RSV pre-f mutants, methods of making and using the same

Abstract

The application belongs to the technical field of biology and relates to an RSV pre-F mutant, a preparation method and application thereof. Compared with a wild-type F0 polypeptide, the RSV pre-F mutant satisfies the following conditions: (1) does not contain a furin enzyme cutting site fragment, a P27 polypeptide, a transmembrane domain and an intracellular domain; (2) has mutations S180C, S186C, A170C, V179C, A241C, Q279C, D486C and A490C; and (3) the C terminal of an F1 polypeptide is not connected or connected with an aggregation motif. The mutant and a trimeric conformation thereof are both highly stable, can self-assemble into a trimer without a heterologous trimeric domain, can induce high levels of binding antibodies and neutralizing antibodies after immunization, avoid high levels of non-neutralizing active binding antibodies produced when immunization is performed on the heterologous trimeric domain, and thus improve the quality of antibodies.

Description

Technical Field

[0001] This application belongs to the field of biotechnology and relates to an RSV pre-F mutant, its preparation method and application. Background Technology

[0002] Respiratory syncytial virus (RSV) is one of the leading viruses causing respiratory infections in infants, young children, and the elderly, and a major cause of death from lower respiratory tract infections in children. RSV is most prevalent in autumn and winter, and its spread is most frequent among young children. RSV is a single-stranded, negative-sense enveloped RNA virus belonging to the Paramyxoviridae family and the Pneumovirus genus. It can cause respiratory symptoms, including cold symptoms, sore throat, cough, sneezing, nasal congestion, and fever. In infants, young children, and those with weakened immune systems, RSV can lead to severe lower respiratory tract infections such as bronchitis and pneumonia.

[0003] The F protein is a type I glycoprotein. The F protein, which mediates viral fusion with the host cell membrane, exists in two forms: pre-fusion and post-fusion. The pre-fusion F protein is expressed on the viral envelope surface in a metastable state. Upon contact with the host cell membrane, the N- and C-terminal ends of the F1 protein undergo a series of rearrangements, forming long α-helical structures. This process leads to the transformation of the F protein from a metastable pre-F structure to a stable post-F structure. This structural change mediates viral entry into the cell during viral infection. Comparative analysis of the structure of the RSV F protein and the neutralizing antibodies it induces reveals that the surface of the pre-fusion F protein differs from that of the post-fusion F protein. The F protein possesses unique high-concentration and active epitopes in its pre-fusion conformation, such as those found in Fibre V. Therefore, constructing a highly stable pre-fusion conformation of the F protein is currently a major direction in RSV vaccine development. For example:

[0004] In 2013, epitopes were discovered and named using antibody D25. To address the lack of D25 antibodies, [the following is a description of a specific site]. To address the instability issue, the engineered stable pre-fusion conformation (DS-Cav1) antigen of RSV F protein was further obtained primarily through disulfide bonding, cavity filling, and charge mutation.

[0005] In 2015, Janssen Pharmaceutical published its research on the construction of stable pre-F protein mutants. By introducing a short-chain linker between F1 and F2, the mutants were modified by amino acid mutations after binding to the fibroin domain to obtain SC-DM and SC-TM.

[0006] GSK further developed DS-Cavl (US_8563002), with the patent focusing on protecting and restricting the furin protease cleavage site and the C-terminal trimerization of the protein. Given its good immunogenicity and ability to induce neutralizing antibodies, GSK announced positive results in Phase III clinical trials of its RSV vaccine in adults aged 60 and older in 2022, and it received marketing authorization on May 3, 2023 (trade name: Arexvy), becoming the first vaccine against the RSV virus.

[0007] Pfizer's stable pre-F protein mutants, based on its structural design, primarily employ mutagenesis strategies involving disulfide bond mutations and cavity filling. The disulfide bond mutation sites include S55C / L188C, S155C / S290C, T103C / I148C, and L142C / N371C, as well as other cavity filling and electrostatic mutation sites to enhance pre-F protein stability. Due to the mutants' excellent immunogenicity and superior vaccine efficacy, Pfizer announced positive results from its Phase III clinical trial of its bivalent RSV vaccine in 2022, and it received marketing authorization in May 2023 (trade name: Abrysvo), becoming the second RSV vaccine after Arexvy.

[0008] Currently, subunit vaccines Arexvy and Abrysvo, and mRNA vaccine mRESVIA have been successfully marketed. They all use a stable mutant F protein as the RSV target antigen and have shown good protective effects in clinical trials.

[0009] However, stability issues still exist in the construction of the pre-fusion conformation of the F protein. The pre-fusion conformation is unstable, which directly affects its immunogenicity and is highly dependent on the heterotrimeric domain. Summary of the Invention

[0010] Based on this, one or more embodiments of this application provide an RSV pre-F mutant, its preparation method, and its application. The technical solutions include the following:

[0011] One or more embodiments of this application provide an RSV pre-F mutant that, relative to the wild-type F0 polypeptide, satisfies the following conditions:

[0012] (1) It does not contain furin protease cleavage site fragments, P27 polypeptide, transmembrane domains and intracellular domains;

[0013] (2) It has mutation combination 1, mutation combination 2, mutation combination 3 and mutation combination 4, among which,

[0014] Mutation combination 1 consists of S180C and S186C.

[0015] Mutation combination 2 consists of A170C and V179C.

[0016] Mutation combination 3 consists of A241C and Q279C.

[0017] Mutation combination 4 is either D486C and A490C, or D486C and D489C; and,

[0018] (3) The C-terminus of the F1 polypeptide is either not linked or is linked with an aggregation motif;

[0019] Optionally, the RSV pre-F mutant also satisfies the following condition:

[0020] (4) The 305th bit is I.

[0021] In some embodiments of this application, the RSV pre-F mutant also has one or more of the following mutations: P102A, I167C, I379V, and M447V.

[0022] In some embodiments of this application, the RSV pre-F mutant does not have one or more of the following mutations: P102A, I167C, I379V, and M447V.

[0023] In some embodiments of this application, the C-terminus of the F2 peptide in the RSV pre-F mutant does not contain NN.

[0024] In some embodiments of this application, in the RSV pre-F mutant, the C-terminus of the F2 polypeptide and the N-terminus of the F1 polypeptide are directly linked by an amide bond or indirectly linked by a flexible short peptide.

[0025] Optionally, the flexible short peptide is GS, G, S, GS, SG, SS, GG, PG, GGG, GGS, SSS, GSG, SGS, GPG, GSGS, GGGS, GPG, GSGS, GGGS, GPGS, GGGG, GGSG, GGSG, SGGG, GSSG, SGSG, GSSG, GGPGG, or GGGGS.

[0026] In some embodiments of this application, the C-terminus of the RSV pre-F mutant is linked to a tag fragment.

[0027] In some embodiments of this application, the aggregation motif has the amino acid sequence shown at positions 475 to 505 of SEQ ID NO:7;

[0028] Optionally, the C-terminus of the F1 polypeptide and the N-terminus of the aggregation motif are directly linked by an amide bond or indirectly linked by a flexible short peptide.

[0029] Optionally, the flexible short peptide is GS, G, S, GS, SG, SS, GG, PG, GGG, GGS, SSS, GSG, SGS, GPG, GSGS, GGGS, GPG, GSGS, GGGS, GPGS, GGGG, GGSG, GGSG, SGGG, GSSG, SGSG, GSSG, GGPGG, or GGGGS.

[0030] In some embodiments of this application, the RSV pre-F mutant has the amino acid sequence shown in positions 1 to 474 of SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:13 and SEQ ID NO:14.

[0031] One or more embodiments of this application provide a nucleic acid molecule that encodes the RSV pre-F mutant.

[0032] One or more embodiments of this application provide a carrier comprising the aforementioned nucleic acid molecule.

[0033] One or more embodiments of this application provide an engineered cell that expresses the RSV pre-F mutant, or that includes the nucleic acid molecule or the vector.

[0034] One or more embodiments of this application provide a method for producing the RSV pre-F mutant, which includes the following steps:

[0035] The engineered cells were cultured, and the RSV pre-F mutant was isolated from the resulting culture supernatant.

[0036] One or more embodiments of this application provide an immune composition comprising the RSV pre-F mutant or the nucleic acid molecule, and an immune adjuvant.

[0037] One or more embodiments of this application provide the application of the RSV pre-F mutant in the preparation of a respiratory syncytial virus antibody detection kit.

[0038] One or more embodiments of this application provide a respiratory syncytial virus antibody detection kit, which includes the RSV pre-F mutant.

[0039] Details of one or more embodiments of this application are set forth in the following description, and other features, objects, and advantages of this application will become apparent from the specification and its claims. Attached Figure Description

[0040] To more clearly illustrate the technical solutions in the embodiments of this application and to more completely understand this application and its beneficial effects, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0041] Figure 1 This is a schematic diagram of the structure of the RSV F protein mutant;

[0042] Figure 2 The results of SDS-PAGE analysis of JW-195(A) under reducing and non-reducing conditions;

[0043] Figure 3 Native-PAGE results for the mutant trimeric protein;

[0044] Figure 4 The results of size exclusion chromatography (SEC-HPLC) analysis of JW-195(A) are shown.

[0045] Figure 5 Statistical results of expression levels of different pre-F trimer proteins;

[0046] Figure 6 The results of stability testing of different mutant proteins at 60℃;

[0047] Figure 7 The results of stability testing of different mutant proteins at 37°C;

[0048] Figure 8 The results of stability testing of different mutant proteins at 4°C;

[0049] Figure 9 The results of bioactivity evaluation for JW-153(A), JW-153(B) and products not containing JW-195(A) and JW-195(B);

[0050] Figure 10 The results show the detection levels of antigen-specific antibodies against pre-F protein in different mutants;

[0051] Figure 11 Results of neutralizing antibody levels in different mutants;

[0052] Figure 12 This is a statistical graph showing the titers of antibody EC50 and neutralizing antibody NT50. Detailed Implementation

[0053] The present application will be further described in detail below with reference to the accompanying drawings, embodiments, and examples. It should be understood that these embodiments and examples are for illustrative purposes only and are not intended to limit the scope of the present application. The purpose of providing these embodiments and examples is to enable a more thorough and comprehensive understanding of the disclosure of the present application. It should also be understood that the present application can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various modifications or alterations without departing from the spirit of the present application, and the equivalent forms obtained also fall within the protection scope of the present application. Furthermore, numerous specific details are set forth in the following description to provide a fuller understanding of the present application. It should be understood that the present application can be implemented without one or more of these details.

[0054] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein in the specification of this application is for descriptive purposes only and is not intended to be limiting of the application.

[0055] the term

[0056] Unless otherwise stated or in case of contradiction, the terms or phrases used herein shall have the following meanings:

[0057] The terms "and / or," "or / and," and "and / or" as used herein include any one of two or more of the related listed items, as well as any and all combinations of the related listed items. These arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or," "or / and," and "and / or," it should be understood that in this application, the technical solution undoubtedly includes technical solutions connected by "logical AND," and also undoubtedly includes technical solutions connected by "logical OR." For example, "A and / or B" includes three parallel solutions: A, B, and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, a technical solution that is connected by "logical OR"), as well as any and all combinations of A, B, C, and D, that is, combinations of any two or three of A, B, C, and D, and also combinations of all four of A, B, C, and D (that is, a technical solution that is connected by "logical AND").

[0058] In this application, the terms "multiple", "various", "multiple times", "multi-dimensional", etc., unless otherwise specified, refer to a quantity greater than or equal to 2. For example, "one or more" means one or more than or equal to two.

[0059] The terms “combinations of,” “any combination of,” and “any combination of” used in this article include all suitable combinations of any two or more of the listed items.

[0060] In this document, the term "suitable" as used in phrases such as "suitable combination," "suitable method," and "any suitable method" refers to the ability to implement the technical solution of this application, solve the technical problem of this application, and achieve the expected technical effect of this application.

[0061] In this document, terms such as “preferred,” “better,” “more suitable,” and “ideal” are merely used to describe implementation methods or examples that achieve better results, and should be understood not to limit the scope of protection of this application.

[0062] In this application, terms such as "further," "even further," and "particularly" are used to describe purposes and indicate differences in content, but should not be construed as limiting the scope of protection of this application.

[0063] In this application, "optionally," "optionally," and "optional" mean that something is optional, that is, it means that it is selected from either "with" or "without." If there are multiple "optional" entries in a technical solution, unless otherwise specified, and there are no contradictions or mutual constraints, each "optional" entry shall be independent.

[0064] In this application, the terms "first aspect," "second aspect," "third aspect," "fourth aspect," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first," "second," "third," "fourth," etc., serve only as a non-exhaustive enumeration and should be understood not to constitute a closed limitation on quantity.

[0065] In this application, the technical features described in an open-ended manner include both closed technical solutions consisting of the listed features and open technical solutions that include the listed features.

[0066] In this application, numerical intervals (i.e., numerical ranges) are involved. Unless otherwise specified, the selected numerical distributions within the aforementioned numerical intervals are considered continuous and include the two endpoints (i.e., the minimum and maximum values) of the numerical range, as well as every value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints. In this document, this is equivalent to directly listing every integer. For example, if t is an integer selected from 1 to 10, it means that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Furthermore, when multiple ranges are provided to describe features or characteristics, these ranges can be merged. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges to which they are included.

[0067] Unless otherwise specified, the temperature parameters in this application are permitted to be either constant-temperature treatment or variations within a certain temperature range. It should be understood that the constant-temperature treatment allows temperature fluctuations within the precision range of the instrument control, such as ±5℃, ±4℃, ±3℃, ±2℃, or ±1℃.

[0068] In this application, % (w / w) and wt% both represent weight percentage, % (v / v) refers to volume percentage, and % (w / v) refers to mass-volume percentage.

[0069] All references to this application are incorporated herein by reference as if each document were individually incorporated herein by reference. Unless they conflict with the purpose and / or technical solution of this application, all cited references are incorporated herein by reference in their entirety and for all purposes. When references are cited in this application, the definitions of relevant technical features, terms, nouns, phrases, etc., are also incorporated herein by reference. Examples and preferred embodiments of the cited technical features may also be incorporated herein by reference, but only to the extent that they enable the implementation of this application. It should be understood that when the cited content conflicts with the description in this application, this application shall prevail or modifications shall be made adaptably to the description in this application.

[0070] RSV: Respiratory Syncytial Virus belongs to the family Pneumoviridae, genus Pneumovirus. The RSV genome is approximately 15 kb in length, containing 10 genes that encode 11 proteins. The virus surface displays three proteins: G protein, F protein, and SH protein.

[0071] F protein: A glycoprotein on the surface of the RSV virus, formed by the digestion of the F0 polypeptide by furin during maturation, resulting in the F1, F2, and P27 polypeptides. The F1 and F2 polypeptides constitute the F protein monomer, which further manifests as a trimer on the viral surface. The F protein exhibits metastable pre-fusion and post-fusion conformations. These two conformations can be distinguished using pre-F specific antibodies (such as D25, AM22, 5C4, and MPE8).

[0072] The construction of pre-F proteins faces stability issues. Furthermore, existing construction strategies involve obtaining a trimeric F protein and attaching a heterologous trimeric domain to its C-terminus. This trimeric domain generates high levels of non-neutralizing binding antibodies during immunization. These non-neutralizing antibodies reduce the proportion of neutralizing antibodies, affecting the quality of effective antibodies and posing potential safety risks. Moreover, the non-neutralizing binding antibodies generated by the heterologous trimeric domain exist as pre-existing antibodies, potentially impacting vaccine efficacy during long-term, repeated immunizations.

[0073] A first aspect of this application provides an RSV pre-F mutant that, relative to the wild-type F0 polypeptide, satisfies the following conditions:

[0074] (2) It has mutation combination 1, mutation combination 2, mutation combination 3 and mutation combination 4, among which,

[0075] Mutation combination 1 consists of S180C and S186C.

[0076] Mutation combination 2 consists of A170C and V179C.

[0077] Mutation combination 3 consists of A241C and Q279C.

[0078] Mutation combination 4 is either D486C and A490C, or D486C and D489C; and,

[0079] (3) The C-terminus of the F1 polypeptide is either not linked or is linked with an aggregation motif;

[0080] Optionally, the RSV pre-F mutant also satisfies the following condition:

[0081] (4) The 305th bit is I.

[0082] In some embodiments of this application, a highly stable RSV pre-F mutant was constructed. The mutation strategy involved introducing S180C / S186C, A170C / V179C, A241C / Q279C, D486C / A490C, and L305I mutations into the RSV F protein. These mutation combinations stabilized the RSV F protein in its pre-fusion conformation and its trimeric form. These mutation combinations are newly discovered and have not been previously reported. This mutant exhibits high stability in both its pre-fusion and trimeric conformations after treatment at 4°C, 37°C, and 60°C. Furthermore, the RSV pre-F mutant of this application exhibits trimeric formation independent of the heterotrimeric domain; it can self-assemble into a highly stable trimer with or without the heterotrimeric domain. This trimeric F protein, which does not contain a heterotrimeric domain, induces high levels of binding and neutralizing antibodies after immunization, avoiding the high levels of non-neutralizing binding antibodies generated during immunization with exogenous trimeric domains, thereby improving the quality of antibodies after final protein immunization. Overall, this application constructs a trimeric pre-F protein that does not contain a heterotrimeric domain. It can stably maintain its pre-fusion conformation and trimer conformation after heat treatment, and can also induce high levels of binding and neutralizing antibodies after mouse immunization, eliminating the potential impact of heterotrimers on vaccine efficacy and safety.

[0083] In some embodiments of this application, the RSV pre-F mutant also has one or more of the following mutations: P102A, I167C, I379V, and M447V.

[0084] In some embodiments of this application, the RSV pre-F mutant does not have one or more of the following mutations: P102A, I167C, I379V, and M447V.

[0085] In some embodiments of this application, the C-terminus of the F2 peptide in the RSV pre-F mutant does not contain NN.

[0086] In some embodiments of this application, in the RSV pre-F mutant, the C-terminus of the F2 polypeptide and the N-terminus of the F1 polypeptide are directly linked by an amide bond or indirectly linked by a flexible short peptide.

[0087] Optionally, the flexible short peptide is GS, G, S, GS, SG, SS, GG, PG, GGG, GGS, SSS, GSG, SGS, GPG, GSGS, GGGS, GPG, GSGS, GGGS, GPGS, GGGG, GGSG, GGSG, SGGG, GSSG, SGSG, GSSG, GGPGG, or GGGGS.

[0088] In some embodiments of this application, the C-terminus of the RSV pre-F mutant is linked to a tag fragment.

[0089] In some embodiments of this application, the aggregation motif has the amino acid sequence shown at positions 475 to 505 of SEQ ID NO:7, or has at least 80% (at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) homology with that amino acid sequence.

[0090] Optionally, the C-terminus of the F1 polypeptide and the N-terminus of the aggregation motif are directly linked by an amide bond or indirectly linked by a flexible short peptide.

[0091] Optionally, the flexible short peptide is GS, G, S, GS, SG, SS, GG, PG, GGG, GGS, SSS, GSG, SGS, GPG, GSGS, GGGS, GPG, GSGS, GGGS, GPGS, GGGG, GGSG, GGSG, SGGG, GSSG, SGSG, GSSG, GGPGG, or GGGGS.

[0092] In some embodiments of this application, the RSV pre-F mutant has the amino acid sequence shown at positions 1 to 474 of SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:13 and SEQ ID NO:14, or has at least 80% (at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) homology with such amino acid sequence.

[0093] The RSV fusion pre-fusion F protein mutant provided in this application has a stable conformation and can induce an immune response in the subject as an antigen / immunogen, producing antibodies.

[0094] The subject is an animal. An animal is a living multicellular vertebrate or invertebrate, including, for example, mammals. The term mammal includes human mammals and non-human mammals. Similarly, the term "subject" includes human and veterinary subjects, such as non-human primates. Therefore, administration to a subject can include administration to human subjects. Non-limiting examples of veterinary subjects include domestic animals (e.g., cats and dogs), livestock (e.g., cattle, horses, pigs, sheep, and goats), and laboratory animals (e.g., mice, rabbits, rats, gerbils, guinea pigs, and non-human primates).

[0095] Antibody: A polypeptide, found in nature, encoded by one or more immunoglobulin genes or fragments thereof, that specifically binds to and recognizes an analyte (e.g., an antigen or immunogen), such as RSVF protein or an antigenic fragment thereof. Immunoglobulin genes include κ, λ, α, γ, 6, ε, and μ constant region genes, as well as numerous immunoglobulin variable region genes. The term "antibody" as used herein includes, for example, modifications of a complete antibody and antibody fragments synthesized de novo using recombinant DNA methods.

[0096] Antibodies exist, for example, in the form of intact immunoglobulins and in the form of various well-characterized antibody fragments. For example, Fab, Fv, and single-chain Fv (SCFv) bound to RSVF proteins will be RSVF protein-specific binders. This includes intact immunoglobulins and variants and portions thereof well-known in the art, such as Fab′ fragments, F(ab)′2 fragments, single-chain Fv proteins (“scFv”), and disulfide-stabilized Fv proteins (“dsFv”). scFv proteins are fusion proteins in which the light chain variable region of an immunoglobulin is linked to the heavy chain variable region of an immunoglobulin by a linker, while in dsFv, the chains have been mutated to introduce disulfide bonds to stabilize chain association. The term also includes genetically engineered forms such as chimeric antibodies (e.g., humanized murine antibodies) and heterologous binding antibodies (e.g., bispecific antibodies). See also Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, IL); Kuby, J., Immunology, 3rd edition, WH Freeman & Co., New York, 1997.

[0097] Antibody fragments are defined as follows: (1) Fab, a fragment containing a monovalent antigen-binding fragment of an antibody molecule produced by digesting an intact antibody with the enzyme papain to obtain a portion of the intact light chain and a heavy chain; (2) Fab′, a fragment of an antibody molecule obtained by treating an intact antibody with pepsin and then reducing it to obtain a portion of the intact light chain and a heavy chain; each antibody molecule yields two Fab′ fragments; (3) (Fab′)2, a fragment of an antibody obtained by treating an intact antibody with the enzyme pepsin without subsequent reduction; (4) F(ab′)2, a dimer in which two Fab′ fragments are held together by two disulfide bonds; (5) Fv, a genetically engineered fragment containing variable regions of the light chain and variable regions of the heavy chain, represented by two chains; and (6) a single-chain antibody (“SCA”), a genetically engineered molecule in the form of a genetically fused single-chain molecule containing variable regions of the light chain and variable regions of the heavy chain linked by suitable polypeptide linkers.

[0098] Typically, naturally occurring immunoglobulins have heavy (H) chains and light (L) chains linked together by disulfide bonds. Two types of light chains exist, λ and κ. Five major heavy chain classes (or isotypes) exist, which determine the functional activity of antibody molecules: IgM, IgD, IgG, IgA, and IgE. Disclosed antibodies can be of different types.

[0099] Each heavy and light chain contains constant and variable regions (also referred to as “domains”). In some embodiments, the heavy and light chain variable domains combine to specifically bind antigens. In other embodiments, only the heavy chain variable domain is required. For example, naturally occurring camel antibodies consisting only of the heavy chain are functional and stable in the absence of the light chain (see, for example, Hamers-Casterman et al., Nature, 363:446-448, 1993; Sheriff et al., Nat. Struct. Biol., 3:733-736, 1996). The light and heavy chain variable domains contain “framework” regions interrupted by three hypervariable regions (also referred to as “complementarity-determining regions” or “CDRs”) (see, for example, Kabat et al., Sequences of Proteins of Immunological Interest, USDapartment of Health and Human Services, 1991). The sequences of the frame regions of different light or heavy chains are relatively conserved within a species. The frame region of an antibody, which is a combined frame region of light and heavy chains, is used to locate and align the CDR in three-dimensional space.

[0100] CDRs are primarily responsible for binding to epitopes of antigens. The amino acid sequence boundaries of a given CDR can be readily determined using any of a number of well-known schemes, including those described by Kabat et al. (“Sequences of Proteins of Immunological Interest,” 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991; “Kabat” numbering scheme), Al-Lazikani et al. (JMB 273, 927-948, 1997; “Chothia” numbering scheme), and Lefranc et al. (“IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains,” Dev. Comp. Immunol., 27: 55-77, 2003; “IMGT” numbering scheme).

[0101] The CDRs for each chain are typically referred to as CDR1, CDR2, and CDR3 (from the N-terminus to the C-terminus) and are usually identified by the chain to which a particular CDR is located. Therefore, VHCDR3 is located in the variable domain of the heavy chain of the antibody in which it is contained, while VLCDR1 is CDR1 from the variable domain of the light chain of the antibody in which it is contained. Light chain CDRs are sometimes referred to as CDRL1, CDRL2, and CDRL3. Heavy chain CDRs are sometimes referred to as CDRH1, CDRH2, and CDRH3.

[0102] Antigen: A compound, composition, or substance that can stimulate the production of an antibody or T-cell response in an animal, including compositions injected or absorbed into the animal. An antigen is a product that reacts with a specific humoral or cellular immune response, including those induced by heterologous antigens, such as the disclosed mutant of the RSV pre-fusion F protein. Examples of antigens include (but are not limited to) polypeptides, peptides, lipids, polysaccharides, combinations thereof (e.g., glycopeptides), and nucleic acids containing antigenic determinants, such as those recognized by immune cells. In some instances, antigens include peptides derived from the pathogen of interest, such as RSV. In specific instances, antigens are derived from RSV, such as antigens comprising modified RSVF proteins stabilized in a pre-fusion conformation. An "epitope" or "antigenic determinant" refers to an antigenic region that reacts with B and / or T cells.

[0103] Immunogen: A protein or portion thereof capable of inducing an immune response in mammals, such as mammals infected with or at risk of infection with pathogens. Administration of an immunogen can result in protective and / or active immunity against the pathogen of interest. Examples include the Pre-F mutant provided in the embodiments of this application.

[0104] Immune response: The response of cells of the immune system, such as B cells, T cells, or monocytes, to a stimulus. In one embodiment, the response is specific to a particular antigen (“antigen-specific response”). In one embodiment, the immune response is a T cell response, such as a CD4+ or CD8+ response. In another embodiment, the response is a B cell response and results in the production of specific antibodies.

[0105] Those skilled in the art will recognize that alterations, additions, or deletions of individual amino acids or small percentages of amino acids (e.g., less than 20%, 15%, 10%, 5%, etc.) in the coding sequence are conserved variations, where the alteration results in the substitution of a chemically similar amino acid. Conserved amino acid substitutions that provide functionally similar amino acids are well known in the art. The following six groups each contain amino acids that are conserved substitutes for each other: 1) alanine (A), serine (S), threonine (T); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); and 6) phenylalanine (F), tyrosine (Y), tryptophan (W).

[0106] Not all residue positions within a protein will allow for normally "conserved" substitutions. For example, if an amino acid residue is essential for protein function, even normally conserved substitutions can disrupt that activity; for instance, the specific binding of an antibody to a target epitope can be disrupted by a conserved mutation in the target epitope.

[0107] Epitopes: Antigenic determinants. These are specific chemical groups or peptide sequences on antigenic molecules that induce specific immune responses; for example, an epitope is an antigenic region that reacts with B and / or T cells. Antibodies bind to specific antigenic epitopes, such as epitopes of RSVF proteins, for example, the D25 or AM22 epitopes present in the pre-fusion conformation of the RSVF protein. Epitopes can be formed from consecutive or discontinuous amino acids juxtaposed through the ternary folding of a protein. Epitopes formed from consecutive amino acids generally remain exposed to denaturing solvents, while epitopes formed through ternary folding are generally lost upon treatment with denaturing solvents. Epitopes typically comprise at least 3 and more usually at least 5, about 9, or about 8-10 amino acids in a distinctive spatial conformation. Methods for determining the spatial conformation of epitopes include, for example, X-ray crystallography and nuclear magnetic resonance. Epitopes may also include post-translational modifications of amino acids, such as N-linked glycosylation.

[0108] A "target epitope" is a specific epitope on an antigen that specifically binds to an antibody of interest, such as a monoclonal antibody. In some instances, a target epitope includes amino acid residues that contact the antibody of interest so that the target epitope can be selected by determining the amino acid residues that contact the antibody of interest.

[0109] Amino acids in peptides, polypeptides, or proteins are typically linked together chemically via amide bonds (CONH). Alternatively, amino acids can be linked together by other chemical bonds. For example, the linkage of amino acids or amino acid analogs can include CH2NH-, -CH2S-, -CH2-CH2-, -CH=CH-- (cis and trans), -COCH2--, -CH(OH)CH2-, and -CHH2SO- (these and others can be found in Spatola, in Chemistry and Biochemistry of Amino Acids, Peptides, and Proteins, ed. B. Weinstein, Marcel Dekker, New York, p. 267 (1983); Spatola, AF, Vega Data (March 1983), Vol. 1, No. 3, Peptide Backbone Modifications (General Review); Morley, Tren dsPharmSci, pp. 463-468, 1980; Hudson et al., Int J Pept Prot Res 14: 177-185, 1979; Spatola et al., Life Sci 38: 1243-1249, 1986; Harm J. Chem. Soc Perkin Trans. 1307-314, 1982; Almquist et al., J. Med. Chem. 23: 1392-1398, 1980; Jennings-White et al., Tetrahedron Lett 23: 2533, 1982; Hollada V et al., Tetrahedron Lett 24: 4401-4404, 1983; and Hruby Life Sci 31: 189-199, 1982).

[0110] Peptide Modification: Peptides, for example, stabilized in their pre-fusion conformation, can be modified, for example by substitution of amino acids comprising a sequence relative to the native RSV protein, or by various chemical techniques to produce derivatives having substantially the same activity and conformation as the unmodified peptide and optionally other desired properties. For example, the carboxylic acid group of the protein, whether carboxyl-terminal or side chain, can be provided or esterified in the form of a pharmaceutically acceptable cationic salt to form a C1-C16 ester, or converted to an amide of the formula NR1R2, wherein R1 and R2 are each independently H or a C1-C16 alkyl group, or combined to form a heterocycle, such as a 5- or 6-membered ring. The amino group of the peptide, whether amino-terminal or side chain, can be in the form of a pharmaceutically acceptable acid addition salt such as HCl, HBr, acetate, benzoate, toluenesulfonate, maleate, tartrate, and other organic salts, or can be modified to a C1-C16 alkyl or dialkylamino group or further converted to an amide.

[0111] The hydroxyl groups on the peptide side chain can be converted to C1-C16 alkoxy or C1-C16 esters using recognized techniques. The phenyl and phenolic rings of the peptide side chain can be substituted with one or more halogen atoms such as F, Cl, Br, or I, or with C1-C16 alkyl, C1-C16 alkoxy, carboxylic acid and its esters, or amides of these carboxylic acids. The methylene group on the peptide side chain can be extended to a homologous C2-C4 alkylene group. Thiols can be protected with any of a variety of recognized protecting groups such as acetamide groups.

[0112] One or more embodiments of this application provide a nucleic acid molecule that encodes the RSV pre-F mutant.

[0113] Nucleic acid molecules, nucleic acids: polymers composed of nucleotide units (ribonucleotides, deoxyribonucleotides, associated naturally occurring structural variants, and their synthetic non-natural analogs) linked by phosphodiester bonds, associated naturally occurring structural variants, and their synthetic non-natural analogs. Therefore, the term includes nucleotide polymers in which the nucleotides and the linkages therebetween include non-natural synthetic analogs such as, but not limited to, phosphate thioesters, aminophosphate esters, methylphosphonates, chiral methylphosphonates, 2-O-methylribonucleotides, peptide-nucleic acid (PNA), etc. These polynucleotides can be synthesized, for example, using an automated DNA synthesizer. The term "oligonucleotide" generally refers to short polynucleotides typically no more than about 50 nucleotides. It should be understood that when the nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), this also includes RNA sequences in which "U" replaces "T" (i.e., A, U, G, C).

[0114] "Nucleotide" includes (but is not limited to) monomers comprising a base linked to a sugar (e.g., pyrimidine, purine, or synthetic analogues thereof) or monomers comprising a base linked to an amino acid (e.g., in peptide nucleic acids (PNA)). A nucleotide is a monomer in a polynucleotide. A nucleotide sequence refers to the base sequence in a polynucleotide.

[0115] "Encoding" refers to the inherent characteristics of a specific sequence of nucleotides in a polynucleotide, such as a gene, cDNA, or mRNA, which acts as a template for the synthesis of other polymers and macromolecules in biological processes, having a defined sequence of nucleotides (e.g., rRNA, tRNA, and mRNA) or a defined sequence of amino acids and the resulting biological characteristics. Thus, if the transcription and translation of mRNA produced by a gene produces a protein in a cell or other biological system, then the gene encodes the protein. The coding strand, whose nucleotide sequence is identical to the mRNA sequence and is generally provided in sequence listing form, and the non-coding strand, which serves as a transcription template for a gene or cDNA, can be referred to as encoding the protein or other product of the gene or cDNA. Unless otherwise stated, "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate from each other and encode the same amino acid sequence. Nucleotide sequences encoding proteins and RNA may include introns. In some instances, nucleic acids encode mutants of this application.

[0116] Taking into account degenerate and conserved variants, this application does not impose any special restrictions on nucleic acid molecules.

[0117] Degenerate variants and conserved variants: Polynucleotides encoding polypeptides that include sequences degenerate due to the genetic code. For example, polynucleotides encoding a disclosed antigen or an antibody that specifically binds to a disclosed antigen that includes a sequence degenerate due to the genetic code. There are 20 naturally occurring amino acids, most of which are designated by more than one codon. Therefore, all degenerate nucleotide sequences are included, provided that the amino acid sequence of the antigen or antibody that binds to the antigen encoded by the nucleotide sequence remains unchanged. Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given polypeptide. For example, the codons CGU, CGC, CGA, CGG, AGA, and AGG all encode the amino acid arginine. Therefore, at each position within a protein-coding sequence that specifies arginine, the codon can be changed to any of the corresponding codons without altering the encoded protein. Such nucleic acid variations are “silent variants,” a type of conserved variant. Each nucleic acid sequence encoding a polypeptide herein also describes each possible silent variant. Those skilled in the art will recognize that each codon in a nucleic acid (except AUG, which is typically the only codon for methionine) can be modified using standard techniques to produce functionally identical molecules. Therefore, each "silent variant" of the nucleic acid encoding the polypeptide is implicit in each of the sequences.

[0118] In some examples, it is codon-optimized for expression in mammalian cells and is operatively linked to a promoter.

[0119] Expression control sequence: A nucleic acid sequence that regulates the expression of a heterologous nucleic acid sequence that is operatively linked. An expression control sequence is operatively linked to a nucleic acid sequence when it controls and regulates transcription and, when appropriate, translation of the nucleic acid sequence. Therefore, an expression control sequence may include a suitable promoter, enhancer, transcription terminator, start codon (ATG) preceding a protein-coding gene, splicing signals for introns, maintaining the appropriate reading frame of the gene to allow proper translation of the mRNA, and a stop codon. The term "control sequence" is intended to include, at a minimum, components whose presence can affect expression, and may also include other components whose presence is advantageous, such as leader sequences and fusion chaperone sequences. An expression control sequence may include a promoter.

[0120] A promoter is the minimal sequence sufficient to initiate transcription. It also includes promoter elements sufficient to make promoter-independent gene expression controllable with respect to cell type specificity, tissue specificity, or induced by external signals or agents; these elements may be located in the 5′ or 3′ region of the gene. This includes constitutive and inducible promoters (see, for example, Bitter et al., Methods in Enzymology 153:516-544, 1987). For example, when cloning in a bacterial system, inducible promoters such as phage λ, plac, ptrp, ptac (ptrp-lac heterozygous promoter), etc., can be used. In one embodiment, when cloning in a mammalian cell system, promoters derived from the mammalian cell genome (e.g., metallothionein promoters) or promoters derived from the mammalian virus genome (e.g., retroviral long terminal repeat sequences; adenovirus late promoters; vaccinia virus 7.5K promoters) can also be used to provide transcription of nucleic acid sequences.

[0121] Polynucleotides can be inserted into expression vectors containing promoter sequences that promote efficient transcription of the inserted genetic sequence in the host. These expression vectors typically contain an origin of replication, a promoter, and specific nucleic acid sequences that allow phenotypic selection in transformed cells.

[0122] RSVF proteins from different RSV subpopulations, the nucleic acid sequences encoding these proteins, and the methods for manipulating and inserting these nucleic acid sequences into vectors are disclosed herein and known in the art (see, for example, Tan et al., PLOSone, 7: e51439, 2011; Sambrook et al., Molecular Cloning, a Laboratory Manual, 2nd ed., Cold Spring Harbor Press, Cold Spring Harbor, NY (1989); Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, New York, NY (1994)).

[0123] One or more embodiments of this application provide a carrier comprising the aforementioned nucleic acid molecule.

[0124] One or more embodiments of this application provide an engineered cell that expresses the RSV pre-F mutant, or that includes the nucleic acid molecule or the vector.

[0125] Expression: Nucleic acids are translated into proteins. Proteins can be expressed and retained inside cells, becoming components of the cell surface membrane, or secreted into the extracellular matrix or culture medium.

[0126] Engineered cell, host cell: a cell in which the vector can reproduce and express its DNA. The cell may be prokaryotic or eukaryotic. The term also includes any progeny of the host cell. It should be understood that all progeny may differ from the parent cell because mutations can occur during replication. However, these progeny are included when the term "host cell" is used.

[0127] One or more embodiments of this application provide a method for producing the RSV pre-F mutant, which includes the following steps:

[0128] The engineered cells were cultured, and the RSV pre-F mutant was isolated from the resulting culture supernatant.

[0129] One or more embodiments of this application provide an immune composition comprising the RSV pre-F mutant or the nucleic acid molecule, and an immune adjuvant.

[0130] Immune adjuvants: Mediators used to enhance antigenicity. Adjuvants include suspensions of minerals (alum, aluminum hydroxide, or phosphate) adsorbed with antigens; or water-in-oil emulsions, for example, in which the antigen solution is emulsified in mineral oil (French incomplete adjuvant), sometimes including bactericidal mycobacteria (French complete adjuvant) to further enhance antigenicity (inhibiting antigen degradation and / or causing macrophage influx). Immunostimulatory oligonucleotides (e.g., those including CpG motifs) can also be used as adjuvants. Adjuvants include biomolecules (“biological adjuvants”), such as co-stimulatory molecules. Exemplary adjuvants include IL-2, RANTES, GM-CSF, TNF-α, IFN-γ, G-CSF, LFA-3, CD72, B7-1, B7-2, OX-40L, 4-1BBL, and Toll-like receptor (TLR) agonists, such as TLR-9 agonists. Adjuvants are well known to those skilled in the art (see, for example, Singh (ed.) Vaccine Adjuvants and Delivery Systems. Wiley-Interscience, 2007). Adjuvants can be used in combination with the disclosed PreF antigen. Optionally, the immune adjuvants in the embodiments of this application are one or more of aluminum salt adjuvants, surfactants, polynucleotides, lipopolysaccharides, liposomes, and oil emulsion adjuvants. Examples include Alum, CpG, Alum+CpG, MF59, AS04, AS01E, etc. MF59 is a water-mixed adjuvant mainly composed of three parts: an oil phase, an emulsifier, and an excipient; the oil phase is a mixture of micronized short-chain triglycerides suitable for human injection; the emulsifier is a surfactant that allows the oil phase and water to mix uniformly; the excipient mainly includes humectants and buffers such as glycerol, threitol, and ATP. AS04 adjuvant is a mixture of AS03 adjuvant and MPL adjuvant. AS03 adjuvant is a mixture of three surfactants: liposomes, TWEEN80, and SORBITAN. AS01E is a nanoscale liposome solution prepared from DOPC, Chol, MPL, and QS-21. The main component of the MPL adjuvant is lipopolysaccharide.

[0131] Immunogenic compositions (immunogenic compositions): Compositions comprising antigens that induce an immune response, such as a measurable CTL response against a virus expressing an antigen, or a measurable B cell response against an antigen (e.g., antibody production). Thus, an immunogenic composition comprises one or more antigens (e.g., peptide antigens) or epitopes. An immunogenic composition may also include one or more additional components capable of inducing or enhancing an immune response, such as excipients, carriers, and / or adjuvants. In some cases, an immunogenic composition is administered to induce an immune response that protects a subject from symptoms or illnesses induced by a pathogen. In some cases, symptoms or illnesses caused by a pathogen are prevented (or reduced or improved) by inhibiting the replication of said pathogen after the subject has been exposed to it (e.g., RSV). In one example, an “immunogenic composition” comprises a recombinant RSVF protein stabilized in a pre-fusion conformation that induces a measurable CTL response against a virus expressing the RSVF protein, or induces a measurable B cell response against the RSVF protein (e.g., antibody production). It further refers to isolated nucleic acids that encode antigens, such as nucleic acids that can be used to express antigens (and thus to induce an immune response against such polypeptides).

[0132] For in vitro use, the immunogenic composition may include an antigen or a nucleic acid encoding an antigen. For in vivo use, the immunogenic composition will typically include a protein, immunogenic peptide, or nucleic acid in a pharmaceutically acceptable carrier and / or other pharmaceutical agent. The ability of any particular peptide, such as a disclosed RSVF protein stabilized in its pre-fusion conformation or a nucleic acid encoding a disclosed RSVF protein stabilized in its pre-fusion conformation, to induce a CTL or B cell response can be readily tested using recognized assays. The immunogenic composition may include adjuvants well known to those skilled in the art.

[0133] Immunological reaction conditions: These include conditions that allow antibodies generated against a specific epitope to bind to said epitope and to a degree that is detectably greater than that of substantially all other epitopes and / or substantially exclude binding to substantially all other epitopes. Immunological reaction conditions depend on the form of antibody binding reaction and are generally those used in immunoassay protocols or encountered in vivo. The immunological reaction conditions used in the methods are “physiological conditions,” which include references to typical conditions (e.g., temperature, molar osmolality, pH) within living mammals or mammalian cells. While some organs are known to experience extreme conditions, the in vivo and intracellular environment is typically around pH 7 (e.g., pH 6.0 to pH 8.0, more typically pH 6.5 to 7.5), contains water as the primary solvent, and exists at temperatures above 0°C and below 50°C. Molar osmolality is within the range supporting cell viability and proliferation.

[0134] One or more embodiments of this application provide the application of the RSV pre-F mutant in the preparation of a respiratory syncytial virus antibody detection kit.

[0135] One or more embodiments of this application provide a respiratory syncytial virus antibody detection kit, which includes the RSV pre-F mutant.

[0136] One or more embodiments of this application provide a method for preventing and treating lower respiratory tract infections caused by respiratory syncytial virus, comprising the following steps: administering a therapeutically effective amount of the immune composition described in the sixth aspect to a subject.

[0137] Administration: The composition is introduced into the subject via a chosen route. Administration can be local or systemic. For example, if the chosen route is intravenous, the composition is administered by introducing it into the subject's vein.

[0138] Effective amount: An amount of an agent, such as the PreF antigen or a nucleic acid or other agent encoding the PreF antigen, sufficient to produce a desired response, such as an immune response against RSVF proteins, or to reduce or eliminate signs or symptoms of a symptom or disease, such as RSV infection. For example, this might be the amount required to inhibit viral replication or to measurably alter the external symptoms of a viral infection. Generally, this amount will be sufficient to measurably inhibit viral (e.g., RSV) replication or infectivity. When administered to a subject, a dose that will reach a target tissue concentration (e.g., in respiratory tissue) that has been shown to achieve in vitro inhibition of viral replication is typically used. In some instances, an "effective amount" is the amount used to treat (including prevent) one or more symptoms and / or underlying causes of any condition or disease, such as the amount used to treat RSV infection. In one instance, an effective amount is a therapeutically effective amount. In one instance, an effective amount is the amount used to prevent the development of one or more signs or symptoms of a particular disease or symptom (e.g., one or more signs or symptoms associated with RSV infection).

[0139] "Prevention" refers to the suppression of the full development of a disease or symptom in subjects at risk of developing a disease (e.g., RSV infection). "Treatment" refers to a therapeutic intervention that improves the signs or symptoms of a disease or pathological condition after it has begun to develop. The term "improvement" in relation to a disease or pathological condition refers to any observable beneficial therapeutic effect. A beneficial effect can be demonstrated, for example, by the delayed onset of clinical symptoms of the disease in susceptible subjects, a reduction in the severity of some or all clinical symptoms of the disease, a slower progression of the disease, an improvement in the overall health or condition of the subject, or other parameters known in the art to be specific to a particular disease. "Prophylactic" treatment is a treatment administered to subjects who do not exhibit signs of disease or only exhibit early signs in order to reduce the risk of developing the lesion. The term "reduction" is relative, meaning that a drug reduces a reaction or symptom if the reaction or symptom is quantitatively reduced after administration, or if it is reduced compared to a reference drug after administration. Similarly, the term "prevention" does not necessarily mean that the drug completely eliminates a reaction or symptom, provided that at least one characteristic of the reaction or symptom is eliminated. Therefore, immunogenic compositions that reduce or prevent infection or reaction (e.g., pathological reaction, such as a vaccine-enhanced viral disease) can, but do not necessarily, completely eliminate such infection or reaction, provided that the infection or reaction is measurably reduced compared to an infection or reaction in the absence of the agent or compared to a reference agent, for example, by at least about 50%, such as at least about 70%, or about 80%, or even about 90% (i.e., reduced to 10% or less).

[0140] In a tenth aspect of this application, an embodiment of this application provides a method for detecting or separating RSVF-binding antibodies in a sample, comprising the following steps:

[0141] The mutant described in the first aspect is contacted with an RSVF-binding antibody in the sample to form an immune complex; and,

[0142] The immune complex is detected to detect or isolate RSV F-binding antibodies in a sample.

[0143] The embodiments of this application will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application. For experimental methods in the following embodiments where specific conditions are not specified, please refer to the guidelines given in this application, or follow experimental manuals or conventional conditions in the art, or follow the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0144] In the specific embodiments described below, the measurement parameters involving raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0145] 1. Preparation of RSV pre-F mutant

[0146] 1.1 Construction of RSV pre-F mutant

[0147] Figure 1 This is a schematic diagram of the constructed RSV preF protein mutant. Amino acids 103 to 145 of the RSV F0 polypeptide are linked using a GS short peptide, deleting the furin cleavage site of F0, thus avoiding the influence of furin protease and resulting in a final F protein that is a trimeric protein composed of single-chain aggregates. Simultaneously, the transmembrane domain and intracellular domain (TM, 514aa-550aa) and the intracellular domain (CT, 551aa-574aa) are deleted, resulting in the final monomeric protein being expressed in a secretory form. Unless otherwise specified, other monomeric RSV F protein mutations involved in the embodiments of this application are designed based on this principle.

[0148] Based on the amino acid sequence of the RSV pre-F mutant, codons were optimized according to the preferred codons of Chinese hamster ovary cells (Cricetulus griseus, CHO cells) to facilitate expression. The optimized nucleotide fragment was then synthesized and finally ligated into the double-digested pcDNA3.1 vector using restriction endonucleases BamHI and XhoI to construct the recombinant vector.

[0149] Gene manipulations, including substitution, insertion, and deletion, were performed on the recombinant vector using the ClonExpress II Recombinant Cloning Kit (Novizan Biotechnology Co., Ltd.). The two fragments at the mutation sites were amplified separately using polymerase chain reaction (PCR) with the high-fidelity enzyme Phanta Max (Novizan Biotechnology Co., Ltd.). After gel recovery, the two fragments were fused by PCR. The recovered fusion fragment was homologously recombined into the pcDNA3.1 vector, which had been double-digested with BamHI and XhoI. The constructed point mutation vector, after being correctly sequenced by Beijing Qingke Biotechnology Co., Ltd., was used for subsequent experiments.

[0150] The sequenced-validated point mutation vector was transformed into *E. coli* and plated on LB (Amp+) plates. Single colonies were picked and cultured in 5 mL of LB (Amp+) liquid medium for 8 h, then inoculated into 300 mL of LB (Amp+) liquid medium and cultured at 37°C and 180 rpm for 16 h. Plasmids were extracted using a large-scale / large-scale plasmid extraction kit and finally stored in 1 mL of sterile TE buffer. All commercial kits and reagents were performed according to the manufacturer's instructions.

[0151] Table 1. Design of RSV F protein mutant protein sequences

[0152]

[0153]

[0154] 1.2 Expression of RSV pre-F mutant

[0155] The mutant protein was constructed using ExpiCHO TM The expression system (Thermofisher) completed the expression process. The expression procedure was implemented entirely according to the manufacturer's standard protocol. In short, the ExpiCHO-S was monitored the day before transfection. TM Cell growth density and viability were assessed, and cells were seeded after counting to achieve a final density of 3 × 10⁻⁶ cells. 6 ~4×10 6 Cells were cultured at 100 viable cells / mL, incubated overnight, under conditions of 37°C, 120 rpm, and humidified air containing 8% CO2. On the day of transfection, after cell counting, cells were diluted to a final density of 6 × 10⁶ cells / mL using fresh expression medium preheated to 37°C. 6 1 live cells / mL, volume 25mL. Prepare Reagent / plasmid DNA complex using cold reagent (4℃): Add 1mL Opti PRO serum-free medium and 20μg plasmid DNA to tube 1, and invert to mix; add 920μL Opti PRO serum-free medium and 80μL Reagent to tube 2, and invert to mix; then combine tubes 1 and 2, and gently invert to mix. Add the Reagent / plasmid DNA complex to the cells dropwise, gently shaking the culture flask during the addition process. After completion, place the culture flask in an incubator for normal incubation. The day after transfection, add 150μL Lenhancer and 6mL Feed to the cells. After 8 days of cell secretion expressing RSV pre-F protein, centrifuge at 4000rpm to remove cells, filter through a 0.45μm filter, and harvest the culture supernatant.

[0156] 1.3 Purification of RSV pre-F mutant

[0157] Protein purification was performed in two steps using affinity chromatography and ion exchange chromatography. Generally, the processed supernatant was added to a Ni-Sepharose 6FF column equilibrated with Buffer A (20 mM PB, 0.15 M NaCl, pH 7.4). Then, linear elution was performed using 0–100% Buffer B (20 mM PB, 0.15 M NaCl, 500 mM imidazole, pH 7.4). The eluent was collected and ultrafiltered to remove imidazole.

[0158] After buffer replacement, the mutant sample was diluted with 20 mM PB buffer to reduce its conductivity to below 4 mS / cm, and the pH was adjusted to 6.0. The protein was loaded into Capto Spimpres packing material equilibrated with Buffer C (20 mM PB, pH 6.0). After loading, the sample was washed with Buffer C until the A280 and conductivity curves were reached. Linear elution was performed using 0–100% Buffer D (20 mM PB, 1 M NaCl, pH 6.0), and the eluent was collected. The pH was adjusted to 7.4–8.0 with 0.2 M Na2HPO4, and the sample was filtered through a 0.45 μm filter to further remove insoluble particles.

[0159] 2. Identification and evaluation of RSV pre-F mutant proteins

[0160] 2.1 Identification of RSV pre-F mutant protein by SDS-PAGE and Native-PAGE

[0161] The purified proteins were detected by SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis). The purified sample was added to a sample buffer containing SDS (sodium dodecyl sulfate) and a reducing agent (dithiothreitol), boiled in a water bath, and then loaded onto a gel. After electrophoresis, the gel was stained with Coomassie Brilliant Blue to stain the proteins. After thorough destaining, gel images were acquired using a protein imaging system.

[0162] Figure 2 Using the representative mutant protein JW-195(A) as an example, SDS-PAGE analysis results under reducing (R) and non-reducing (NR) conditions are presented. The results show that the mutant protein can be obtained with high purity after purification. Taking JW-195(A) as an example, it exists as a monomer in the gel under reducing conditions, while under non-reducing conditions, it is maintained in a trimer conformation by its interchain disulfide bonds, and therefore appears as a trimer in electrophoresis.

[0163] The purified proteins were detected by Native-PAGE. The purified sample was loaded into a gel using sample buffer. After electrophoresis, the gel was stained with Coomassie Brilliant Blue, and after thorough destaining, gel images were acquired using a protein imaging system.

[0164] Figure 3Native-PAGE results of mutant trimer proteins are shown, displaying mutant proteins containing (JW-153(A) and JW-153(B)) and those without (JW-195(A) and JW-195(B)) exogenous trimeric domains. The results indicate that even mutant proteins without exogenous trimeric domains can maintain their trimeric conformation by interchain disulfide bonds under non-denaturing conditions. Figure 3 In this context, the monomeric F protein is shown in SEQ ID NO: 17.

[0165] 2.2 Purity and homogeneity of RSV pre-F mutant protein

[0166] The integrity and homogeneity of mutant proteins were determined using high-performance liquid chromatography (HPLC) (Thermo U3000). A TSKgel G3000PWXL size exclusion column (TOSOH) was used with a UV detector at 280 nm and PBS as the mobile phase at a flow rate of 1 mL / min. Peak values ​​of the sample in the column were detected. The protein elution peaks at different retention times were integrated by area analysis, and the purity of the target protein was calculated based on the percentage of the target protein elution peak area relative to the total peak area.

[0167] Figure 4 Taking the representative mutant protein JW-195(A) as an example, the results of size exclusion chromatography (SEC-HPLC) analysis of the mutant protein are shown. The results show that after purification, a protein with high purity and exhibiting a uniform characteristic single peak in SEC-HPLC can be obtained. Furthermore, the retention time is reduced to 16.920 min compared to the monomer (18.9 min). Figure 4 In this context, the monomeric F protein is shown in SEQ ID NO: 17.

[0168] 2.3 Expression level and thermostability of RSV pre-F mutant

[0169] The expression level of RSV pre-F mutant was detected using a double-antibody sandwich ELISA method. Palivizumab, which can simultaneously recognize both pre-F and post-F proteins, was used as the coating antibody to detect RSV pre-F. Horseradish peroxidase-labeled specific neutralizing antibody D25 monoclonal antibody and horseradish peroxidase-labeled AM14 monoclonal antibody recognizing RSV pre-F trimer were used as detection antibodies. Standard curves were established for each mutant protein using purified, serially diluted samples. The concentration of the sample was obtained by measuring its absorbance at OD450 and converting it to the standard curve based on the dilution factor. The sample detection procedure was as follows: Palizumab was diluted to 1 μg / L with carbonate buffer, 100 μL per well, and coated overnight at 2–8°C. After coating, the sample was washed three times with 20 mM PBS, followed by adding 200 μL of blocking buffer (4% bovine serum albumin) to each well and blocking for 60 min at room temperature. The sample was then washed three times with 20 mM PBS-T solution. A series of 3-fold diluted mutant F proteins were then added to 96-well microplates. PBS was used as the negative control. The plates were incubated at 37°C for 60 min. Then wash three times with 20mM PBS-T solution; take HRP-conjugated D25 antibody and AM14 antibody, dilute them 1:2000 with enzyme conjugate dilution buffer, add 100μL to each well of a 96-well microplate, and incubate at 37℃ for 10 min; remove the secondary antibody from the wells, wash three times with 20mM PBS-T solution, add 100μL of TMB chromogenic solution to each well, and stop the reaction by adding 50μL of stop solution after 10 min. Use a microplate reader to measure the absorbance values ​​of A450 and A630.

[0170] Figure 5 The expression levels of different pre-F trimer proteins in the culture supernatant on day 8 of expression were shown; the results indicate that the constructed pre-F trimer mutant protein can be expressed normally. Epitopes and trimer epitopes. Further addition of interchain disulfide bonds can effectively increase the proportion of trimerized pre-F protein, and it still exhibits trimerization even after removal of the exogenous trimeric domain T4.

[0171] The thermal stability of the samples was mainly evaluated by assessing the change in the Pre content of the antigen protein over time during storage at different temperatures. Specific storage temperatures and sampling times are as follows;

[0172] (a) Store at 60°C for 1 hour;

[0173] (b) Store at 37°C for 1, 3, 5, or 7 days;

[0174] (c) Store at 4°C for 1 week, 2 weeks, 3 weeks, and 4 weeks;

[0175] The methods for evaluating the stability of pre-F protein monomers and trimers were the same as those for detecting the expression levels of RSV pre-F mutants. The concentrations of pre-fusion conformation F protein and trimer conformation F protein in samples at different heat-treated time points were detected using D25 and AM14 monoclonal antibodies, respectively. Standard curves were established using serially diluted mutant proteins of known concentrations as standards. The pre-treatment mutant protein concentration was taken as 100%, and the remaining percentages of pre-fusion conformation F protein and trimer conformation F protein for each mutant F protein after treatment were calculated.

[0176] Figure 6 The results show the percentage of Pre-F and Trimeric F proteins remaining after storage at 60°C for 1 hour for different mutant proteins. The results indicate that, except for JW-189, other mutant F proteins can still stably retain about 95% of their pre-fusion or trimeric conformation after heat treatment at 60°C for 1 hour.

[0177] Figure 7 The results show the remaining percentages of Pre-F and trimer F proteins after storage at 37°C for 1, 3, 5, and 7 days, respectively. The results indicate that after 7 days of treatment at 37°C, more than 90% of the Pre-F and trimer F conformations of the F protein can still be stably retained, indicating that the mutant protein has good thermal stability, and the removal of the T4 trimerization motif has no effect on the thermal stability of the mutant protein.

[0178] Figure 8 The study also showed the remaining percentages of Pre-F and Trimeric F proteins after storage at 4°C for 1, 2, 3, and 4 weeks, respectively. The results indicated that the stabilized mutant Trimeric F protein could still retain more than 90% after treatment at 4°C for 4 weeks, suggesting that the mutant protein has good thermal stability before and after the removal of the T4 foldon trimerization motif.

[0179] Table 2 presents the evaluation results of the JW-195(A) mutation site. JW-195(A) contains two sets of intra-chain disulfide bonds, two sets of inter-chain disulfide bonds, and one single-point mutation. Among these mutants, there are mutants without the single-point mutation (JW-183), mutants without inter-chain disulfide bonds (JW-189(A) / JW-196(A)), and a single-sided mutant without intra-chain disulfide bonds (JW-201(A)). The expression levels and stability of the five mutant proteins were compared. The results show that the single-point mutation at amino acid position 305 increases protein expression; the deletion of inter-chain disulfide bonds significantly affects the trimer stability of the mutant protein. The deletion of intra-chain disulfide bonds drastically reduces protein expression and pre-fusion conformational stability.

[0180] Table 2

[0181]

[0182] 2.4 Evaluation of the biological activity of RSV pre-F mutant protein

[0183] The mutant RSV F protein was analyzed using an ELISA method. Antigenicity evaluation of epitopes, epitopes II, V, and trimeric epitopes. The method is as follows: Serially diluted concentrations of the test protein were diluted to 2 μg / mL using carbonate coating buffer, followed by 3-fold serial dilutions, and 100 μL was added to each well of a 96-well plate. The plates were incubated overnight at 4°C. The next day, the overnight coated plates were washed and dried. Blocking buffer was added at 150 μL / well, and the plates were incubated at 37°C for 2 hours to block non-specific binding sites. After blocking, the plates were washed and dried using a plate washer. The detection antibodies (D25 monoclonal antibody) were then added to each well. Epitope antibodies (Palivizumab (II epitope antibody), ADI-15568 monoclonal antibody (V epitope antibody), and AM14 (trimeric F protein antibody)) were diluted 5000-fold and added to each well of the ELISA plate at 100 μL / well, and incubated at 37°C for 1 h. After incubation, the ELISA plates were washed and dried. HRP-conjugated goat anti-human secondary antibody was diluted to an appropriate concentration and added to the ELISA plate, and incubated at 37°C for 30 min. After antibody incubation, TMB single-component chromogenic solution (warmed to room temperature) was added, and incubated at 37°C for 5 min. After chromogenic development, 50 μL / well of stop solution was added to terminate the reaction. The absorbance at OD450 / OD630 was read using a microplate reader. Data processing was performed using a four-parameter fitting method.

[0184] from Figure 9 The results show that JW-153(A) and JW-153(B) containing the T4 trimer domain, and JW-195(A) and JW-195(B) without the trimer domain, can all be... The specific antibody recognition of epitopes II, V and trimeric epitopes indicates that this trimeric pre-F protein can normally display the relevant epitopes.

[0185] Immunogenicity evaluation of 2.5RSV pre-F mutant protein

[0186] Mice were immunized with mutant proteins to evaluate the immunogenicity of RSV pre-F mutants with different pre-fusion conformations. Female BalB / c mice (14–16 g) were immunized with 6 μg of vaccine antigen in aluminum hydroxide + CpG as adjuvant. PostF protein was used as a control. Intramuscular injections were administered at weeks 0 and 3. Serum was collected after the first and second immunizations to determine total IgG antibody titers and neutralizing antibody titers.

[0187] Table 3

[0188]

[0189] 2.6 Detection of antigen-specific antibody levels

[0190] Antibody detection was performed using an indirect ELISA method. Simply put, the corresponding immune proteins for each group were used as capture proteins. The antigen was diluted to 2 μg / mL and added to the ELISA plate at 100 μL / well, and incubated overnight at 4°C. The overnight coated ELISA plate was washed and dried. Blocking buffer was added at 150 μL / well, and the plate was incubated at 37°C for 2 hours to block non-specific binding sites. After blocking, the ELISA plate was washed and dried. The serum samples to be tested were diluted proportionally and added to the ELISA plate at 100 μL / well, and incubated at 37°C for 1 hour. After incubation, the ELISA plate was washed and dried. The serum samples to be tested were serially diluted and added to the ELISA plate at 100 μL / well, and incubated at 37°C for 60 minutes. After incubation, wash and dry the ELISA plate. Dilute the enzyme-labeled secondary antibody (goat anti-mouse-AP) 1:3000 and add 100 μL / well to the plate, incubating at 37°C for 60 min. Weigh 0.1% Phosphatase substrate, add substrate buffer and mix until the chromogenic agent is completely dissolved. Wash the plate five times after avidin incubation and dry. Add 100 μL / well of the chromogenic solution to the plate and incubate at 37°C for 60 min. Add 50 μL / well of stop solution to the 96-well plate after chromogenic incubation to terminate the reaction. Preheat the ELISA reader 15 min before use and read the values ​​at OD405. Set 2.1 times the cutoff value for the negative group. Calculate the serum titers and perform four-parameter fitting on the results to calculate EC50.

[0191] Figure 10 The results shown in the figure indicate that the pre-F protein in different mutants can induce high levels of binding antibody titers after immunization. It is worth noting that the binding antibody titers of the mutants JW-195(A) and JW-195(B) after removing the T4 trimer domain are numerically lower than those of the JW-153(A) mutant containing the T4 trimer domain.

[0192] 2.7 Neutralizing antibody level detection

[0193] Neutralizing antibody titers were detected using a high-throughput microplate assay. Specifically, Hep-2 cells were seeded into 96-well plates and incubated overnight in a cell culture incubator (37°C, 5% CO2). The seeding density was adjusted to achieve approximately 90% confluence on the second day. The serum to be tested was then inactivated in a water bath at 56°C for 30 min. Subsequently, the initial well of the virus sample was diluted 100-fold, followed by a 3-fold serial dilution, resulting in 8 dilutions (including the initial well) and 2 replicates. The appropriate amount of virus was added based on the PFU value. Diluted virus was added to the sample wells and virus control wells, and the return drop wells were serially diluted 2-fold down to 4 dilutions. All wells were incubated at 37°C, 5% CO2 for 1 h. Then, the virus, serum neutralization products, positive control, and return drop well viruses were added to the prepared cells, with 100 μL of culture medium added to each well, and the cells were incubated for approximately 22 h. The supernatant was discarded, the cells were fixed, and fluorescently labeled detection antibodies were added. The plate was read using a CTL instrument. The neutralizing antibody titer was set at the serum dilution factor (NT50) that resulted in a 50% reduction in infected units.

[0194] Figure 11 The results showed that the serum of mice after booster immunization in each experimental group could efficiently neutralize both type A and type B wild-type live viruses, indicating that the RSV pre-F protein mutant without exogenous trimeric domain provided by this invention has good immunogenicity and can induce cross-protection against both type A and type B strains.

[0195] The proportion of neutralizing antibodies in total antigen-specific binding antibodies is evaluated by comparing the titers of binding antibody EC50 and neutralizing antibody NT50, thus assessing antibody quality. Figure 12 .

[0196] The antigen design strategy in this application effectively improves the pre-fusion conformational stability, trimer stability, and expression level of the trimerized RSV pre-F protein through a series of mutation strategies. Furthermore, the exogenous trimeric domain is removed, resulting in a T4-free trimeric F protein that exhibits good stability and immunogenicity.

[0197] This application describes the construction of a highly stable RSV pre-F mutant. By introducing two pairs of intrachain disulfide bonds (A170C / V179C, S180C / S186C), two pairs of interchain disulfide bonds (A241C / Q279C, D486C / A490C), and a single-point mutation (L305I) into the RSV F protein, the RSV F protein is stabilized in its pre-fusion conformation and exists in a trimer form. During the stabilization process, the two pairs of intrachain disulfide bonds (A170C / V179C, S180C / S186C) are respectively linked to (α3 / β3 and β3 / β4) within the monomer, stabilizing the RSV F protein in its pre-fusion conformation and providing a foundation for constructing the trimerized RSV pre-F protein. During stabilization, two pairs of intrachain disulfide bonds and two pairs of interchain disulfide bonds (A241C / Q279C, D486C / A490C) link the three monomers of the trimer in pairs at the middle and tail of the F protein, respectively. This provides a basis for the stable existence of the trimer and ensures that the mutant F protein still has good trimer stability even without the presence of exogenous trimeric domains. The introduction of L305I, present in the wild-type B subtype, during stabilization effectively increased the expression level of the trimerized RSV pre-F protein.

[0198]

[0199]

[0200]

[0201] The technical features of the above-described embodiments and examples can be combined in any suitable manner. For the sake of brevity, not all possible combinations of the technical features in the above-described embodiments and examples are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0202] The embodiments described above are merely illustrative of several implementation methods of this application, intended to facilitate a detailed understanding of the technical solutions of this application, but should not be construed as limiting the scope of protection of the patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the scope of protection of this application. Furthermore, it should be understood that after reading the above teachings of this application, those skilled in the art can make various alterations or modifications to this application, and the equivalent forms obtained also fall within the scope of protection of this application. It should also be understood that technical solutions obtained by those skilled in the art based on the technical solutions provided in this application through logical analysis, reasoning, or limited experimentation are all within the scope of protection of the appended claims. Therefore, the scope of protection of this patent application should be determined by the content of the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. An RSV pre-F mutant, characterized in that, The amino acid sequence of the RSV pre-F mutant is shown as positions 1 to 474 in SEQ ID NO:13 or SEQ ID NO:

14.

2. An RSV pre-F mutant, characterized in that, The RSV pre-F mutant consists of the fragment shown in positions 1 to 474 of SEQ ID NO:13 or SEQ ID NO:14 and a tag fragment connected to the C-terminus of the fragment.

3. The RSV pre-F mutant according to claim 2, characterized in that, The amino acid sequence of the RSV pre-F mutant is shown in SEQ ID NO:13 or SEQ ID NO:

14.

4. A nucleic acid molecule encoding the RSV pre-F mutant as described in any one of claims 1 to 3.

5. A vector comprising the nucleic acid molecule of claim 4.

6. An engineered cell expressing the RSV pre-F mutant of any one of claims 1 to 3, or comprising the nucleic acid molecule of claim 4 or the vector of claim 5.

7. A method for producing the RSV pre-F mutant according to any one of claims 1 to 3, comprising the following steps: The engineered cells of claim 6 are cultured, and the RSV pre-F mutant is isolated from the resulting culture supernatant.

8. An immune composition comprising the RSV pre-F mutant of any one of claims 1 to 3 or the nucleic acid molecule of claim 4, and an immune adjuvant.

9. The use of the RSV pre-F mutant according to any one of claims 1 to 3 in the preparation of a respiratory syncytial virus antibody detection kit.

10. A respiratory syncytial virus antibody detection kit comprising the RSV pre-F mutant according to any one of claims 1 to 3.

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