraarterial, intra-articular, intraperitoneal, intranasal, sublingual, tonsillar, oropharyngeal, or other parenteral and mucosal routes.In some embodiments, a pharmaceutical composition comprising one or more immunogens disclosed is an immunogenic composition. Practical methods for preparing administerable compositions are known or obvious to those skilled in the art and are described in detail in publications such as Remington's Pharmaceutical Sciences, 19th edition, Mack Publishing Company, Easton, Pa., 1995.

[0174] Therefore, immunogens, such as recombinant RSV F antigen, such as the trimer described herein, and proteins, can be formulated with pharmaceutically acceptable carriers to help retain biological activity and improve stability during storage within an acceptable temperature range. Possible carriers include, but are not limited to, physiologically balanced media, phosphate-buffered saline, water, emulsions (e.g., oil / water or water / oil emulsions), various types of wetting agents, cryogenic additives or stabilizers, such as proteins, peptides or hydrolysates (e.g., albumin, gelatin), sugars (e.g., sucrose, lactose, sorbitol), amino acids (e.g., monosodium glutamate), or other protective agents. The resulting aqueous solutions can be packaged as is or lyophilized. The lyophilized formulations are prepared for single or multiple doses in combination with a sterile solution before administration.

[0175] Formulated compositions, particularly liquid formulations, may contain bacteriostatic agents to prevent or minimize degradation during storage, which may include, but are not limited to, benzyl alcohol, phenol, m-cresol, chlorobutanol, methylparaben, and / or propylparaben in an effective concentration (usually 1% w / v). Bacteriostatic agents may be contraindicated in some patients; therefore, lyophilized formulations may be reconstituted in solutions containing or without such components.

[0176] The immunogenic compositions of this disclosure may optionally contain pharmaceutically acceptable vehicle substances, such as pH adjusters and buffers, osmotic regulators, and wetting agents, to approximate physiological conditions, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, and triethanolamine oleate. The immunogenic compositions may optionally contain adjuvants to enhance the host immune response. Suitable adjuvants include, for example, Toll-like receptor agonists, aluminum agents, AlPO4, aluminum aldehyde hydrogels, 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 polyoxylpropylene (POP), such as POE-POP-POE block copolymers, MPLs. TM (3-O-deacylated monophosphoryl lipid A; Corixa, Hamilton, Ind.) and IL-12 (Genetics Institute, Cambridge, Mass.) and 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 help stimulate the immune system in a nonspecific manner, thereby having the advantage of enhancing the immune response to the drug. In some embodiments, the immunogenic compositions of the Disclosure may contain or be administered with one or more adjuvants. In some embodiments, the immunogenic compositions of the Disclosure may contain or be administered with two adjuvants. In some embodiments, the immunogenic compositions of the Disclosure may contain or be administered with multiple adjuvants. For example, in some cases, a vaccine comprising an immunogenic composition according to this specification may contain multiple adjuvants or may be administered together with multiple adjuvants.

[0177] For vaccine compositions, suitable adjuvants include, for example, aluminum hydroxide, lecithin, Freund's adjuvant, and MPL. TM 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 time-release formulations. This can be achieved in compositions containing sustained-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.

[0178] In some embodiments, the immunogenic composition of the present invention may include an adjuvant formulation comprising a metabolizable oil (e.g., squalene) in the form of an oil-in-water emulsion, α-tocopherol, and polyoxyethylene sorbitan monooleate (Tzeen-80). In some embodiments, the adjuvant formulation may contain about 2% to about 10% squalene, about 2% to about 10% α-tocopherol (e.g., D-α-tocopherol), and about 0.3% to about 3% polyoxyethylene sorbitan monooleate. In some embodiments, the adjuvant formulation may contain about 5% squalene, about 5% tocopherol, and about 0.4% polyoxyethylene sorbitan monooleate. In some embodiments, the immunogenic compositions of the present disclosure may comprise 3-O-deacylated monophosphoryl lipid A (3D-MPL) and an adjuvant in the form of an oil-in-water emulsion, the adjuvant comprising a metabolite oil, α-tocopherol, and polyoxyethylene sorbitan monooleate. In some embodiments, the immunogenic compositions of the present disclosure may comprise QS21 (Quillaja saponaria Molina extract: fraction 21), 3D-MPL, and an oil-in-water emulsion, wherein the oil-in-water emulsion comprises a metabolite oil, for example, squalene, α-tocopherol, and Zween-80. In some embodiments, the immunogenic compositions of this disclosure may contain an adjuvant in the form of a liposome composition.

[0179] In some embodiments, the immunogenic compositions of the present disclosure may contain an adjuvant formulation comprising a metabolizable oil (e.g., squalene), polyoxyethylene sorbitan monooleate (Tzeen-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.

[0180] In some embodiments, the immunogenic compositions of the present disclosure may contain, for example, an adjuvant formulation in the form of a nanoparticle composition, the adjuvant formulation comprising quillaja saponin, cholesterol, and phospholipids. In some embodiments, the immunogenic compositions of the present disclosure may contain a mixture of separately purified quillaja fractions, which are then formulated together with cholesterol and phospholipids.

[0181] In some embodiments, the immunogenic compositions of this disclosure are MF59 TM Matrix-A TM Matrix-C TM Matrix-M TM It may also contain an adjuvant selected from AS01, AS02, AS03, and AS04.

[0182] In some embodiments, the immunogenic compositions of this disclosure may contain a Toll-like receptor 9 (TLR9) agonist, wherein the TLR9 agonist is an oligonucleotide having a length of 8 to 35 nucleotides and containing a non-methylated cytidine-phospho-guanosine (also known as CpG or cytosine-phosphate-guanosine) motif, and the amounts of the RSV antigen and the oligonucleotide present in the immunogenic composition can effectively stimulate an immune response to the RSV antigen in a mammalian subject requiring it, such as a human subject. TLR9 (CD289) recognizes the non-methylated cytidine-phospho-guanosine (CpG) motif found in microbial DNA, the latter of which can be mimicked using synthesized 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 typically contain a palindrome sequence following the following general formulas: 5'-purine-purine-CG-pyrimidine-pyrimidine-3' or 5'-purine-purine-CG-pyrimidine-pyrimidine-CG-3'. See U.S. Patent No. 6,589,940, which is incorporated herein by reference in its entirety. In some embodiments, the CpG oligonucleotide is linear. In other embodiments, the CpG oligonucleotide is cyclic or contains a hairpin loop. The CpG oligonucleotide may be single-stranded or double-stranded. In some embodiments, the CpG oligonucleotide may contain modifications. Modifications include, but are not limited to, modifications of 3'OH or 5'OH groups, modifications of nucleotide bases, modifications of sugar components, and modifications of phosphate groups. The modified base may be included in the palindromic sequence of the CpG oligonucleotide, provided that the modified base retains the same specificity to its native complement through Watson-Crick base pairing (for example, the palindromic portion remains self-complementary). In some embodiments, the CpG oligonucleotide contains a non-standard base.In some embodiments, the CpG oligonucleotide comprises a modified nucleoside. In some embodiments, the modified nucleoside is selected from 2'-deoxy-7-deazaguanosine, 2'-deoxy-6-thioguanosine, arabinoguanosine, 2'-deoxy-2'-substituted arabinoguanosine, and 2'-O-substituted arabinoguanosine. The CpG oligonucleotide may include phosphate group modifications. For example, in addition to phosphodiester bonds, phosphate modifications further include, but are not limited to, methylphosphonates, phosphorothioates, phosphoramides (crosslinked or uncrosslinked), phosphotryesters, and phosphorodithioates, and any combination may be used. Other non-phosphoester bonds may also be used. In some embodiments, the oligonucleotide comprises only a phosphorothioate skeleton. In some embodiments, the oligonucleotide comprises only a phosphodiester skeleton. In some embodiments, the oligonucleotide comprises a combination of phosphoester bonds to the phosphoester skeleton, for example, a combination of a phosphodiester bond and a phosphorothioate bond. Oligonucleotides having a phosphorothioate skeleton may be more immunogenic and more resistant to degradation after injection into the host than oligonucleotides having a phosphodiester skeleton (Braun et al., J Immunol, 141:2084-2089, 1988, and Latimer et al., Mol Immunol, 32:1057-1064, 1995). The CpG oligonucleotides of this disclosure contain at least one, two, or three internucleotide phosphorothioate ester bonds. 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 bonds of the CpG oligonucleotide are phosphorothioate bonds, or in other words, the CpG oligonucleotide has a phosphorothioate skeleton.

[0183] Any suitable CpG oligodeoxynucleotide (ODN) or combination thereof can be used as an adjuvant in this disclosure. For example, K-type ODNs (also called B-type) encode multiple CpG motifs on a phosphorothioate backbone. K-type ODNs can be based on the sequence TCCATGGACGTTCCTGAGCGTT. Compared to native phosphodiester nucleotides, the use of phosphorothioate nucleotides results in increased resistance to nuclease digestion and a substantially longer in vivo half-life. K-type ODNs induce pDC differentiation and TNF-α production, as well as B cell proliferation and IgM secretion. D-type ODNs (also called A-type) are constructed on a mixed phosphodiester / phosphorothioate backbone and contain a single CpG motif with palindromic sequences on both sides and poly-G tails (structural motifs that readily form concatemers) at the 3' and 5' ends. D-type ODNs can be based on the sequence GGTGCATCGATGCAGGGGGG. D-type ODNs induce pDC maturation and IFN-α secretion, but do not affect B cells. C-type ODNs are similar to K-type ODNs in that they are composed entirely of phosphorothioate nucleotides, but are similar to D-type ODNs in that they contain a palindromic CpG motif. C-type ODNs can be based on the sequence TCGTCGTTCGAACGACGTTGAT. This class of ODNs stimulates B cells to secrete IL-6 and stimulates pDCs to produce IFN-α. P-type ODNs contain two palindromes, which allows them to form a higher-order, ordered structure. P-type ODNs can be based on the sequence TCGTCGACGATCGGCGCGCGCCG. P-type ODNs activate both B cells and pDCs and induce higher IFN-α production compared to C-type ODNs. In this paragraph, bold text in ODN sequences indicates a self-complementary palindrom, and CpG motifs are underlined.

[0184] Exemplary CpG ODNs, such as CpG7909 (5’-TCGTCGTTTTGTCGTTTTGTCGTT-3’) and CpG1018 (5’-TGACTGTGAACGTTCGAGATGA-3’), are known and are described in U.S. Pat. Nos. 7,255,868, 7,491,706, 7,479,285, 7,745,598, 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, Application Publication US2020 / 0002704, and Bode et al., “CpG DNA as a vaccine adjuvant,” Expert Rev Vaccines (2011), 10(4):499-511, and all of these documents are incorporated herein by reference in their entirety for all purposes.

[0185] One or more adjuvants can be used in combination and include, but are not limited to, aluminum hydroxide (aluminum salts), oil-in-water emulsions, water-in-oil emulsions, liposomes and microparticles, such as poly(lactide-coglycolide) microparticles (Shah et al., Methods Mol, 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 of amorphous aluminum hydroxyphosphate sulfate, aluminum hydroxide, aluminum phosphate and potassium aluminum sulfate. In some embodiments, the aluminum salt adjuvant comprises one or two of aluminum hydroxide and aluminum phosphate. In some embodiments, the aluminum salt adjuvant comprises aluminum hydroxide. In some embodiments, the unit dose of the immunogenic composition is about 0.25 to about 0.50 mg Al 3+ or about 0.35 mg Al 3+This includes, but is not limited to, squalene-type emulsions in water (e.g., MF59 or AS03), TLR3 agonists (e.g., polyIC or polyICLC), TLR4 agonists (e.g., bacterial lipopolysaccharide derivatives, e.g., monophosphoryl lipid A (MPL) and / or saponins, e.g., Quil A or QS-21, e.g., in AS01 or AS02), TLR5 agonists (bacterial flagellin), and TLR7, TLR8 and / or TLR9 agonists (imidazoquinoline derivatives, e.g., imiquimod and resiquimod) (Coffman et al., Immunity, 33:492-503, 2010). In some embodiments, other adjuvants include MPL and aluminum agents (e.g., AS04). For veterinary applications and antibody production in non-human animals, mitotic components of Freund's adjuvants (complete and incomplete) can be used.

[0186] In some embodiments, the immunogenic composition includes pharmaceutically acceptable excipients, such as solvents, bulking agents, buffers, osmotic regulators, and preservatives (Pramanick et al., Pharma Times, 45:65-77, 2013). In some embodiments, the immunogenic composition may include excipients that function as one or more of solvents, bulking agents, buffers, and osmotic regulators (for example, sodium chloride in physiological saline can function as both an aqueous carrier and an osmotic regulator).

[0187] In some embodiments, the immunogenic composition comprises an aqueous vehicle as a solvent. Suitable vehicles include, for example, sterile water, physiological saline solution, phosphate-buffered physiological saline, and Ringer's solution. In some embodiments, the composition is isotonic.

[0188] The immunogenic composition may contain a buffer. The buffer controls the pH to inhibit the degradation of the activator during processing, storage, and optional reconstitution. Suitable buffers include, for example, salts containing acetate, citrate, phosphate, or sulfate. Other suitable buffers include, for example, amino acids such as arginine, glycine, histidine, and lysine. The buffer may further contain hydrochloric acid or sodium hydroxide. In some embodiments, the buffer maintains the pH of the composition within the range of 6 to 9. In some embodiments, the pH is greater than 6 (lower limit) and is 7 or 8. In some embodiments, the pH is less than 9 (upper limit) and is 8 or 7. That is, the pH is within the range of about 6 to 9, where the lower limit is less than the upper limit.

[0189] The immunogenic composition may contain an osmotic regulator. Suitable osmotic regulators include, for example, dextrose, glycerin, sodium chloride, glycerol, and mannitol.

[0190] The immunogenic composition may contain a volume extender. Volume extenders are particularly useful when the pharmaceutical composition is freeze-dried before administration. In some embodiments, the volume extender is a protective agent that helps stabilize and prevent the degradation of the activator during freeze-drying or spray-drying and / or storage. Suitable volume extenders are sugars (monosaccharides, disaccharides and polysaccharides), such as sucrose, lactose, trehalose, mannitol, sorbitol, glucose and raffinose.

[0191] The immunogenic composition may contain a preservative. Suitable preservatives include, for example, antioxidants and antimicrobial agents. However, in preferred embodiments, the immunogenic composition is prepared under sterile conditions and is in a single-use container, so it does not need to contain a preservative.

[0192] In some embodiments, the composition may be provided as a sterile composition. The pharmaceutical composition typically contains an effective amount of the disclosed immunogen and can be prepared by conventional art. Typically, the amount of immunogen in each dose of the immunogenic composition is selected as an amount that induces an immune response without serious adverse side effects. In some embodiments, the composition may be provided in unit dosage forms to induce an immune response in a subject. The unit dosage forms include a single pre-selected dose suitable for administration to a subject, or two or more pre-selected unit doses, or a suitable mark or measured multiple of a unit dose, and / or a measuring mechanism for administration in a unit dose or a multiple thereof. In other embodiments, the composition further comprises an adjuvant.

[0193] IV. Methods for inducing an immune response In some embodiments, this specification provides a method for inducing an immune response to an RSV surface antigen in a subject, wherein the method comprises administering to the subject an effective amount of a complex containing a recombinant polypeptide selected from SEQ ID NO: 1-16, 64-71. In some embodiments, this specification provides a method for inducing an immune response to an RSV surface antigen in a subject, wherein the surface antigen comprises an F protein or an antigenic fragment thereof, and the method comprises administering to the subject an effective amount of a complex containing a recombinant polypeptide selected from SEQ ID NO: 1-16, 64-71. In some embodiments, this specification provides a method for inducing an immune response to an RSV surface antigen in a subject, wherein the surface antigen comprises a sequence selected from SEQ ID NO: 17-47 and 72-79, and the method comprises administering to the subject an effective amount of a complex containing a recombinant polypeptide selected from SEQ ID NO: 1-16 and 64-71. In some embodiments, this specification provides a method for inducing an immune response in a subject to an RSV surface antigen, wherein the surface antigen comprises the RSV F protein or an antigenic fragment thereof, and optionally, the surface antigen comprises one or more sequences or antigenic fragments thereof from SEQ ID NO: 17-47 and 72-79, and the method comprises administering to the subject an effective amount of a complex comprising a recombinant polypeptide containing one of the sequences shown in SEQ ID NO: 1-16 and 64-71.

[0194] In some embodiments, this specification provides a method for inducing an immune response in a subject to an RSV surface antigen, wherein the surface antigen comprises an F protein or a fragment thereof, and the method comprises administering to the subject a complex or any combination of two or more complexes comprising a recombinant polypeptide containing a sequence selected from SEQ ID NO: 1 to 16 in an effective amount.

[0195] The disclosed immunogens (e.g., recombinant RSV F antigen, e.g., the trimer, protein, nucleic acid molecule (e.g., RNA molecule) or vector encoding a protocol of the disclosed recombinant RSV F antigen, or protein nanoparticles or virus-like particles containing the disclosed recombinant RSV F antigen) can be administered to a subject to induce an immune response to the corresponding RSV F antigen in the subject. In one particular example, the subject is a human. The immune response may be a protective immune response, e.g., a response that inhibits subsequent infection by the corresponding RSV. Induction of an immune response can also be used to treat or inhibit infections and diseases associated with the corresponding RSV.

[0196] In some embodiments, subjects who are already infected with RSV or are at risk of becoming infected with RSV (e.g., due to exposure to or potential exposure to RSV) can be selected and treated. After administration of the disclosed immunogen, the subject's infection status, RSV-related symptoms, or both can be monitored.

[0197] The therapeutic agents and methods of the Disclosure are intended to treat typical subjects, including humans, non-human primates, and other animals. To identify subjects to be prevented or treated according to the methods of the Disclosure, certified screening methods are used to determine the target or suspected disease or disease-related risk factors, or to determine the subject's existing disease or disease status. These screening methods include, for example, routine tests to determine the target or suspected disease or environmental, familial, occupational, and other such risk factors, as well as diagnostic methods to detect and / or characterize RSV infection, such as various ELISAs and other immunoassays. These and other conventional methods enable clinicians to select patients in need of treatment using the methods and pharmaceutical compositions of the Disclosure. According to these methods and principles, the compositions may be administered as an independent preventive or therapeutic scheme, or as a follow-up, adjunct, or coordinated treatment regimen to other treatments, according to the teachings herein or other conventional methods.

[0198] The disclosed immunogen, e.g., RSV F antigen, e.g., trimer, protein, can be used for prophylactic or therapeutic purposes. When provided prophylactically, the disclosed therapeutic agent is provided before any symptoms appear, e.g., before infection. Prophylactic administration of the disclosed therapeutic agent is used to prevent or improve any secondary infection. When provided therapeutically, the disclosed treatment is provided at or after the onset of symptoms of the disease or infection, e.g., after the appearance of RSV infection symptoms corresponding to the RSV F antigen, or after a diagnosis of RSV infection. Thus, the therapeutic agent may be provided before exposure to RSV is expected, thereby reducing the expected severity, duration, or degree of symptoms of the infection and / or related disease after exposure to the virus, after suspected exposure, or after the actual onset of infection.

[0199] The immunogens and immunogenic compositions described herein are provided to a subject (preferably human) in an amount effective in inducing or enhancing an immune response to the RSV F antigen. The actual dose of the disclosed immunogen will vary depending on many factors, such as the subject's disease signs and specific conditions (e.g., the subject's age, build, health status, severity of symptoms, susceptibility factors, etc.), the time and route of administration, other drugs or treatments administered concurrently, and the specific pharmacology of the composition for inducing the desired activity or biological response in the subject. The dose regimen can be adjusted to provide an optimal prophylactic or therapeutic response.

[0200] An immunogenic composition comprising one or more disclosed immunogens can be used in coordinated (or primary-booster) immunization protocols or combination formulations. In some embodiments, novel combination immunogenic compositions and coordinated immunization protocols use separate immunogens or formulations, each immunogen or formulation inducing an antiviral immune response, e.g., an immune response to the RSV F antigen. The separate immunogenic compositions that induce an antiviral immune response can be combined into a multivalent immunogenic composition administered to the subject in a single immunization step, or they can be administered separately (in a monovalent immunogenic composition) in a coordinated (or primary-booster) immunization protocol.

[0201] Multiple booster immunizations may be administered, and each booster immunization may use a different disclosed immunogen. In some cases, a booster immunization may use a different booster immunization or the same immunogen as the initial immunization. The initial immunization and booster immunizations may be administered as a single dose or multiple doses, for example, two, three, four, five, six, or more doses to a subject over several days, weeks, or months. Multiple booster immunizations may also be administered, for example, one to five times (e.g., one, two, three, four, or five booster immunizations) or more. Different doses may be used in a series of sequential immunizations. For example, a relatively large dose may be used for the first immunization, and a relatively small dose for the booster immunizations.

[0202] In some embodiments, the booster immunization may be administered approximately 2 weeks, 3–8 weeks, or 4 weeks after the initial immunization, or several months after the initial immunization. In some embodiments, the booster immunization may be administered approximately 5, 6, 7, 8, 10, 12, 18, or 24 months after the initial immunization, or more or less than that period after the initial immunization. Booster immunizations may be used regularly and additionally at appropriate times to enhance the subject's "immunological memory." The appropriateness of selected vaccination parameters, such as formulation, dosage, and schedule, can be determined by taking aliquots of serum from the subject and measuring antibody titers during the course of the immunization program. The clinical condition of the subject can also be monitored to obtain the desired effect, such as prevention of infection or improvement of disease condition (e.g., reduction of viral load). If such monitoring indicates that vaccination is not optimal, the subject can be boost-immunized with an additional dose of immunogenic composition, and the vaccination parameters can be modified in a manner that is expected to enhance the immune response.

[0203] In some embodiments, the primary-booster immunization method may include an inoculation protocol that provides a subject with a DNA primary immunization and a protein booster immunization vaccine. The method may include two or more doses of nucleic acid molecules or proteins.

[0204] In the case of protein-based therapies, each human dose typically contains 1 to 1000 μg, for example, about 1 μg to about 100 μg, for example, about 1 μg to about 50 μg, for example, 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.

[0205] The amount used in an immunogenic composition is selected based on the subject population (e.g., infants or the elderly). Standard studies, including observation of the subject's antibody titer and other responses, can determine the optimal dose of a particular composition. As recognized, the therapeutically effective amount of the disclosed immunogen in an immunogenic composition, e.g., the disclosed recombinant RSV F antigen (e.g., trimer, protein), viral vector, or nucleic acid molecule, may include amounts that, for example, in an initial-additional immunization protocol, are not effective in inducing an immune response with a single dose but are effective after multiple doses.

[0206] Following administration of the immunogens disclosed herein, the subject's immune system typically responds to the immunogenic composition by producing antibodies specific to the RSV F protein peptide contained in the immunogen. Such a response signifies that an immunoeffective dose has been delivered to the subject.

[0207] In some embodiments, the antibody response of a subject is determined in the context of evaluating an effective dose / immunization protocol. In most cases, it is sufficient to assess the antibody titer in serum or plasma obtained from the subject. The decision of whether or not to administer an additional immunization and / or to change the amount of therapeutic agent administered to the individual can be based at least in part on the antibody titer level. The antibody titer level can be based, for example, on an immunobinding assay, which measures the concentration of antibodies in serum that bind to an antigen (e.g., recombinant RSV F antigen, e.g., trimer, protein).

[0208] The method is effective even without completely eliminating, mitigating, or preventing RSV infection. For example, inducing an immune response to RSV with one or more of the disclosed immunogens compared to RSV infection in the absence of immunogens can reduce or inhibit RSV infection by a desired amount, e.g., 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%, and even at least 100% (eliminating or preventing detectable infected cells). In other embodiments, the disclosed method can reduce or inhibit RSV replication. The method is effective even without completely eliminating RSV replication. For example, compared to RSV replication in the absence of an immunogen, an immune response with one or more disclosed immunogens can reduce the replication of the corresponding RSV by a desired amount, e.g., 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%, and even at least 100% (eliminating or preventing detectable RSV replication).

[0209] In some embodiments, an adjuvant is administered to the subject along with the disclosed immunogen. In other embodiments, the disclosed immunogen is administered to the subject within a sufficient time after the administration of the adjuvant to induce an immune response.

[0210] An approach to administering a single nucleic acid is direct immunization using plasmid DNA, such as a mammalian expression plasmid. Immunotherapy using nucleic acid constructs is well known in the art and is disclosed, for example, in U.S. Patent No. 5,643,578 (which describes a method for immunizing vertebrates by introducing DNA encoding a desired antigen to induce a cell-mediated or humoral response), and U.S. Patents No. 5,593,972 and 5,817,637 (which describe the operable linking of a nucleic acid sequence encoding an antigen to a regulatory sequence that enables its expression). U.S. Patent No. 5,880,103 describes several methods for delivering immunogenic peptides or nucleic acids encoding other antigens to organisms. The methods involve the nucleic acid (or the synthetic peptide itself) and an immunostimulatory construct or ISCOMS. TM , in other words, cholesterol and Quill A TM It contains liposome delivery of a negatively charged 30-40 nm cage-like structure that spontaneously forms after mixing with (saponin). ISCOMS TM The vehicle was delivered using the antigen to generate protective immunity (Mowat and Donachie, Immunol. Today 12:383, 1991). ISCOMS TM Low doses of antigen, approximately 1 μg, encapsulated within the material were found to induce a class I-mediated CTL response (Takahashi et al., Nature 344:873, 1990).

[0211] In some embodiments, the disclosed immunogen is expressed in a subject using a plasmid DNA vaccine. For example, a nucleic acid molecule encoding the disclosed immunogen can be administered to a subject to induce an immune response to the RSV F antigen. In some embodiments, the nucleic acid molecule may be contained in a plasmid vector for DNA immunization, such as the pVRC8400 vector (described in Barouch et al., J. Virol, 79, 8828-8834, 2005, which is incorporated herein by reference).

[0212] In another method of immunization using nucleic acids, the disclosed recombinant RSV F antigen, e.g., trimer, protein, can be expressed by an attenuated viral host or vector or bacterial vector. Recombinant vaccinia virus, adeno-associated virus (AAV), herpesvirus, retrovirus, cytogmegrovirus, or other viral vectors may be used to induce a CTL response by expressing the peptide or protein. For example, U.S. Patent No. 4,722,848 describes a vaccinia virus vector and method that can be used in an immunization protocol. BCG (Bacillus calmette-Guérin) has provided another vector for expressing the peptide (see Stover, Nature 351:456-460, 1991).

[0213] In one embodiment, the nucleic acid encoding the disclosed recombinant RSV F antigen is directly introduced into cells. For example, the nucleic acid is loaded into gold microspheres by a standard method and placed in Bio-Rad's HELIOS TM It is introduced into the skin using devices such as gene guns. The nucleic acid may be "naked" and consists of plasmids under the control of a strong promoter. Typically, the DNA is injected into the muscle, but it may also be injected directly into other sites. The injection dose is usually about 0.5 μg / kg to about 50 mg / kg, and typically about 0.005 mg / kg to about 5 mg / kg (see, for example, U.S. Patent No. 5,589,466).

[0214] For example, nucleic acids are loaded into gold microspheres using a standard method, and Bio-Rad's HELIOS TM It is introduced into the skin using devices such as gene guns. The nucleic acid may be "naked" and consists of plasmids under the control of a strong promoter. Typically, the DNA is injected into the muscle, but it may also be injected directly into other sites. The injection dose is usually about 0.5 μg / kg to about 50 mg / kg, and typically about 0.005 mg / kg to about 5 mg / kg (see, for example, U.S. Patent No. 5,589,466).

[0215] In another embodiment, an mRNA-based immunization protocol can be used to directly deliver the nucleic acid encoding the disclosed recombinant RSV F antigen into cells. In some embodiments, mRNA-based nucleic acid-based vaccines can provide an effective alternative to the aforementioned approaches. mRNA vaccines eliminate safety concerns regarding the integration of DNA into the host genome and can be translated directly in the host cytoplasm. Furthermore, the simple cell-free in vitro synthesis of RNA avoids the manufacturing complexities associated with viral vectors. Two exemplary forms of RNA-based vaccination that may be used to deliver the nucleic acids encoding the recombinant RSV F antigen disclosed are 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," Naturebiotechnology, 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 Heterologous Subtype Virus Infections"). This includes Heterosubtypic Viral Challenge, PLoSOne, 11(8):e0161193, 2016, and Brito et al., "Self-amplifying mRNA vaccines," AdvGenet., 89:179-233, 2015).

[0216] In some embodiments, a neutralizing immune response was induced in subjects by administering one or more disclosed immunogens in therapeutically effective doses. To evaluate neutralizing activity, serum can be collected from subjects at an appropriate time after immunization, frozen, stored, and tested for neutralization. Assay methods for neutralizing activity are known to those skilled in the art and are further described herein, and include, but are 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 group of RSV pseudoviruses.

[0217] In some embodiments, a neutralizing immune response was induced in subjects by administering one or more disclosed immunogens in therapeutically effective doses. To evaluate neutralizing activity, serum can be collected from subjects at an appropriate time after immunization, frozen, stored, and tested for neutralization. Assay methods for neutralizing activity are known to those skilled in the art and are further described herein, and include, but are 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 group of RSV pseudoviruses.

[0218] In some embodiments, the neutralizing immune response induced by the immunogens disclosed herein produced neutralizing antibodies against RSV. In some embodiments, the neutralizing antibodies herein bind to cellular receptors or co-receptors of RSV or its components. Nucleolins are co-receptors of RSV entry and also mediate cellular entry of influenza viruses, parainfluenza viruses, certain enteroviruses, and tularemia-causing bacteria. The binding of pre-fusion RSV-F glycoprotein to insulin-like growth factor 1 receptor (IGF1R) can also mobilize nucleolins from the cell nucleus to the cytoplasmic membrane and bind to RSV-F on the viral particle by causing activation of protein kinase Cζ (PKCζ). In some embodiments, the viral receptor or co-receptor is a paramyxovirus receptor or co-receptor, preferably a pneumonia virus receptor or co-receptor, and more preferably an RSV receptor or co-receptor. For example, CCR1, CCR2, CCR3, CCR4, CCR5 and / or CCR8 receptors may relate to human RSV infection. RhoA is another example of a host cell RSV receptor or co-receptor. In some embodiments, the neutralizing antibodies herein modulate, reduce, antagonize, mitigate, block, inhibit, resolve and / or interfere with at least one RSV activity or binding or RSV receptor activity or binding, e.g., RSV release, RSV receptor signaling, membrane RSV cleavage, RSV activity, RSV production and / or synthesis, in vitro, in situ and / or in vivo. In some embodiments, the immunogens disclosed herein induce a neutralizing antibody against RSV, which modulates, reduces, antagonizes, mitigates, blocks, inhibits, resolves and / or interferes with the binding of RSV to RSV receptors or co-receptors, e.g., nucleolin, IGF1R, CCR1, CCR2, CCR3, CCR4, CCR5, CCR8 and / or RhoA.

[0219] V. Products or Kits Products or kits comprising recombinant polypeptides, proteins, and immunogenic compositions are also provided. The product may include a container and a label or packaging insert located on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, test tubes, and IV infusion bags. The container may be formed from a variety of materials, such as glass or plastic. In some embodiments, the container has a sterile access port. Exemplary containers include intravenous infusion bags, vials, and containers equipped with stoppers that can be punctured with an injection needle. The product or kit may further include a packaging insert indicating that the composition can be used to treat a particular disease, such as the disease described herein (e.g., RSV infection). Alternatively, or additionally, the product or kit may further include another or the same container of a pharmaceutically acceptable buffer. It may further include other materials, such as other buffers, diluents, filters, needles, and / or syringes.

[0220] The label or packaging insert may indicate that the composition is used to treat an individual's RSV infection. The label or packaging insert on or associated with the container may indicate instructions regarding the recomposition and / or use of the formulation. The label or packaging insert may also indicate that the formulation is used or intended to treat or prevent an individual's RSV infection by subcutaneous, intravenous or other means of administration.

[0221] In some embodiments, the container contains a composition or a combination of the composition with another composition that can effectively treat, prevent and / or diagnose the disease. The product or kit comprises (a) a first container containing a composition (i.e., a first drug), wherein the composition comprises the immunogenic composition or its protein or recombinant polypeptide, and (b) a second container containing a composition (i.e., a second drug), wherein the composition comprises another drug, such as an adjuvant or another therapeutic agent, and the product or kit further includes a description on the label or packaging insert relating to the treatment of the subject with an effective amount of the second drug.

[0222] term Unless otherwise defined, all technical terms, symbols, and other technical and scientific terms used herein are intended to have the same meaning as those generally understood by those skilled in the art in the field to which the claimed subject matter pertains. Where applicable, for clarity and / or convenience of reference, this specification defines terms that have a generally understood meaning, and such definitions contained herein should not necessarily be construed as representing a substantial difference from those generally understood in the art.

[0223] The terms “polypeptide” and “protein” are used interchangeably to refer to polymers of amino acid residues and do not limit the minimum length. Polypeptides (including the receptor and other polypeptides, e.g., linkers or peptides) may contain amino acid residues such as native and / or non-native amino acid residues. The terms further include post-expression modifications of polypeptides, e.g., glycosylation, sialylation, acetylation, and phosphorylation. In some embodiments, polypeptides may contain modifications relating to the natural or native sequence, provided that the protein retains the required activity. These modifications may be intentional, such as through site-directed mutagenesis, or accidental, resulting from mutations in the host producing the protein or errors in PCR amplification.

[0224] As used herein, “subject” is a mammal, e.g., human or other animal, and is usually human. In some embodiments, the subject (e.g., patient) to whom one or more drugs, cells, cell groups or compositions are administered is a mammal, and is usually a primate, e.g., human. In some embodiments, the primate is a monkey or ape. The subject may be male or female and may be of any appropriate age, including infants, juveniles, adolescents, adults and elderly subjects. In some embodiments, the subject may be a non-primate mammal, e.g., a rodent.

[0225] As used herein, “treatment” (and its grammatical variations) means the complete or partial improvement or reduction of a disease or disorder, or any symptoms, adverse reactions or outcomes, or phenotype associated therewith. The ideal effects of treatment include, but are not limited to, prevention of the onset or recurrence of the disease, reduction of symptoms, reduction of any direct or indirect pathological consequences of the disease, prevention of metastasis, slowing of the rate of disease progression, improvement or reduction of the disease state, and remission or mitigation of prognosis. The foregoing terms do not imply a complete cure of the disease, or complete elimination of any symptoms or consequences, or effectiveness against all symptoms or outcomes.

[0226] As used herein, “delaying disease progression” means slowing, hindering, slowing, delaying, stabilizing, inhibiting, and / or postponing the progression of a disease (e.g., cancer). This delay can be of varying lengths depending on the disease history and / or the individual being treated. In some embodiments, delaying sufficiently or significantly so that the individual does not develop the disease can effectively encompass prevention. For example, terminal cancer, such as the development of metastases, can be delayed.

[0227] As used herein, “preventing” includes providing prevention with respect to the onset or recurrence of a disease in an individual who may be susceptible to the disease but has not been diagnosed with the disease. In some embodiments, the cells and compositions provided are used to delay the onset of the disease or to slow the progression of the disease.

[0228] As used herein, “suppressing” a function or activity means reducing its function or activity when compared to the same condition or to a different condition in a manner other than the target condition or parameter. For example, cells that suppress tumor growth reduce the rate of tumor growth compared to the rate of tumor growth in the absence of such cells.

[0229] In the context of administration, the “effective dose” of a drug, such as a drug formulation, cells, or composition, refers to the amount in terms of dosage / quantity and duration required to effectively achieve the desired outcome, such as a therapeutic or prophylactic result.

[0230] The “therapeutic effective dose” of a drug, such as a drug formulation, cells, or composition, refers to the amount in dose and duration necessary to effectively achieve the desired therapeutic outcome, e.g., the treatment of a disease, disorder, or disability, and / or the pharmacokinetic or pharmacodynamic effects of the treatment. The therapeutic effective dose can vary depending on many factors, e.g., the disease state of the subject, age, sex, and weight, and the population of cells administered. In some embodiments, the methods provided involve administering the cells and / or composition in an effective dose (e.g., a therapeutic effective dose).

[0231] The "prophylactic effective dose" refers to the amount in dose and time required to effectively achieve the desired prophylactic effect. Typically, though not always, the prophylactic effective dose is smaller than the therapeutic effective dose because the prophylactic dose is administered to the subject before or in the early stages of the disease. In some embodiments, the prophylactic effective dose may be greater than the therapeutic effective dose when the tumor burden is low.

[0232] As used herein, the term “approximately” refers to the normal range of error for each value, which is readily apparent to those skilled in the art. “Approximately” numerical values ​​or parameters referred to herein include (described) embodiments relating to the numerical value or parameter itself.

[0233] As used herein, unless otherwise clearly indicated by the context, the singular forms “one,” “one kind,” and “the” include multiple referents. For example, “one” or “one kind” means “at least one (kind)” or “one (kind) or more (kinds).”

[0234] Throughout this disclosure, various aspects of the claimed subject matter are presented in the form of scopes. It should be understood that these scope descriptions are for convenience and brevity only and should not be interpreted as inflexible limitations on the scope of the claimed subject matter. Therefore, the scope descriptions should be considered to clearly disclose all possible sub-scopes and the individual numerical values ​​within those scopes. For example, where a range of values ​​is provided, it should be understood that each intermediate value between the upper and lower limits of that range, and any other description or intermediate value within that range, are included in the claimed subject matter. These smaller upper and lower limits may independently be included in smaller ranges and are also included in the scope of the claimed subject matter, subject to any limitations explicitly excluded within those ranges. If the range includes one or more limit values, the scope excluding any one or both of those limit values ​​is also included in the claimed subject matter. This applies regardless of the breadth of the range.

[0235] As used herein, a composition refers to any mixture of two or more products, substances, or compounds containing cells. It may be a solution, suspension, liquid, powder, paste, aqueous solution, non-aqueous solution, or any combination thereof.

[0236] As used herein, the term “vector” refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is ligated. The term includes vectors as self-replicating nucleic acid structures, and vectors incorporated into the genome of a host cell into which they are introduced. Some vectors can guide the expression of nucleic acids to which they are operably ligated. Such vectors are referred herein to as “expression vectors.”

[0237] 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 the C-terminal propeptide of collagen, wherein the C-terminal propeptide of the recombinant polypeptide forms an interpolypeptide disulfide bond.

[0238] Embodiment 2. The protein according to Embodiment 1, wherein the RSV belongs to subtype A or subtype B.

[0239] Embodiment 3. The protein according to Embodiment 1 or 2, wherein the epitope is a linear epitope or a conformational epitope.

[0240] Embodiment 4. The protein according to any one of Embodiments 1 to 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.

[0241] Embodiment 5. The protein according to any one of Embodiments 1 to 4, wherein the F protein peptide comprises a signal peptide, a heptarepeat sequence C (HRC) peptide, a pep27 peptide, a fusion peptide (FP), a heptarepeat sequence A (HRA) peptide, a domain I peptide, a domain II peptide, or a heptarepeat sequence B (HRB) peptide, or any combination thereof.

[0242] Embodiment 6. The protein according to any one of Embodiments 1 to 5, wherein the F protein peptide includes the F1 subunit of the F protein but does not include the F2 subunit, or vice versa.

[0243] Embodiment 7. The protein according to any one of Embodiments 1 to 6, wherein the F protein peptide comprises an F1 subunit and an F2 subunit of the F protein, optionally lacking pep27, and optionally linking the F1 subunit and the F2 subunit via a disulfide bond or an artificially introduced linker.

[0244] Embodiment 8. The protein according to any one of Embodiments 1 to 7, wherein the F protein peptide does not include a transmembrane (TM) domain peptide and / or a cytoplasmic (CP) domain peptide.

[0245] Embodiment 9. The protein according to any one of Embodiments 1 to 8, wherein the F protein peptide includes a protease cleavage site, where the protease is optionally furin protease, trypsin, factor Xa, or cathepsin L.

[0246] Embodiment 10. The protein according to any one of Embodiments 1 to 8, wherein the F protein peptide does not contain a protease cleavage site, and the protease is optionally furin protease, trypsin, factor Xa, or cathepsin L.

[0247] Embodiment 11. The protein according to any one of Embodiments 1 to 10, wherein the F protein peptide is soluble or does not directly bind to a lipid bilayer, such as a membrane or viral envelope.

[0248] Embodiment 12. The protein according to any one of Embodiments 1 to 11, wherein the F protein peptide between the recombinant polypeptides of the protein is the same or different.

[0249] Embodiment 13. The protein according to any one of Embodiments 1 to 12, wherein the F protein peptide is directly fused to the C-terminal propeptide or linked to the C-terminal propeptide via a linker, for example, a linker comprising a glycine-XY repeat sequence, where X and Y are independently any amino acid and optionally proline or hydroxyproline.

[0250] Embodiment 14. The protein according to any one of Embodiments 1 to 13, wherein the protein is soluble or does not directly bind to a lipid bilayer, such as a membrane or viral envelope.

[0251] Embodiment 15. The protein according to any one of Embodiments 1 to 14, wherein the protein can form a rosette-like oligomer containing an F protein peptide trimer.

[0252] Embodiment 16. The protein according to any one of Embodiments 1 to 15, wherein the protein can bind to cell surface adhesion factors or receptors of a subject, and optionally the subject is a mammal, such as a primate, such as a human.

[0253] Embodiment 17. The C-terminal propeptide is a protein belonging to human collagen, as described in any one of Embodiments 1 to 16.

[0254] Embodiment 18. The protein according to any one of Embodiments 1 to 17, wherein the C-terminal propeptide comprises a C-terminal polypeptide or fragment thereof of pro α1(I), pro α1(II), pro α1(III), pro α1(V), pro α1(XI), pro α2(I), pro α2(V), pro α2(XI), or pro α3(XI).

[0255] Embodiment 19. The protein according to any one of Embodiments 1 to 18, wherein the C-terminal propeptide between the recombinant polypeptides is the same or different.

[0256] Embodiment 20. The protein according to any one of Embodiments 1 to 19, wherein the C-terminal propeptide comprises an amino acid sequence having SEQ ID NO: 48 or at least 90% identity thereto, which can form a polypeptide-to-polypeptide disulfide bond and trimerize the recombinant polypeptide.

[0257] Embodiment 21. The protein according to any one of Embodiments 1 to 19, wherein the C-terminal propeptide comprises an amino acid sequence having SEQ ID NO: 49 or at least 90% identity thereto, which can form a polypeptide-to-polypeptide disulfide bond and trimerize the recombinant polypeptide.

[0258] Embodiment 22. The protein according to any one of Embodiments 1 to 19, wherein the C-terminal propeptide comprises an amino acid sequence having SEQ ID NO: 50 or at least 90% identity thereto, which can form a polypeptide-to-polypeptide disulfide bond and trimerize the recombinant polypeptide.

[0259] Embodiment 23. The protein according to any one of Embodiments 1 to 19, wherein the C-terminal propeptide comprises an amino acid sequence having SEQ ID NO: 51 or at least 90% identity thereto, which can form a polypeptide-to-polypeptide disulfide bond and trimerize the recombinant polypeptide.

[0260] Embodiment 24. The protein according to any one of Embodiments 1 to 19, wherein the C-terminal propeptide comprises an amino acid sequence having SEQ ID NO: 52 or at least 90% identity thereto, which can form a polypeptide-to-polypeptide disulfide bond and trimerize the recombinant polypeptide.

[0261] Embodiment 25. The protein according to any one of Embodiments 1 to 19, wherein the C-terminal propeptide comprises an amino acid sequence having SEQ ID NO: 53 or at least 90% identity thereto, which can form a polypeptide-to-polypeptide disulfide bond and trimerize the recombinant polypeptide.

[0262] Embodiment 26. The protein according to any one of Embodiments 1 to 19, wherein the C-terminal propeptide comprises an amino acid sequence having SEQ ID NO: 54 or at least 90% identity thereto, which can form a polypeptide-to-polypeptide disulfide bond and trimerize the recombinant polypeptide.

[0263] Embodiment 27. The protein according to any one of Embodiments 1 to 19, wherein the C-terminal propeptide comprises an amino acid sequence that can form a polypeptide-to-polypeptide disulfide bond and trimerize the recombinant polypeptide, having at least 90% identity with any one of SEQ ID NO: 55 to 59.

[0264] Embodiment 28. The protein according to any one of Embodiments 1 to 19, wherein the C-terminal propeptide comprises an amino acid sequence having SEQ ID NO: 60 or at least 90% identity thereto, which can form a polypeptide-to-polypeptide disulfide bond and trimerize the recombinant polypeptide.

[0265] Embodiment 29. The protein according to any one of Embodiments 1 to 19, wherein the C-terminal propeptide comprises an amino acid sequence that can form a polypeptide disulfide bond and trimerize the recombinant polypeptide, which is one of SEQ ID NO: 61 to 63 or has at least 90% identity thereto.

[0266] Embodiment 30. The protein according to any one of Embodiments 1 to 29, wherein the C-terminal propeptide comprises an amino acid sequence containing a glycine-XY repeat sequence linked to the N-terminus of any one of SEQ ID NO: 48 to 63, where X and Y are independently any amino acids, and optionally proline or hydroxyproline, or an amino acid sequence having at least 90% identity thereto, which can form an interpolypeptide disulfide bond and trimerize the recombinant polypeptide.

[0267] Embodiment 31. The protein according to any one of Embodiments 1 to 30, wherein the F protein peptide in each recombinant polypeptide exhibits a pre-fusion conformation or a post-fusion conformation, and optionally, the protein comprises a rosette-like oligomer containing a cane-shaped rod-shaped F protein peptide trimer.

[0268] Embodiment 32. The protein according to any one of Embodiments 1 to 31, wherein the F protein peptide in each recombinant polypeptide contains one of SEQ ID NO: 72 to 79 or an amino acid sequence having at least 80% identity thereto.

[0269] Embodiment 33. The recombinant polypeptide comprises one of SEQ ID NO: 76-79 or an amino acid sequence having at least 80% identity thereto, as described in any one of Embodiments 1-31.

[0270] Embodiment 34. An immunogen comprising the protein described in any one of Embodiments 1 to 33.

[0271] Embodiment 35. Protein nanoparticles comprising a protein according to any one of Embodiments 1 to 33, which is directly or indirectly linked to the nanoparticles.

[0272] Embodiment 36. Virus-like particles (VLPs) comprising the protein described in any one of Embodiments 1 to 33.

[0273] Embodiment 37. Isolated nucleic acids encoding one, two, three or more recombinant polypeptides of the protein described in any one of Embodiments 1 to 33.

[0274] Embodiment 38. The isolated nucleic acid according to Embodiment 37, wherein the polypeptide encoding the F protein peptide is fused in frame with the polypeptide encoding the C-terminal propeptide of collagen.

[0275] Embodiment 39. The isolated nucleic acid is the isolated nucleic acid according to Embodiment 37 or 38, which is operably linked to a promoter.

[0276] Embodiment 40. The isolated nucleic acid is the isolated nucleic acid according to any one of Embodiments 37 to 39, which is a DNA molecule.

[0277] Embodiment 41. The isolated nucleic acid is an RNA molecule, and optionally, an mRNA molecule, for example, a nucleoside-modified mRNA, a non-amplified mRNA, a self-amplifying mRNA or a trans-amplified mRNA, which is the isolated nucleic acid according to any one of Embodiments 37 to 39.

[0278] Embodiment 42. A vector comprising the isolated nucleic acid according to any one of Embodiments 37 to 41.

[0279] Embodiment 43. The vector is a viral vector, which is the vector according to Embodiment 42.

[0280] Embodiment 44. A virus, pseudovirus or cell comprising the vector according to Embodiment 42 or 43, and optionally, the virus or cell has a recombinant genome, which is a virus, pseudovirus or cell.

[0281] Embodiment 45. An immunogenic composition comprising the 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 agent.

[0282] Embodiment 46. A vaccine comprising the immunogenic composition according to Embodiment 45 and an optional adjuvant, and optionally, the vaccine is a subunit vaccine, and / or optionally, the vaccine is a prophylactic and / or therapeutic vaccine.

[0283] Embodiment 47. The vaccine comprises a plurality of different adjuvants, which is the vaccine according to Embodiment 46.

[0284] Embodiment 48. A method for producing a protein, the method comprising expressing an isolated nucleic acid or vector described in any one of Embodiments 37 to 43 in a host cell to produce a protein described in any one of Embodiments 1 to 33, and purifying the protein.

[0285] Embodiment 49. A protein produced by the method described in Embodiment 48.

[0286] Embodiment 50. A method for inducing an immune response in a subject to an RSV F protein peptide or a fragment or epitope thereof, the method comprising administering to the subject an effective amount of a protein, immunogen, protein nanoparticles, VLP, isolated nucleic acid, vector, virus, pseudovirus, cell, immunogenic composition or vaccine described in any one of Embodiments 1 to 47 and 49 to induce the immune response.

[0287] Embodiment 51. The method according to Embodiment 50, wherein the method is for treating or preventing RSV infection.

[0288] Embodiment 52. The method according to Embodiment 50 or 51, wherein the immune response causes the replication of RSV in the subject to be suppressed or reduced.

[0289] Embodiment 53. The method according to any one of Embodiments 50 to 52, wherein the immune response comprises a cell-mediated response and / or a humoral response, and optionally comprises the production of one or more neutralizing antibodies, such as polyclonal antibodies or monoclonal antibodies.

[0290] Embodiment 54. The method according to any one of Embodiments 50 to 53, wherein the immune response is against the F protein peptide or a fragment or epitope thereof of the RSV, but not against the C-terminal propeptide.

[0291] Embodiment 55. The method according to any one of Embodiments 50 to 54, wherein the administration does not result in antibody-dependent enhancement (ADE) due to the subject's prior exposure to one or more RSVs.

[0292] Embodiment 56. The method according to any one of Embodiments 50 to 55, wherein the administration does not result in antibody-dependent enhancement (ADE) when the subject is subsequently exposed to one or more RSVs.

[0293] Embodiment 57. The method according to any one of Embodiments 50 to 56, further comprising a priming step and / or a boosting step.

[0294] Embodiment 58. The method according to any one of Embodiments 50 to 57, wherein the administration step is carried out by local, 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, intravitreous, subretinal, intra-articular, peri-articular, local, or surface administration.

[0295] Embodiment 59. The method according to any one of Embodiments 50 to 58, wherein the effective amount is administered as a single dose or in a schedule of multiple doses with one or more intervals between doses.

[0296] Embodiment 60. The method according to any one of Embodiments 50 to 59, wherein the effective amount is administered without adjuvant.

[0297] Embodiment 61. The method according to any one of Embodiments 50 to 59, wherein the effective amount is administered together with an adjuvant.

[0298] Embodiment 62. A method comprising administering an effective amount of the protein described in any one of Embodiments 1 to 33 to a subject to produce a neutralizing antibody or neutralizing antiserum against RSV in the subject.

[0299] Embodiment 63. The method according to embodiment 62, wherein the subject is a mammal, optionally a human or non-human primate.

[0300] Embodiment 64. The method according to embodiment 62 or 63, further comprising isolating the neutralizing antibody or neutralizing antiserum from the subject.

[0301] Embodiment 65. The method according to embodiment 64, comprising administering an effective amount of the isolated neutralizing antibody or neutralizing antiserum to a human subject by passive immunization to prevent or treat RSV infection.

[0302] Embodiment 66. The neutralizing antibody or neutralizing antiserum against RSV comprises a polyclonal antibody against the RSV F protein peptide or a fragment or epitope thereof, and optionally, the neutralizing antibody or neutralizing antiserum does not contain or substantially does not contain an antibody against the collagen C-terminal propeptide. The method according to any one of embodiments 62 to 65.

[0303] Embodiment 67. The neutralizing antibody comprises a monoclonal antibody against the RSV F protein peptide or a fragment or epitope thereof, and optionally, the neutralizing antibody does not contain or substantially does not contain an antibody against the collagen C-terminal propeptide. The method according to any one of embodiments 62 to 65.

[0304] Embodiment 68. 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 inducing an immune response against RSV in a subject and / or for treating or preventing RSV infection.

[0305] Embodiment 69. Uses of the protein, immunogen, protein nanoparticles, VLP, isolated nucleic acid, vector, virus, pseudovirus, cell, immunogenic composition, or vaccine according to 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 RSV infection.

[0306] Embodiment 70. Uses of proteins, immunogens, protein nanoparticles, VLPs, isolated nucleic acids, vectors, viruses, pseudoviruses, cells, immunogenic compositions, or vaccines according to any one of Embodiments 1 to 47 and 49 for inducing an immune response to RSV in a subject and / or for producing a drug or prophylactic agent for treating or preventing RSV infection.

[0307] Embodiment 71. A method for analyzing a sample, the method comprising contacting the sample with a protein described in any one of Embodiments 1 to 33, and detecting a binding between the protein and an analyte that can specifically bind to the F protein peptide or a fragment or epitope thereof of the RSV.

[0308] Embodiment 72. The method according to Embodiment 71, wherein the analyte is an antibody, receptor, or cell that recognizes the F protein peptide or a fragment thereof or epitope.

[0309] Embodiment 73. The method according to Embodiment 71 or 72, wherein the binding indicates the presence of the analyte in the sample and / or infection of the RSV in the subject from which the sample originates.

[0310] Embodiment 74. A kit comprising a protein according to any one of Embodiments 1 to 33 and a substrate, pad, or vial for containing or immobilizing the protein, wherein the kit is optionally an ELISA or lateral flow assay kit.

[0311] Examples The following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

[0312] Example 1: Recombinant polypeptide containing RSV F protein peptide The RSV F glycoprotein construct is derived from the RSV A2 strain (registration number AAC55970). The coding sequence of residues 1-520 of the F protein peptide was codon-optimized and synthesized, and subcloned into the mammalian expression vector pTPRIMER-T0D, which encodes the Hind III and Bgl II sites of human α1 collagen C-propeptide. Figure 1 is a schematic diagram showing exemplary recombinant polypeptides. A: SCB-N25 fusion protein, B: SCB-N25C, C: SCB-N25A, D: SCB-N25T, E: SCB-N25Y, F: SCB-N25G, G: SCB-N25S, H: RSV-WT-F. Here, SCB-N25: SEQ ID NO: 8 SCB-N20: SEQ IQ NO: 12 SCB-N25C:SEQ ID NO:64 SCB-N25A: SEQ ID NO: 80 SCB-N25T:SEQ ID NO:81 SCB-N25Y:SEQ ID NO:82 SCB-N25G:SEQ ID NO:83 SCB-N25S: SEQ ID NO: 84 RSV-WT-F:SEQ ID NO:2

[0313] Example 2: Expression of pre-fusion F protein of recombinant polypeptide containing RSV F protein peptide On day 3, the expression level of the pre-fusion F protein in the HEK-293T cell supernatant was determined by measuring the optical density using an ELISA kit (Figure 2). HEK-293T cells were transiently transfected with a plasmid containing the variant shown on the horizontal axis, and after three days, the culture supernatant was collected and centrifuged to remove cells and cell debris.

[0314] 293T cells stably subcultured in DMEM (10% FBS) were diluted to 0.75 million / mL, and 0.5 mL of cells and 1.5 mL of DMEM were seeded into a 6-well plate. The cells were incubated overnight at 37°C in a CO2 incubator. 300 μL of DMEM (-FBS) was placed in a 1.5 mL centrifuge tube, 9 μL of FuGene6 was added, and the mixture was thoroughly mixed. After standing at room temperature for 5 minutes, 100 μL of the DMEM-FuGene6 mixture was placed in three 1.5 mL centrifuge tubes, and 1 μg of recombinant plasmid was added to each. After thoroughly mixing, the mixture was left to stand at room temperature for 20 minutes. 1 mL of culture medium was removed from each well of the 6-well plate used for 293T cell culture. All of the plasmid-FuGene6 mixture was then added to each well, and after shaking well to ensure homogeneity, the plates were incubated. After three days, the cell supernatant was collected and protein expression was detected. Subsequently, the OD value was determined by analyzing the supernatant after centrifugation at OD280 using Synagis and AM22 antibody. A: Shows a comparison between high optical density (OD) values ​​of the variants; B: Shows a comparison between intermediate OD values ​​of the variants. N25C showed a higher expression level than the pre-fusion F protein of the other variants. The publicly available US10,899,800B2 of SCB-N20 was used with the E161P / S215P mutation as a control.

[0315] On day 3, the expression level of the pre-fusion F protein in the CHO cell supernatant was determined by measuring the optical density using an ELISA kit (Figure 3). A: Comparison of high optical density (OD) values ​​among the mutants; B: Comparison of intermediate OD values ​​among the mutants. N25C showed a high expression level of the pre-fusion F protein in the variant.

[0316] On day 7, the expression level of the pre-fusion F protein in the CHO cell supernatant was determined by measuring the optical density using an ELISA kit (Figure 4). A: Shows a comparison between high OD values ​​between variants; B: Shows a comparison between intermediate OD values ​​between variants. N25C showed a high expression level of the pre-fusion F protein in the variant.

[0317] Example 3: Production of recombinant polypeptide containing RSV F protein peptide This specification provides a secreted form of recombinant polypeptide containing RSV F protein peptide as a candidate vaccine.

[0318] The RSV F glycoprotein construct was derived from the RSVA2 strain (registration number AAC55970). The sequence encoding residues 1-520 of the F protein peptide was codon-optimized, synthesized, and subcloned into the Hind III and Bgl II sites of a mammalian expression vector encoding human α1 collagen C propeptide. Figure 1 is a schematic diagram showing an exemplary recombinant polypeptide.

[0319] Recombinant plasmids were transfected into GH-CHO(dfhr-) cells, selected to be free of hypoxanthine and thymidine (HT) (Invitrogen), and the gene was gradually amplified with increasing MTX (Sigma) concentration, enabling high titer expression of the fusion protein under serum-free culture conditions using CD007-4TM1 medium (Jianshun Biosciences). Exemplary recombinant polypeptides were affinity-bound to Endo180, initially purified using salt gradient elution, and further purified using a Superdex 200 gel filtration column (GE Healthcare). The purity of the exemplary recombinant polypeptides containing the RSV F peptide was measured by size exclusion chromatography (SEC-HPLC) according to the manufacturer's instructions (Sepax Technologies) (Figure 6). The main peak area of ​​the SCB-N25 fusion protein was 92.8%, and the main peak area of ​​the SCB-N25C protein was 81.7%.

[0320] In serum-free fed-batch culture, the production titer of fusion peptides linked by disulfide bonds (e.g., trimers) was confirmed to reach approximately 0.15 g / L (Figure 1B). Conditional media containing the trimerized recombinant polypeptide were first purified by affinity binding with the Fc-labeled collagen receptor uPARAP / Endo180 (a member of the mannose receptor family) pre-captured by protein A chromatography column (Thomas et al., (2005) J. Biol. Chem. 280, 22596-22605), followed by gel filtration chromatography. SEC-HPLC analysis showed that the concentration of exemplary recombinant polypeptide trimers was approximately 95% (Figure 6).

[0321] Figure 5 shows the expression levels of the fusion peptide analyzed by 8% SDS-PAGE using exemplary fusion peptide expression from serum-free fed-batch cell cultures. SCB-N25C: Cell-free culture media from day 1 to day 13 were separated under non-reducing and reducing conditions, and stained with Coomassie brilliant blue, with a sample volume of 26 μl. SCB-N25: Cell-free culture media from day 1 to day 13 were separated under non-reducing and reducing conditions, and stained with Coomassie brilliant blue, with a sample volume of 26 μl. The theoretical molecular weight of the fusion protein is 91 kD for the monomer and 273 kD for the trimer.

[0322] Figure 7 shows the affinity kinetics study. Figures 7A and 7B show binding studies using biolayer interference to exemplary fusion peptides, including Synagis and RSV F protein peptides.

[0323] For the affinity analysis in this experiment, a Fortebio Octet molecular interaction analyzer was used, and five Protein A probes were used, which were immersed in PBS before use.

[0324] Reagent preparation: Regeneration solution (0.01M glycine solution): 0.03g of glycine was accurately weighed, dissolved in water up to 40mL, mixed uniformly, the pH was adjusted to approximately 1.5 with 6M hydrochloric acid, and the solution was stored at room temperature.

[0325] Sample addition order: A clean 96-well measurement plate was taken, and the samples were added at a rate of 200 μL / well in the following order. [Table 1]

[0326] After adding the reagents and samples as described above, the data acquisition software was launched, sample and reagent information was entered, and the program was executed. The experiment was conducted in two groups: the first group had the affinity between the target protein sample and the Synagis antibody measured, and the second group had the affinity between the target protein sample and the D25 antibody measured. After the execution was complete, the obtained Kon, Kdis, and KD results were analyzed using Octet data analysis software.

[0327] The binding affinity of Synagis to purified exemplary SCB-N25 and SCB-N25C recombinant polypeptides is shown in Figure 7 and labeled in Figure 7. Figures 7C and 7D show affinity kinetic studies of binding between D25 and exemplary fusion peptides containing RSV F protein peptides. Corresponding KD, Kon, and Kdis from antibody D25 affinity experiments are shown labeled in Figure 7. SCB-N25C showed high affinity for both palivizumab and antibody D25.

[0328] Example 4: Functional characterization of recombinant polypeptides containing RSV F protein peptide To evaluate the immunogenicity and protective effects of the exemplary recombinant polypeptide produced as described in Example 1, BALB / c mice were randomly assigned to groups and immunized twice intramuscularly on days 0 and 21 with an exemplary fusion polypeptide containing one of three doses (1, 6, and 30 μg) of a different adjuvant. Another group immunized with PBS served as a control group.

[0329] Figures 8A–8D show the results of immunization studies using exemplary fusion peptides containing RSV F protein peptides.

[0330] Figure 8A is a schematic diagram showing the experimental method for immunization. Mice were immunized on day 0 and day 21, and serum was collected on day 0 and day 35. 6-8 week old SPF grade BALB / c female mice were randomly divided into 16 groups of 10 mice each. Each group of mice received an intramuscular injection of 50 μL of immunizing PBS or antigen. A booster immunization was administered 3 weeks later, and after two immunizations, blood was collected from the orbital vein at 2 weeks, centrifuged to obtain serum, and along with the serum, four mice were selected from each group, their spleens removed, and used for the Elispot experiment.

[0331] Figure 8B shows the serum anti-FIgG ELISA potency of a purified exemplary fusion peptide, the fusion peptide comprising the RSV F protein peptide and the adjuvant Alum, Alum+CpG1018, or CAS-1. Animal serum immunized with an hRSV candidate vaccine was mixed with hRSV A2 true virus, and Hep2 cells were added to the serum-virus mixture and incubated. The RSV A2 virus was able to infect Hep2 cells, and after incubation for 3-4 days (determined according to the status of cytopathic effect (CPE), when CPE in the virus control well reached 60% or more), newly proliferated viruses could be captured by biotin-labeled Synagis antibody, stained using SA-HRP secondary antibody, and the amount of viral proliferation could be indirectly reflected by the magnitude of the absorbance at OD450nm. If the added serum sample contains neutralizing antibodies capable of neutralizing the hRSV virus, the neutralized virus becomes unable to infect cells, which in turn affects the absorbance. A lower OD450nm value indicates a higher neutralizing antibody titer in the serum. Fusion proteins with Alum + CpG1018 as an adjuvant showed higher potency than the corresponding fusion proteins with Alum or CAS-1 as an adjuvant. SCB-N25C showed higher potency than other antigens.

[0332] Figure 8C shows the competitive IgG titers of D25 and palivizumab that provide 50% inhibition of binding of D25 and palivizumab to heat-inactivated RSV (HI-RSV) particles, as measured using serum sample dilutions. Values ​​are expressed as log2 and mean ± SEM. DCA:D25 was added as a coating antibody to a high-adsorption ELISA 96-well plate and coated overnight under conditions of 2–8°C. Then, gradient-diluted serum samples and an SCB-N20-biotin (N20-biotin) antigen-labeled mixture (1:1) were added. DCA in the immunoserum competitively bound to the site zero site of the N20-biotin antigen marker F protein with D25. Subsequently, a secondary antibody (SA-HRP) was added to recognize N20-biotin, and unbound portions were eluted and removed after each step. Finally, TMB was added to develop color, and the reaction was stopped with H2SO4. The lower the OD450nm value, the higher the DCA titer of the competitive antibody in the serum. PCA: Synagis was added as a coating antibody to a 96-well plate of high-adsorption ELISA and coated overnight at 2-8°C. Then, gradient-diluted serum samples and an SCB-N20-biotin (N20-biotin) antigen-labeled mixture (1:1) were added. PCA in the immunoserum competitively bound to the Site II site of the N20-biotin antigen marker F protein with Synagis. Subsequently, a secondary antibody (SA-HRP) was added to recognize N20-biotin, and unbound portions were eluted and removed after each step. Finally, TMB was added to develop color, and the reaction was stopped with H2SO4. The lower the OD450nm value, the higher the PCA titer of the competing antibody in the serum. All antigens that bound to Alum showed a lower antibody (DCA and PCA) response than the other two adjuvants. When used in combination with the Alum+CpG1018 adjuvant, both DS-CAV1-Trimer and N20 exhibited high DCA efficacy and PCA titer, with no significant difference between the two. N25 showed the lowest. None of the four candidate antigens used in combination with the CAS-1 adjuvant showed effective DCA titer. N20 and N25 showed high PCA titer, while DS-CAV1-Trimer showed the lowest.

[0333] Figure 8D shows the results of the Elispot test. After immunizing mice with the vaccine, spleen cells were stimulated to locally secrete cytokines, which were captured by specific monoclonal antibodies. The captured cytokines bound to biotin-labeled secondary antibodies and then to alkaline phosphatase-labeled avidin. After incubation with BCIP / NBT substrate, blue-violet spots appeared on the PVDF well plate, indicating that the cells had secreted specific cytokines. The results were obtained after analyzing the spots using the ELISpot enzyme-conjugated spot analysis system. The N25C antigen, in combination with the Alum adjuvant, induced a strong Th1 cell immune response. The antigen, in combination with the Alum+CpG1018 adjuvant, both induced a low Th2 cell immune response. The antigen, in combination with the CAS-1 adjuvant, both induced high Th1 and Th2 cell immune responses.

[0334] Serum was evaluated by enzyme-linked immunosorbent assay (ELISA). Briefly, a 96-well plate was coated overnight with 2 μg / mL of purified exemplary fusion peptide (in PBS) at 4°C and blocked with 1 mg / mL of BSA. The plate was 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. The bound antibody was detected for 1 hour at room temperature using HRP-conjugated goat anti-mouse IgG (Southern Biotech). The enzymatic reaction was performed using TMB (Thermo), and the reaction was stopped by adding 2M HCl, after which the absorbance at 450 nm was recorded. Mouse serum immunized with the same dilution of PBS was used as a negative control group, and antibody titer was defined as the serum dilution that produced a ratio of ODRSV F trimer to ODPBS of 2.0.

[0335] A micro-neutralization assay for RSV was performed using HeLa cells and two RSVA strains. Serum was heated and inactivated at 56°C for 30 minutes and serially diluted in serum-free DMEM in a 96-well cell culture plate (50 μL / well). The same volume of virus (1,000 pfu / mL, prepared in serum-free DMEM) was added to the plate, and the serum / virus mixture was incubated at 37°C for 1 hour. Approximately 5 × 10⁻⁶ 4 100 μL of DMEM supplemented with 10% FBS, containing 100% HeLa cells, was added to a plate and incubated at 37°C until the positive control (virus only) well showed 100% CPE. The plate was washed with PBST and fixed in PBS containing 80% pre-cooled acetone for 10 minutes. An appropriate amount of palivizumab was added to the wells, blocked with 1 mg / mL BSA for 1 hour, and incubated at room temperature for 2 hours. After three washes, HRP-conjugated goat anti-human IgG (Southern Biotech) was added, and the enzymatic reaction was performed, recording the OD at 450 nm. The dilution that yielded 50% inhibition of CPE formation was determined as the neutralizing antibody titer.

[0336] Since antigen site II is exposed in the exemplary recombinant polypeptide, competitive ELISA with Paliviz and D25 monoclonal antibody was performed to determine whether the antibody induced by the exemplary recombinant polypeptide targeted this site.

[0337] 5 x 10 6Competitive ELISA for palivizumab and D25 monoclonal antibody 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 of serum mixtures (1:32 to 1:4,096) were added to the plates along with appropriate amounts of palivizumab and D25 monoclonal antibody, and incubated at room temperature for 2 hours. Binding palivizumab was detected using HRP-conjugated goat anti-human IgG (Southern Biotech) and TMB substrate. Wells containing PBS-immunized mouse serum represented the non-competitive positive control group, and the inhibition percentage was calculated as ((ODPBS-ODRSV F trimer) / ODPBS) × 100%. Competitive binding titer is expressed as the dilution that causes 50% inhibition.

[0338] The present invention is not intended to be limited to the scope of any particular disclosed embodiment, which is provided, for example, to illustrate various aspects of the invention. Various modifications of the compositions and methods will become apparent from the description and teachings herein. These modifications may be made without departing from the true scope and spirit of the disclosure and are intended to be within the scope of the disclosure.

[0339] array JPEG2026519175000004.jpg254158JPEG2026519175000005.jpg254157JPEG2026519175000006.jpg253157JPEG2026519175000007.jpg255156JPEG2026519175000008.jpg255158JPEG2026519175000009.jpg254159JPEG2026519175000010.jpg253159JPEG2026519175000011.jpg254158JPEG2026519175000012.jpg254156JPEG2026519175000013.jpg255158JPEG2026519175000014.jpg255158JPEG2026519175000015.jpg254157JPEG2026519175000016.jpg253156JPEG2026519175000017.jpg254158JPEG2026519175000018.jpg254159JPEG2026519175000019.jpg252157JPEG2026519175000020.jpg254158JPEG2026519175000021.jpg254158JPEG2026519175000022.jpg255156JPEG2026519175000023.jpg254158JPEG2026519175000024.jpg254160JPEG2026519175000025.jpg40159

Claims

1. An application of a recombinant subunit vaccine in the manufacture of a drug for preventing respiratory syncytial virus (RSV) infection in mammals, wherein the recombinant subunit vaccine comprises a soluble RSV virus surface antigen, the soluble RSV virus surface antigen comprises an F1 peptide or a fragment or epitope having one or more mutation sites, and the one or more mutation sites of the F1 peptide or a fragment or epitope are amino acids containing sulfide bonds or amino acid derivatives containing sulfide bonds, and the soluble RSV virus surface antigen is linked to collagen via in-frame fusion to form a trimer fusion protein linked by disulfide bonds.

2. The RSV belongs to subtype A or subtype B, as per the use described in claim 1.

3. The use according to claim 1 or 2, wherein the RSV virus surface antigen comprises an F2 peptide or a fragment or epitope thereof.

4. The use according to any one of claims 1 to 3, wherein the RSV virus surface antigen comprises a mutant F2 peptide or a fragment or epitope thereof.

5. The use according to any one of claims 1 to 4, wherein one or more mutation sites of the F1 peptide or its fragment or epitope are R106C and / or R108C.

6. The RSV virus surface antigen comprises the pep27 peptide or a fragment or epitope thereof, as described in any one of claims 1 to 5.

7. The use according to claim 6, wherein the pep27 peptide or its fragment or epitope comprises one or more mutation sites.

8. The use according to claim 6 or 7, wherein one or more mutant sites contained in the pep27 peptide or its fragment or epitope are amino acids containing a sulfide bond.

9. The use according to any one of claims 6 to 8, wherein one or more mutation sites of the pep27 peptide or its fragment or epitope are freely selected from R133C, R135C, R136C or any combination thereof.

10. The use according to any one of claims 1 to 9, wherein the fusion protein comprises a sequence freely selected from those shown in SEQ ID NO: 64 to 79.

11. The use according to any one of claims 1 to 10, wherein the fusion protein comprises a sequence freely selected from those shown in SEQ ID NO: 76 to 79, and is selectively SEQ ID NO: 64, 68, 72, or 76.

12. The recombinant subunit vaccine is administered by intramuscular injection, as described in any one of claims 1 to 11.

13. The recombinant subunit vaccine is administered by intranasal spray, as described in any one of claims 1 to 11.

14. The use according to any one of claims 1 to 12, wherein the recombinant subunit vaccine is administered as a single dose or in a schedule of multiple doses spaced several weeks or months apart.

15. The recombinant subunit vaccine is administered without an adjuvant, as described in any one of claims 1 to 13.

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

17. The use according to any one of claims 1 to 16, wherein the recombinant subunit vaccine comprises a pharmaceutically acceptable adjuvant, the adjuvant being one or more selected from buffers, osmotic regulators, stabilizers, and bacteriostatic agents.

18. An application for producing a reagent used in a method for detecting antibodies against respiratory syncytial virus (RSV) from mammalian serum, wherein the method comprises the step of contacting the serum with the soluble RSV surface antigen, the soluble RSV virus surface antigen comprising an F1 peptide or a fragment or epitope having one or more mutation sites, wherein the mutation sites are amino acids containing sulfide bonds, and the soluble RSV surface antigen is linked to collagen via in-frame fusion to form a trimer fusion protein linked by disulfide bonds.

19. An application of a recombinant subunit vaccine containing a soluble surface antigen from respiratory syncytial virus (RSV) in the production of a neutralizing antibody for treating patients infected with the RSV virus by passive immunization, wherein the production comprises a method of immunizing a mammal and purifying the neutralizing antibody produced, wherein the soluble RSV virus surface antigen comprises an F1 peptide or a fragment or epitope having one or more mutation sites, and the mutation sites are amino acids containing sulfide bonds, and wherein the soluble surface antigen is linked to collagen via in-frame fusion to form a trimer fusion protein linked by disulfide bonds.

20. The use according to claim 19, wherein the neutralizing antibody comprises a polyclonal antibody and / or a monoclonal antibody, wherein the neutralizing antibody is a monoclonal antibody against an F protein or peptide.

21. One or more polynucleotides encoding a soluble RSV virus surface antigen, comprising sequences freely selected from those shown in SEQ ID NO: 64-79.

22. One or more vectors comprising one or more polynucleotides as described in claim 21.

23. A host cell comprising one or more polynucleotides according to claim 21, or one or more vectors according to claim 22.

24. The host cell according to claim 23, wherein it is GH-CHO.

25. The vector according to claim 23, which is pTPRIMER-TOD.

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