RSV pre-F mutant as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biology, and relates to an RSV pre-F mutant as well as a preparation method and application thereof. Relative to a wild type F0 polypeptide, the RSV pre-F mutant meets the following conditions: (1) the RSV pre-F mutant does not contain a furin enzyme cutting site fragment, a P27 polypeptide, a transmembrane structural domain and an intracellular structural domain; (2) the gene has mutations of S180C, S186C, A170C, V179C, A241C, Q279C, D486C and A490C; and (3) the C end of the F1 polypeptide is not connected or is connected with an aggregation motif. The conformations of the mutant and the trimer thereof are highly stable, the mutant and the trimer thereof can be self-assembled to form the trimer without depending on a heterotrimer structural domain, a high-level binding antibody and a neutralizing antibody can be induced after immunization, and the situation that the high-level binding antibody with non-neutralizing activity is generated when the heterotrimer structural domain is immunized is avoided, so that the quality of the antibody is improved.

Description

Technical Field

[0001] The present application belongs to the field of biotechnology and relates to a RSV pre-F mutant and a preparation method and application thereof. Background Art

[0002] Respiratory syncytial virus (RSV) is one of the main viruses that cause respiratory infections in infants, young children and the elderly, and is also one of the main causes of death from lower respiratory tract infections in children. RSV is most prevalent in autumn and winter, and is most frequently transmitted among young children. RSV is a single-stranded negative-sense enveloped RNA virus of 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 and people with weakened immune systems, RSV can cause severe lower respiratory tract infections such as bronchitis and pneumonia.

[0003] The F protein belongs to type I glycoprotein. The F protein that mediates the fusion of the virus and the host cell membrane exists in two forms: pre-fusion (preFusion) and post-fusion (postFusion). The F protein is expressed on the surface of the viral envelope as a metastable pre-fusion F protein. After contacting the host cell membrane, the N and C termini of the F1 protein undergo a series of rearrangements to form a long α-helical structure. This series of processes causes the F protein to transform from a metastable pre-F structure to a stable post-F structure. This structural change mediates the entry of the virus into the cell during viral infection. By analyzing and comparing the structure of the RSV F protein and the neutralizing antibodies it induced, it was found that the surface of the F protein in the pre-fusion conformation exists compared to the post-fusion conformation F protein. Therefore, the construction of a highly stable pre-fusion conformation of F protein is the main direction of RSV vaccine research and development at this stage, for example:

[0004] In 2013, the epitope was discovered and the site was named using antibody D25 To solve the problem of lack of D25 antibody, the site To solve the instability problem, we further modified and engineered a stable RSV F protein prefusion conformation (DS-Cav1) antigen mainly through disulfide bonds, cavity filling and charge mutation.

[0005] In 2015, Janssen Pharmaceutical published research on its construction of stabilized pre-F protein mutants, introducing a short-chain linker between F1 and F2. After the mutants bound to the fibrin domain, amino acid mutations were performed to obtain SC-DM and SC-TM.

[0006] GSK conducted secondary development based on DS-Cavl (US_8563002), and the patent focused on protecting and restricting the furin enzyme cleavage site and protein C-terminal trimerization. In view of its good immunogenicity and ability to induce neutralizing antibodies, GSK announced in 2022 that its RSV vaccine achieved positive results in Phase III clinical trials for adults over 60 years old, and obtained marketing authorization on May 3, 2023 (trade name: Arexvy), which is also the first vaccine against RSV virus.

[0007] The mutation strategies involved in the stabilized pre-F protein mutants designed by Pfizer based on its structure mainly include disulfide bond mutations and cavity filling. The disulfide bond mutation sites involved include: S55C / L188C, S155C / S290C, T103C / I148C, L142C / N371C, and other cavity filling and electrostatic mutation sites that enhance the stability of the pre-F protein. Due to the good immunogenicity and excellent vaccine efficacy of the mutants, Pfizer announced in 2022 that its bivalent RSV vaccine Phase III clinical trial achieved positive results and obtained marketing authorization in May 2023 (trade name: Abrysvo), becoming the second vaccine against RSV virus after Arexvy.

[0008] At present, the subunit vaccines Arexvy and Abrysvo, and the mRNA vaccine mRESVIA have been successfully launched on the market. They all use stabilized mutated F protein as RSV target antigen, and have shown good protective effects in clinical trials.

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

[0010] Based on this, one or more embodiments of the present application provide a RSV pre-F mutant and a preparation method and application thereof, including the following technical solutions:

[0011] One or more embodiments of the present application provide a RSV pre-F mutant, relative to the wild-type F0 polypeptide, the RSV pre-F mutant meets the following conditions:

[0012] (1) Not containing the furin cleavage site fragment, P27 polypeptide, transmembrane domain and intracellular domain;

[0013] (2) having mutation combination 1, mutation combination 2, mutation combination 3 and mutation combination 4, wherein:

[0014] Mutation combination 1 is S180C and S186C,

[0015] Mutation combination 2 is A170C and V179C,

[0016] Mutation combination 3 is A241C and Q279C,

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

[0018] (3) the C-terminus of the F1 polypeptide is not connected or is connected to an aggregation motif;

[0019] Optionally, the RSV pre-F mutant further satisfies the following conditions:

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

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

[0022] In some embodiments of the present 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 the present application, in the RSV pre-F mutant, the C-terminus of the F2 polypeptide does not contain NN.

[0024] In some embodiments of the present application, in the RSV pre-F mutant, the C-terminus of the F2 polypeptide and the N-terminus of the F1 polypeptide are directly connected by an amide bond or indirectly connected 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, GPGS, GGGG, GSGG, GGSG, SGGG, GSSG, SGSG, GSSG, GGPGG or GGGGS.

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

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

[0028] Optionally, the C-terminus of the F1 polypeptide and the N-terminus of the aggregation motif are directly connected via an amide bond or indirectly connected via 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, GPGS, GGGG, GSGG, GGSG, SGGG, GSSG, SGSG, GSSG, GGPGG or GGGGS.

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

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

[0032] One or more embodiments of the present application provide a vector comprising the nucleic acid molecule.

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

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

[0035] The engineered cells are cultured, and the RSV pre-F mutant is isolated from the obtained culture supernatant.

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

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

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

[0039] The details of one or more embodiments of the present application are set forth in the description which follows, and other features, objects, and advantages of the present application will be apparent from the description and its claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] In order to more clearly illustrate the technical solutions in the embodiments of the present application and to more completely understand the present application and its beneficial effects, the following is a brief introduction to the drawings required for the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present application, and those skilled in the art can obtain other drawings based on these drawings without creative work.

[0041] Figure 1 Schematic diagram of the structure of RSV F protein mutants;

[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 of mutant trimer protein;

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

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

[0046] Figure 6 The stability test results of different mutant proteins at 60°C;

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

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

[0049] Fig. 9 The results of biological activity evaluation of JW-153(A), JW-153(B) and JW-195(A) and JW-195(B) are shown in Table 1.

[0050] Fig.10 The results are for the detection of antigen-specific antibody levels of different mutant pre-F proteins;

[0051] Fig.11 The results of neutralizing antibody level testing for different mutants;

[0052] Fig.12 It is a statistical chart of antibody EC50 and neutralizing antibody NT50 titer. DETAILED DESCRIPTION

[0053] The present application will be further described in detail below in conjunction with the accompanying drawings, embodiments and examples. It should be understood that these embodiments and examples are only used to illustrate the present application and are not used to limit the scope of the present application. The purpose of providing these embodiments and examples is to make the understanding of the disclosure of the present application more thorough and comprehensive. It should also be understood that the present application can be implemented in many different forms, is not limited to the embodiments and examples described herein, and those skilled in the art can make various changes or modifications without violating the connotation of the present application, and the equivalent form obtained also falls within the protection scope of the present application. In addition, in the description below, a large number of specific details are given in order to provide a more comprehensive understanding of the present application, and 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 those commonly understood by those skilled in the art to which this application belongs. The terms used herein in the specification of this application are only for the purpose of describing implementation modes and embodiments and are not intended to limit this application.

[0055] the term

[0056] Unless otherwise specified or incompatible herewith, the terms and phrases used herein shall have the following meanings:

[0057] The terms "and / or", "or / and", and "and / or" used in this article include any one of two or more related listed items, and also include any and all combinations of related listed items, and the 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 that are all connected by "logical and", and undoubtedly includes technical solutions that are all connected by "logical or". For example, "A and / or B" includes three parallel solutions of 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, the technical solution that is all connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, the combination of any two or any three of A, B, C, and D, and also includes the combination of four of A, B, C, and D (that is, the technical solution that is all connected by "logical AND").

[0058] In the present application, "plurality", "multiple", "multiple times", "multiples", etc., unless otherwise specified, refer to a number greater than 2 or equal to 2. For example, "one or more" means one or greater than or equal to two.

[0059] As used herein, "combination thereof", "any combination thereof", "any combination thereof" etc. include all suitable combinations of any two or more of the listed items.

[0060] Herein, the “suitable” mentioned in “suitable combination”, “suitable method”, “any suitable method”, etc., shall be based on the ability to implement the technical solution of this application, solve the technical problems of this application, and achieve the expected technical effects of this application.

[0061] Herein, “preferred”, “better”, “more preferred” and “suitable” are merely used to describe implementation methods or examples with better effects, and it should be understood that they do not constitute limitations on the scope of protection of this application.

[0062] In the present application, "further", "further", "particularly" and the like are used for descriptive purposes to indicate differences in content, but should not be construed as limiting the scope of protection of the present application.

[0063] In this application, "optionally", "optional", and "optional" mean optional or dispensable, that is, any one of the two parallel schemes of "yes" or "no". If multiple "options" appear in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "optional" is independent.

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

[0065] In the present application, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0066] In the present application, when it comes to numerical intervals (i.e., numerical ranges), unless otherwise specified, the optional numerical distribution is considered continuous within the above numerical interval, and includes the two numerical endpoints (i.e., the minimum and maximum values) of the numerical range, and each numerical value between the two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to the integers within the numerical interval, it includes the two endpoint integers of the numerical range, and each integer between the two endpoints. In this article, it is equivalent to directly listing each integer, such as t is an integer selected from 1 to 10, indicating that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. In addition, when multiple ranges are provided to describe features or characteristics, these ranges can be combined. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges included therein.

[0067] The temperature parameters in this application, unless otherwise specified, are allowed to be either constant temperature treatment or to vary within a certain temperature range. It should be understood that the constant temperature treatment allows the temperature to fluctuate within the accuracy range controlled by the instrument. Fluctuations within the range of ±5°C, ±4°C, ±3°C, ±2°C, and ±1°C are allowed.

[0068] In the present 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 documents mentioned in this application are cited as references in this application, just as each document is cited as reference separately. Unless they conflict with the application purpose and / or technical solution of this application, the cited documents involved in this application are cited with all contents and all purposes. When the cited documents are involved in this application, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited. When the cited documents are involved in this application, the examples and preferred methods of the cited relevant technical features can also be incorporated into this application as references, but are limited to the implementation of this application. It should be understood that when the cited content conflicts with the description in this application, the present application shall prevail or be modified adaptively according to the description of this application.

[0070] RSV: Respiratory Syncytial Virus belongs to the Pneumoviridae family and the Pneumovirus genus. The RSV genome is about 15Kb long, contains 10 genes, and encodes 11 proteins. The virus displays three proteins on its surface: G protein, F protein, and SH protein.

[0071] F protein: A glycoprotein on the surface of RSV virus, which is formed by the F0 polypeptide being digested by furin during maturation to form F1 polypeptide, F2 polypeptide and P27 polypeptide. Among them, F1 polypeptide and F2 polypeptide constitute F protein monomer, and F protein is further presented as a trimer on the surface of the virus. F protein has a metastable pre-fusion conformation (Pre-Fusion) and a post-fusion conformation (PostFusion). The two protein conformations can be distinguished by Pre-F specific antibodies (such as D25, AM22, 5C4, MPE8, etc.).

[0072] The pre-F protein faces stability issues during construction. In addition, the existing construction strategy is to obtain a trimeric F protein by connecting a heterologous trimeric domain at the C-terminus. The trimeric domain produces a high level of non-neutralizing binding antibodies during immunization. This non-neutralizing antibody will reduce the proportion of neutralizing antibodies, affect the quality of effective antibodies, and pose potential safety risks. In addition, the non-neutralizing binding antibodies produced by the heterologous trimeric domain will exist in the form of pre-existing antibodies, affecting the efficacy of the vaccine in the subsequent long-term and repeated immunization process.

[0073] In a first aspect of the embodiments of the present application, a RSV pre-F mutant is provided. Compared with the wild-type F0 polypeptide, the RSV pre-F mutant satisfies the following conditions:

[0074] (2) having mutation combination 1, mutation combination 2, mutation combination 3 and mutation combination 4, wherein:

[0075] Mutation combination 1 is S180C and S186C,

[0076] Mutation combination 2 is A170C and V179C,

[0077] Mutation combination 3 is A241C and Q279C,

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

[0079] (3) the C-terminus of the F1 polypeptide is not connected or is connected to an aggregation motif;

[0080] Optionally, the RSV pre-F mutant further satisfies the following conditions:

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

[0082] In some embodiments of the present application, a highly stable RSV pre-F mutant is constructed, and its mutation strategy is to introduce S180C / S186C, A170C / V179C, A241C / Q279C, D486C / A490C and L305I mutations into the RSV F protein. These mutation combinations stabilize the RSV F protein in a prefusion conformation and exist in a trimer form. These mutation combinations are discovered for the first time and have not been reported. After treatment at 4°C, 37°C and 60°C, the mutant has highly stable prefusion conformations and trimer conformations, showing high stability; and the trimer of the RSV pre-F mutant of the present application does not rely on a heterotrimeric domain, and can self-assemble to form a highly stable trimer with or without a heterotrimeric domain. This trimeric F protein without heterotrimeric domains induces high levels of binding antibodies and neutralizing antibodies after immunization, avoiding the high level of non-neutralizing binding antibodies produced during immunization with exogenous trimer domains, thereby improving the antibody quality after final protein immunization. Overall, the present application constructs a trimeric pre-F protein that may not contain heterotrimeric domains, which can stably maintain the pre-fusion conformation and the trimeric conformation after heat treatment, and can also induce high levels of binding antibodies and neutralizing antibodies after mouse immunization, eliminating the potential impact of heterotrimers on vaccine efficacy and safety.

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

[0084] In some embodiments of the present 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 the present application, in the RSV pre-F mutant, the C-terminus of the F2 polypeptide does not contain NN.

[0086] In some embodiments of the present application, in the RSV pre-F mutant, the C-terminus of the F2 polypeptide and the N-terminus of the F1 polypeptide are directly connected by an amide bond or indirectly connected 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, GPGS, GGGG, GSGG, GGSG, SGGG, GSSG, SGSG, GSSG, GGPGG or GGGGS.

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

[0089] In some embodiments of the present application, the aggregation motif has the amino acid sequence shown in 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 the amino acid sequence.

[0090] Optionally, the C-terminus of the F1 polypeptide and the N-terminus of the aggregation motif are directly connected via an amide bond or indirectly connected via 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, GPGS, GGGG, GSGG, GGSG, SGGG, GSSG, SGSG, GSSG, GGPGG or GGGGS.

[0092] In some embodiments of the present application, the RSV pre-F mutant has the amino acid sequence shown at positions 1 to 474 in 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 the amino acid sequence.

[0093] The mutant of RSV pre-fusion F protein provided in the examples of the present application has a stable conformation and can trigger an immune response in a subject as an antigen / immunogen to produce antibodies.

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

[0095] Antibody: A polypeptide substantially encoded by an immunoglobulin gene or multiple immunoglobulin genes or fragments thereof in nature, which specifically binds and recognizes an analyte (e.g., an antigen or immunogen), such as an RSV F protein or an antigenic fragment thereof. Immunoglobulin genes include kappa, lambda, alpha, gamma, 6, epsilon, and mu constant region genes, as well as numerous immunoglobulin variable region genes. The term "antibody" as used herein includes antibody fragments produced, for example, by modification of intact antibodies and by de novo synthesis using recombinant DNA methods.

[0096] Antibodies exist, for example, in the form of complete immunoglobulins and in the form of a variety of well-characterized antibody fragments. For example, Fab, Fv and single-chain Fv (scFv) bound to RSV F protein will be RSV F protein-specific binding agents. This includes complete 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 protein is a fusion protein in which the light chain variable region of an immunoglobulin is bound to the heavy chain variable region of an immunoglobulin by a linker, and in dsFv, the chain has been mutated to introduce a disulfide bond to stabilize the association of the chain. The term also includes genetically engineered forms such as chimeric antibodies (e.g., humanized murine antibodies), heterologous binding antibodies (e.g., bispecific antibodies). See also Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, IL); Kuby, J., Immunology, 3rd ed., 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 an intact light chain and a portion of one heavy chain; (2) Fab', a fragment of an antibody molecule obtained by treating an intact antibody with the enzyme pepsin followed by reduction to obtain an intact light chain and a portion of a heavy chain; two Fab' fragments are obtained per antibody molecule; (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 of two Fab' fragments held together by two disulfide bonds; (5) Fv, a genetically engineered fragment containing the variable region of a light chain and the variable region of a heavy chain represented as two chains; and (6) single-chain antibody ("SCA"), a genetically engineered molecule containing the variable region of a light chain and the variable region of a heavy chain connected by a suitable polypeptide linker in the form of a genetically fused single chain molecule.

[0098] Typically, naturally occurring immunoglobulins have heavy (H) chains and light (L) chains interconnected by disulfide bonds. There are two types of light chains, λ and κ. There are five major heavy chain classes (or isotypes) that determine the functional activity of the antibody molecule: IgM, IgD, IgG, IgA, and IgE. The disclosed antibodies can be class-switched.

[0099] Each heavy and light chain contains a constant region and a variable region, (the regions are also referred to as "domains"). In several embodiments, the heavy and light chain variable domains combine to specifically bind to an antigen. In other embodiments, only the heavy chain variable domain is required. For example, naturally occurring camel antibodies consisting only of heavy chains are functional and stable in the absence of light chains (see, e.g., 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 a "framework" region interrupted by three hypervariable regions (also referred to as "complementarity determining regions" or "CDRs") (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, 1991). The sequences of the framework regions of different light or heavy chains are relatively conserved within a species. The framework region of an antibody, the combined framework regions of the constituent light and heavy chains, is used to position and align the CDRs in three-dimensional space.

[0100] The CDR is primarily responsible for binding to the epitope of the antigen. The amino acid sequence boundaries of a given CDR can be readily determined using any of a variety 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 of each chain are usually referred to as CDR1, CDR2 and CDR3 (from N-terminus to C-terminus), and are usually also identified by the chain to which a particular CDR is located. Thus, VHCDR3 is located in the variable domain of the heavy chain of the antibody in which it is present, while VLCDR1 is the CDR1 of the variable domain of the light chain of the antibody in which it is present. 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 antibodies or T cell responses in an animal, including compositions that are injected or absorbed into an animal. Antigens react with products of specific humoral or cellular immunity, including those induced by heterologous antigens, such as mutants of the disclosed RSV prefusion 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 examples, antigens include peptides derived from pathogens of interest, such as RSV. In a specific example, the antigen is derived from RSV, such as an antigen comprising a modified RSV F protein stabilized in a prefusion conformation. "Epitope" or "antigenic determinant" refers to an antigenic region that reacts with B and / or T cells.

[0103] Immunogen: A protein or portion thereof that is capable of inducing an immune response in a mammal, such as a mammal infected with a pathogen or at risk of infection with a pathogen. Administration of an immunogen can result in protective immunity and / or active immunity against the pathogen of interest. Pre-F mutants such as those provided in the examples of the present 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 for a particular antigen (an "antigen-specific response"). In one embodiment, the immune response is a T cell response, such as a CD4+ response or a CD8+ response. In another embodiment, the response is a B cell response and results in the production of specific antibodies.

[0105] One of ordinary skill in the art will recognize that individual substitutions, deletions or additions that alter, add or delete a single amino acid or a small percentage of amino acids (e.g., less than 20%, 15, 10, 5%, etc.) in a coding sequence are conservative variations, wherein the alteration results in the replacement of an amino acid with a chemically similar amino acid. Conservative 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 conservative substitutions 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

[0182] 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

[0106] Not all residue positions within a protein will tolerate an otherwise "conservative" substitution. For example, if the amino acid residue is essential for protein function, even an otherwise conservative substitution may destroy the activity, e.g., specific binding of an antibody to a target epitope may be destroyed by a conservative mutation in the target epitope.

[0107] Epitope: Antigenic determinant. These are specific chemical groups or peptide sequences on antigenic molecules so that they induce specific immune responses, for example, epitopes are antigenic regions that react with B and / or T cells. Antibodies bind to specific antigenic epitopes, such as epitopes of RSVF proteins, for example, D25 or AM22 epitopes present in the pre-fusion conformation of RSVF proteins. Epitopes can be formed by continuous amino acids or discontinuous amino acids juxtaposed by the tertiary folding of proteins. Epitopes formed by continuous amino acids are usually kept exposed to denaturing solvents, while epitopes formed by tertiary folding are usually lost when treated with denaturing solvents. Epitopes generally include at least 3 and more generally at least 5, about 9 or about 8-10 amino acids in a unique 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 particular epitope on an antigen that specifically binds to an antibody of interest, such as a monoclonal antibody. In some examples, the target epitope includes amino acid residues that contact the antibody of interest such that the target epitope can be selected by determining the amino acid residues that contact the antibody of interest.

[0109] The amino acids in a peptide, polypeptide or protein are usually chemically bound together via an amide bond (CONH). In addition, the amino acids may be bound together by other chemical bonds. For example, the linkage of amino acids or amino acid analogs may 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, B. Weinstein, ed., 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 are stabilized in a pre-fusion conformation, for example, the mutants of the present application embodiments can be modified, for example, to include amino acid substitutions compared to the natural RSV protein sequence, or by a variety of chemical techniques to produce derivatives having substantially the same activity and conformation as the unmodified peptide and optionally having other desired properties. For example, the carboxylic acid group of the protein, whether it is the carboxyl terminus or the side chain, can be provided in the form of a pharmaceutically acceptable cationic salt or esterified to form a C1-C16 ester, or converted to an amide of the formula NR1R2, wherein R1 and R2 are each independently H or C1-C16 alkyl, or combined to form a heterocycle, such as a 5-membered or 6-membered ring. The amino group of the peptide, whether it is the amino terminus or the side chain, can be in the form of a pharmaceutically acceptable acid addition salt such as HCl, HBr, acetate, benzoate, tosylate, maleate, tartrate and other organic salts, or can be modified to a C1-C16 alkyl or dialkylamino or further converted to an amide.

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

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

[0113] Nucleic acid molecule, nucleic acid: the polymer consisting of nucleotide units (ribonucleotides, deoxyribonucleotides, related naturally occurring structural variants and analogs of non-naturally occurring synthesized thereof) connected by phosphodiester bonds, related naturally occurring structural variants, and analogs of non-naturally occurring synthesized thereof. Therefore, the term includes nucleotides and connections therebetween including nucleotide polymers of non-naturally occurring synthetic analogs such as and not limited to thiophosphates, phosphoramidates, methylphosphonates, chiral methylphosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNA), etc. These polynucleotides can be synthesized, for example, using an automated DNA synthesizer. The term "oligonucleotide" generally refers to a short polynucleotide that is usually not 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 (i.e., A, U, G, C) in which "U" replaces "T".

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

[0115] "Coding" refers to the inherent properties of a specific sequence of nucleotides such as genes, cDNA or mRNA in a polynucleotide, which serve as templates for synthesizing other polymers and macromolecules in a biological process, and have nucleotides (for example, rRNA, tRNA and mRNA) or amino acids of a limited sequence and the resulting biological properties. Therefore, if the transcription and translation of the mRNA produced by a gene produce protein in a cell or other biological system, the gene encodes the protein. Its nucleotide sequence is identical to the mRNA sequence and the coding strands provided in a sequence table form and the non-coding strands used as the transcription template of a gene or cDNA can be referred to as encoding the protein or other products of the gene or cDNA. Unless otherwise indicated, "nucleotide sequences encoding amino acid sequences" include all nucleotide sequences that are in degenerate form and encode the same amino acid sequence to each other. The nucleotide sequences of coded proteins and RNA may include introns. In some instances, nucleic acid encodes the mutant of the application.

[0116] Taking into account degenerate variants and conservative variants, the present application does not impose any particular limitation on nucleic acid molecules.

[0117] Degenerate variants and conservative variants: polynucleotides encoding polypeptides including sequences that are degenerate due to the genetic code. For example, polynucleotides encoding disclosed antigens or antibodies that specifically bind to disclosed antigens that include sequences that are degenerate due to the genetic code. There are 20 natural amino acids, most of which are specified by more than one codon. Therefore, all degenerate nucleotide sequences are included, as long as the amino acid sequence of the antigen or antibody that binds 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 specifying arginine within the protein coding sequence, the codon can be changed to any of the corresponding codons without changing the encoded protein. Such nucleic acid variations are "silent variations", which are a type of conservative variations. Each nucleic acid sequence encoding a polypeptide herein also describes each possible silent variation. Those skilled in the art will recognize that each codon in a nucleic acid (except AUG, which is usually the only codon for methionine) can be modified by standard techniques to produce functionally identical molecules. Accordingly, each "silent variation" of a nucleic acid that encodes a polypeptide is implicit in each described sequence.

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

[0119] Expression control sequence: a nucleic acid sequence that regulates the expression of an operably connected heterologous nucleic acid sequence. When the expression control sequence controls and regulates the transcription and appropriate translation of the nucleic acid sequence, the expression control sequence is operably connected to the nucleic acid sequence. Therefore, the expression control sequence may include a suitable promoter, an enhancer, a transcription terminator, a start codon (ATG) in front of a protein-coding gene, a splicing signal of an intron, a suitable reading frame of the gene to allow appropriate translation of mRNA, and a stop codon. The term "control sequence" is intended to minimally include components whose presence may affect expression, and may also include other components whose presence is advantageous, for example, a leader sequence and a fusion partner sequence. The expression control sequence may include a promoter.

[0120] A promoter is a minimal sequence sufficient to direct transcription. Also included are those promoter elements sufficient to make promoter-independent gene expression controllable with respect to cell type specificity, tissue specificity, or inducible by external signals or agents; these elements may be located in the 5′ or 3′ region of the gene. Constitutive and inducible promoters are included (see, e.g., Bitter et al., Methods in Enzymology 153: 516-544, 1987). For example, when cloning in a bacterial system, inducible promoters such as pL of bacteriophage λ, plac, ptrp, ptac (ptrp-lac hybrid promoter) and the like may be used. In one embodiment, when cloning in a mammalian cell system, promoters derived from the genome of mammalian cells (e.g., metallothionein promoters) or promoters derived from the genome of mammalian viruses (e.g., retroviral long terminal repeats; adenovirus late promoters; vaccinia virus 7.5K promoters) may be used. Promoters produced by recombinant DNA or synthetic techniques may also be used to provide transcription of nucleic acid sequences.

[0121] The polynucleotide can be inserted into an expression vector containing a promoter sequence that promotes efficient transcription of the inserted genetic sequence of the host. The expression vector usually contains an origin of replication, a promoter, and specific nucleic acid sequences that allow phenotypic selection of transformed cells.

[0122] RSV F proteins from different RSV subgroups, as well as nucleic acid sequences encoding these proteins and methods for manipulating and inserting these nucleic acid sequences into vectors are disclosed herein and known in the art (see, e.g., Tan et al., PLOS one, 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 the present application provide a vector comprising the nucleic acid molecule.

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

[0125] Expression: The translation of nucleic acid into protein. Proteins can be expressed and retained within the cell, become components of the cell surface membrane, or be secreted into the extracellular matrix or culture medium.

[0126] Engineered cell, host cell: A cell in which a vector can propagate and express its DNA. The cell can be prokaryotic or eukaryotic. The term also includes any progeny of the subject host cell. It should be understood that all progeny may not be identical to the parent cell because mutations may occur during replication. However, these progeny are included when the term "host cell" is used.

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

[0128] The engineered cells are cultured, and the RSV pre-F mutant is isolated from the obtained culture supernatant.

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

[0130] Immunological adjuvant: a vehicle for enhancing antigenicity. Adjuvants include suspensions of minerals (alum, aluminum hydroxide or phosphate) adsorbed with antigens; or water-in-oil emulsions, for example, wherein the antigen solution is emulsified in mineral oil (Freund's incomplete adjuvant), sometimes including killed mycobacteria (Freund's complete adjuvant) to further enhance antigenicity (inhibit antigen degradation and / or cause macrophage influx). Immunostimulatory oligonucleotides (e.g., those including CpG motifs) can also be used as adjuvants. Adjuvants include biological molecules ("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 the present application are one or more of aluminum salt adjuvants, surfactants, polynucleotides, lipopolysaccharides, liposomes, and oil emulsion adjuvants. For example, Alum, CpG, Alum+CpG, MF59, AS04, AS01E, etc. MF59 is a water-mixed adjuvant, mainly composed of three parts: oil phase, emulsifier and adjuvant; the oil phase is a mixture of micronized short-chain triglycerides suitable for human injection; the emulsifier is a surfactant that can evenly mix the oil phase and water; the adjuvant mainly includes glycerol, threitol, ATP and other moisturizers and buffers. 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 nano-liposome solution prepared from DOPC, Chol, MPL and QS-21. The main component of MPL adjuvant is lipopolysaccharide.

[0131] Immune composition (immunogenic composition): A composition comprising an antigen that induces an immune response, such as a measurable CTL response to a virus expressing an antigen or a measurable B cell response (e.g., antibody production) to an antigen. Thus, an immunogenic composition comprises one or more antigens (e.g., polypeptide antigens) or antigenic epitopes. The 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 the subject from symptoms or conditions induced by a pathogen. In some cases, symptoms or diseases caused by a pathogen (e.g., RSV) are prevented (or reduced or improved) by inhibiting the replication of the pathogen after the subject is exposed to the pathogen. In one example, an "immunogenic composition" comprises a recombinant RSV F protein stabilized in a prefusion conformation that induces a measurable CTL response to a virus expressing an RSV F protein, or induces a measurable B cell response (e.g., antibody production) to an RSV F protein. It further refers to an isolated nucleic acid encoding an antigen, such as a nucleic acid that can be used to express the antigen (and thus to induce an immune response against such a polypeptide).

[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 generally include a protein, immunogenic peptide, or nucleic acid in a pharmaceutically acceptable carrier and / or other agent. Any particular peptide, such as a disclosed RSV F protein stabilized in a prefusion conformation or a nucleic acid encoding a disclosed RSV F protein stabilized in a prefusion conformation, can be easily tested for its ability to induce a CTL or B cell response by a recognized assay. The immunogenic composition may include adjuvants well known to those skilled in the art.

[0133] Immunological reaction conditions: including reference to conditions that allow antibodies produced for a specific epitope to bind to the epitope and the extent of their binding is detectably greater than substantially all other epitopes and / or substantially excludes binding to substantially all other epitopes. Immunological reaction conditions depend on the antibody binding reaction format and are typically those used in immunoassay protocols or those encountered in vivo. The immunological reaction conditions used in the method are "physiological conditions", which include reference to typical conditions (e.g., temperature, molar osmotic pressure concentration, pH) inside living mammals or mammalian cells. Although it is recognized that some organs are subjected to extreme conditions, the intracellular and intracellular environment is typically about pH 7 (e.g., pH 6.0 to pH 8.0, more typically pH 6.5 to 7.5), contains water as the main solvent, and exists at a temperature above 0°C and below 50°C. The molar osmotic pressure concentration is within the range of supporting cell viability and proliferation.

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

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

[0136] One or more embodiments of the present application provide a method for preventing and treating lower respiratory tract infection 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: Introducing a composition into a 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 a vein of a subject.

[0138] Effective amount: An amount of an agent, such as a PreF antigen or a nucleic acid encoding a PreF antigen, or other agent, sufficient to produce a desired response, such as an immune response to RSV F protein, or to reduce or eliminate a sign or symptom of a condition or disease, such as RSV infection. For example, this may be an amount required to inhibit viral replication or measurably change an outward symptom of viral infection. Generally, such an amount will be sufficient to measurably inhibit viral (e.g., RSV) replication or infectivity. When administered to a subject, a dose that will achieve a target tissue concentration (e.g., in respiratory tissue) that has been demonstrated to achieve in vitro inhibition of viral replication will generally be used. In some examples, an "effective amount" is an amount that treats (including prevents) one or more symptoms and / or underlying causes of any condition or disease, such as an amount that treats RSV infection. In one example, an effective amount is a therapeutically effective amount. In one example, an effective amount is an amount that prevents the development of one or more signs or symptoms of a particular disease or condition, such as one or more signs or symptoms associated with RSV infection.

[0139] Prevention, prevention, treatment means: for example, in a subject at risk of a disease (e.g., RSV infection), inhibiting the full development of a disease or condition. "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" with respect to a disease or pathological condition refers to any observable beneficial therapeutic effect. The beneficial effect may be demonstrated, for example, by the delayed onset of clinical symptoms of the disease in a susceptible subject, 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 health of the subject, or other parameters that are well known in the art to be specific to a particular disease. "Preventive" treatment is a treatment administered to a subject who does not show signs of the disease or only shows early signs in order to reduce the risk of developing a lesion. The term "reduce" is a relative term, such that if the reaction or condition is quantitatively reduced after the administration of the agent, or if it is reduced after the administration of the agent compared to a reference agent, the agent reduces the reaction or condition. Similarly, the term "prevention" does not necessarily mean that the agent completely eliminates the reaction or condition, as long as at least one feature of the reaction or condition is eliminated. Thus, an immunogenic composition that reduces or prevents an infection or reaction (e.g., a pathological reaction, e.g., a vaccine-enhanced viral disease) can, but need not completely eliminate, such an infection or reaction, as long as the infection or reaction is measurably attenuated, e.g., by at least about 50%, e.g., by at least about 70%, or about 80%, or even about 90% (i.e., to 10% or less) compared to the infection or reaction in the absence of the agent or compared to a reference agent.

[0140] In the tenth aspect of the embodiments of the present application, the embodiments of the present application provide a method for detecting or isolating RSV F binding antibodies in a sample, comprising the following steps:

[0141] contacting the mutant described in the first aspect with an RSV F binding antibody in a sample to form an immune complex; and,

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

[0143] The embodiments of the present application will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods for which specific conditions are not specified in the following examples are preferably referred to the guidance provided in the present application, and can also be based on the experimental manual or normal conditions in this area, can also be based on the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0144] In the following specific embodiments, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

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

[0146] 1.1 Construction of RSV pre-F mutant

[0147] Figure 1 The schematic diagram of the structure of the RSV preF protein mutant constructed. The 103rd to 145th amino acids of the RSV F0 polypeptide are connected using a GS short peptide, and the furin cleavage site of F0 is deleted, which is not affected by furin, so that the final F protein is a trimer protein composed of a single chain. At the same time, the transmembrane domain and the intracellular domain (TM, 514aa-550aa) and the intracellular domain (CT, 551aa-574aa) are deleted, and the final monomer protein is expressed in a secretory form; unless otherwise specified, other monomer RSV F protein mutations involved in the invention of the present application embodiment are designed on this basis.

[0148] According to the amino acid sequence of RSV pre-F mutant, the codons preferred by Chinese hamster ovary cells Cricetulusgriseus (CHO cells) were optimized to facilitate expression in . The optimized nucleotide fragments were gene synthesized and finally connected to the double-enzyme-cut pcDNA3.1 vector by restriction endonucleases BamHI and XhoⅠ to construct a recombinant vector.

[0149] The recombinant vector was subjected to gene manipulations such as substitution, insertion and deletion using the ClonExpress II Recombination Cloning Kit (Novagene Biotech Co., Ltd.). The fragments at both ends of the mutation site were amplified by polymerase chain reaction (PCR) using the high-fidelity enzyme Phanta Max (Novagene Biotech Co., Ltd.). The amplified products were recovered by gel and then the two fragments were fused by PCR. The recovered fusion fragments were homologously recombined into the pcDNA3.1 vector after double digestion with BamHⅠ and XhoⅠ. The constructed point mutation vector was sequenced correctly by Beijing Qingke Biotech Co., Ltd. and used for subsequent experiments.

[0150] The point mutation vector after sequencing verification was transformed into E. coli and spread on LB (Amp+) plates. A single clone was picked and cultured in 5 mL LB (Amp+) liquid medium for 8 hours, then inoculated into 300 mL LB (Amp+) liquid medium, cultured at 37°C and 180 rpm for 16 hours, and the plasmid was extracted using a large / large plasmid extraction kit; finally stored in 1 mL sterile TE Buffer. All commercial kits or reagents were performed according to the manufacturer's instructions.

[0151] Table 1. RSV F protein mutant protein sequence design

[0152]

[0153]

[0154] 1.2 Expression of RSV pre-F mutant

[0155] ExpiCHO TM Expression was performed using the ExpiCHO-S expression system (Thermofisher). The expression process was carried out in full accordance with the manufacturer's standard protocol. Briefly, the day before transfection, the ExpiCHO-S TM The cell growth density and viability were measured. After cell counting, the cells were divided to make the final density 3×10 6 ~4×10 6 viable cells / mL, grown overnight, the cell culture conditions were 37°C, 120 rpm, humidified air containing 8% CO2. On the day of transfection, after counting the cells, dilute the cells to a final density of 6×10 using fresh expression medium preheated to 37°C. 6 viable cells / mL, volume 25mL. Use cold reagent (4℃) to prepare Reagent / plasmid DNA complex: add 1mL Opti PRO serum-free medium, 20μg plasmid DNA to tube 1, and invert to mix; add 920μL Opti PRO serum-free medium, 80μL Lessagent to tube 2, invert to mix; then mix tubes 1 and 2 and gently invert to mix. Add Reagent / plasmid DNA complex to the cells, gently shake the culture bottle during the addition process, and place the culture bottle in the incubator for normal culture after the end. The day after transfection, add 150μL Enhancer and 6mL Feed to the cells. After the cells secrete and express RSV pre-F protein for 8 days, centrifuge at 4000rpm to remove the cells and filter through a filter with a pore size of 0.45μm to harvest the culture supernatant.

[0156] 1.3 Purification of RSV pre-F mutant

[0157] Protein purification was completed in two steps using affinity chromatography and ion exchange chromatography. In general, the treated supernatant was added to a Ni-Sepharose 6FF column that had been equilibrated with Buffer A (20 mM PB, 0.15 M NaCl, pH 7.4). Then, the column was linearly eluted using 0-100% Buffer B (20 mMPB, 0.15 M NaCl, 500 mM imidazole, pH 7.4) elution buffer, and the eluate was collected and ultrafiltered to remove imidazole.

[0158] After the liquid change, the mutant sample was diluted with 20mM PB buffer to make its conductivity lower than 4ms / cm, and the pH was adjusted to 6.0. The protein was loaded into the Capto SPImpres filler balanced with Buffer C (20mM PB, pH6.0). After loading, it was rinsed with Buffer C to the level of A280 and conductivity curve, and linearly eluted with 0-100% Buffer D (20mM PB, 1MNaCl, pH6.0), and the eluate was collected. The pH was adjusted to 7.4-8.0 with 0.2M Na2HPO4, and insoluble particles were further removed by filtration through a 0.45μm filter membrane.

[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 protein was detected by SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide GelElectrophoresis). The purified test sample was added to a sample buffer containing SDS (sodium dodecyl sulfate) and a reducing agent (dithiothreitol), and then loaded into the gel after boiling in a water bath. After the electrophoresis, the gel was stained with Coomassie Brilliant Blue staining method for protein, and the gel image was obtained using a protein imaging system after sufficient decolorization.

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

[0163] The purified protein was detected by Native-PAGE. The purified test sample was added to the sample buffer and loaded into the gel. After electrophoresis, the gel was stained with Coomassie Brilliant Blue. After sufficient decolorization, the gel image was obtained using a protein imaging system.

[0164] Figure 3The Native-PAGE results of mutant trimeric proteins are shown, showing mutant proteins containing (JW-153(A) and JW-153(B)) and not containing (JW-195(A) and JW-195(B)) exogenous trimeric domains; the results show that under non-denaturing conditions, even the interchain disulfide bonds of the mutant proteins without exogenous trimeric domains can maintain them in a trimeric conformation. Figure 3 In the present invention, 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 detected by high performance liquid chromatography (HPLC) (ThermoU3000). A TSKgel G3000PWXL molecular exclusion column (TOSOH) was used, the UV detector wavelength was 280nm, and the mobile phase was PBS, 1mL / min. The peak of the sample in the chromatographic column was detected. The protein elution peaks with different retention times were processed by area integration, and the purity of the target protein was calculated based on the percentage of the target protein elution peak area to the total peak area.

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

[0168] 2.3 Expression and thermal stability of RSV pre-F mutants

[0169] The expression level of RSV pre-F mutant was detected using double antibody sandwich ELISA. Palivizumab, which can recognize both pre-F and post-F proteins, was used as the coating antibody. The horseradish peroxidase-labeled specific neutralizing antibody D25 monoclonal antibody for the site and the horseradish peroxidase-labeled AM14 monoclonal antibody for recognizing the trimeric RSV pre-F were used as detection antibodies. At the same time, the purified gradient diluted mutant proteins were used to establish their respective standard curves. The content of the sample to be tested was obtained by detecting its absorbance at OD450 and the dilution multiple through the standard curve conversion. The sample detection process is as follows: take palivizumab and dilute it to 1μg / L using carbonate buffer, 100μL per well, and coat overnight at 2-8℃; after coating, wash 3 times with 20mM PBS, then add 200μL blocking solution (4% bovine serum albumin) to each well, and block at room temperature for 60min; then wash 3 times with 20mM PBS-T solution, and add a series of 3-fold diluted mutant F proteins to a 96-well ELISA plate, and the negative control is PBS, and react at 37℃ for 60min. Then wash three times with 20mM PBS-T solution; take HRP-coupled D25 antibody and AM14 antibody respectively, dilute them with enzyme conjugate diluent 1:2000, add them to the 96-well ELISA plate, 100μL per well, and react at 37℃ for 10min; aspirate the secondary antibody in the well, wash three times with 20mM PBS-T solution, add 100μL TMB colorimetric solution to each well, add 50μL stop solution to terminate the reaction after 10min, and use an ELISA reader to measure the A450 and A630 absorbance values.

[0170] Figure 5 The expression levels of pre-F and trimer F proteins in the culture supernatant of different pre-F trimer proteins on the 8th day of expression are shown; the results show that the constructed pre-F trimer mutant protein can be normally displayed Further addition of interchain disulfide bonds can effectively increase the proportion of trimerized pre-F protein and still present it as a trimer after removing the exogenous trimer domain T4.

[0171] The thermal stability of the sample is mainly evaluated by evaluating the changes in the Pre content of the antigen protein over time during the storage of the antigen protein at different temperatures. The specific storage temperature and sampling time are as follows;

[0172] (a) at 60°C for 1 hour;

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

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

[0175] The pre-F protein monomer and trimer stability evaluation detection method is the same as the RSV pre-F mutant expression detection method. D25 monoclonal antibody and AM14 monoclonal antibody were used to detect the concentration of prefusion conformation F protein and trimer conformation F protein of samples at different time points of heat treatment. A standard curve was established by gradient dilution using mutant proteins of known concentration as standards. The mutant protein concentration before treatment was taken as 100%, and the remaining percentage of prefusion conformation F protein and trimer conformation F protein of each mutant F protein after treatment was calculated.

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

[0177] Figure 7 The remaining percentages of Pre-F and trimeric F proteins of different mutant proteins after being stored at 37°C for 1, 3, 5 and 7 days were shown; the results showed that after treatment at 37°C for 7 days, the Pre-F and trimeric F conformation F proteins could still be stably retained at more than 90%, indicating that the mutant protein has good thermal stability and that removal of the T4 trimerization motif has no effect on the thermal stability of the mutant protein.

[0178] Figure 8 The remaining percentages of Pre-F and trimeric F proteins of different mutant proteins after being stored at 4°C for 1, 2, 3, and 4 weeks were also shown; the results showed that the stabilized mutant trimeric F protein could still stably retain more than 90% after treatment at 4°C for 4 weeks, indicating that the mutant protein had good thermal stability before and after the removal of the T4 foldon trimerization motif.

[0179] Table 2 shows the results of the evaluation of the mutation sites of JW-195(A), which contains two sets of intrachain disulfide bonds, two sets of interchain disulfide bonds and one single point mutation. Among these mutants, there are mutants without single point mutations (JW-183), mutants without interchain disulfide bonds (JW-189(A) / JW-196(A)) and mutants without intrachain disulfide bonds (JW-201(A)). The expression and stability of the five mutant proteins were compared respectively. The results showed that the single point mutation of amino acid 305 increased the protein expression; the loss of interchain disulfide bonds significantly affected the trimer stability of the mutant protein. The loss of intrachain disulfide bonds greatly reduced the protein expression and pre-fusion conformational stability.

[0180] Table 2

[0181]

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

[0183] The mutant RSV F protein was tested by ELISA. Antigenicity evaluation of epitope, II epitope, V epitope and trimer epitope. The method is as follows: dilute the gradient dilution concentration of the protein to be tested to 2μg / mL using carbonate coating solution, then dilute it 3 times and add 100μL to a 96-well plate. Coat overnight at 4℃. The next day, wash and pat dry the overnight coated ELISA plate, add 150μL / well of blocking solution, and incubate at 37℃ for 2h to block nonspecific binding sites. Wash and pat dry the blocked ELISA plate with a plate washer, and add the detection antibodies (D25 monoclonal antibody ( Epitope antibody), Palivizumab (Ⅱ epitope antibody), ADI-15568 monoclonal antibody (Ⅴ epitope antibody) and AM14 (trimeric F protein antibody) were diluted 5000 times and added to the ELISA plate at 100 μL / well respectively, and incubated at 37°C for 1 hour. The ELISA plate after the incubation of the sample was washed and patted dry. HRP-conjugated goat anti-human secondary antibody was diluted to an appropriate concentration, added to the ELISA plate, and incubated at 37°C for 30 minutes. After the enzyme-labeled antibody incubation was completed, TMB single-component colorimetric solution was added to room temperature and incubated at 37°C for 5 minutes. After the color development was completed, 50 μL / well stop solution was added to terminate the reaction. The absorbance value at OD450 / OD630 wavelength was read using an ELISA reader. Data processing was performed according to four-parameter fitting.

[0184] from Fig. 9 The results showed that JW-153 (A) and JW-153 (B) containing the T4 trimerization domain and JW-195 (A) and JW-195 (B) without the trimerization domain could be Specific antibodies recognized epitope Ⅱ, epitope Ⅴ and trimer epitope, indicating that this trimer pre-F protein can normally display the relevant epitopes.

[0185] 2.5 Evaluation of the immunogenicity of RSV pre-F mutant protein

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

[0187] Table 3

[0188]

[0189] 2.6 Antigen-specific antibody level detection

[0190] The binding antibody detection was completed using the indirect ELISA method. In short, the corresponding immune protein of each group was used as the capture protein, the antigen was diluted to 2μg / mL and added to the ELISA plate at 100μL / well, and coated at 4°C overnight. The overnight coated ELISA plate was washed and patted dry with a plate washer, and the blocking solution was added at 150μL / well and incubated at 37°C for 2h to block nonspecific binding sites. The blocked ELISA plate was washed and patted dry with a plate washer, and the serum to be tested was diluted proportionally. Add 100μL / well to the ELISA plate and incubate at 37°C for 1h. The ELISA plate at the end of the incubation sample was washed and patted dry with a plate washer. After the serum sample to be tested was diluted in a gradient ratio, it was added to the ELISA plate at 100μL / well and incubated at 37°C for 60min. After the incubation is completed, the ELISA plate is cleaned and patted dry. The enzyme-labeled secondary antibody (sheep anti-mouse-AP) is diluted 1:3000 and added to the ELISA plate at 100 μL / well, and incubated at 37°C for 60 minutes. Weigh 0.1% Phosphatase substrate, add substrate buffer and mix until the colorant is completely dissolved. Wash the ELISA plate after the incubation with avidin five times, pat dry, add the color development solution to the ELISA plate at 100 μL / well, and incubate at 37°C for 60 minutes. Add 50 μL / well of the stop solution to the 96-well plate after the color development to terminate the reaction. Turn on the preheating instrument 15 minutes in advance before use, and read the value at the OD405 wavelength. Set 2.1 times the negative group as the Cutoff value. Calculate the titer of each serum, and perform four-parameter fitting on the test results to calculate EC50.

[0191] Fig.10 The results in the figure show that different mutant pre-F proteins 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 the JW-153(A) mutation containing the T4 trimer domain.

[0192] 2.7 Neutralizing antibody level detection

[0193] The neutralizing antibody titer was detected by high-throughput microplate method. Specifically, Hep-2 cells were inoculated into 96-well plates and placed in a cell culture incubator (37°C, 5% CO2) for overnight culture. The inoculation density was adjusted so that the cell confluence on the second day was about 90%. The serum to be tested was then inactivated in a water bath at 56°C for 30 minutes. Subsequently, the first well of the test virus strain sample was diluted 100 times, and a 3-fold gradient dilution was performed, with a total of 8 dilutions (including the first well) and 2 replicates; then, according to the PFU value of the virus, the appropriate amount of virus was added. The diluted virus was added to the sample well and the virus control well, and the back-drip well was diluted downward in a 2-fold gradient for a total of 4 dilutions, and incubated in a 37°C, 5% CO2 incubator for 1 hour. Then, the above-mentioned virus and serum neutralization product and the positive and back-drip well viruses were added to the cells prepared in advance, and 100 μL of culture medium was added to each well to continue culturing for about 22 hours. The supernatant was discarded, the cells were fixed, and the fluorescently labeled detection antibody was added, and the plate was read using a CTL instrument. Neutralizing antibody titers were set as the serum dilution that resulted in a 50% reduction in infectious units (NT50).

[0194] Fig.11 The results showed that the sera of mice in each test group after booster immunization could effectively neutralize both type A and type B wild-type live viruses, indicating that the RSV pre-F protein mutant without exogenous trimer domain provided by the present invention has good immunogenicity and can induce cross-protection against both A / B strains.

[0195] By comparing the EC50 of binding antibodies and the NT50 titer of neutralizing antibodies, the proportion of neutralizing antibodies in the total antigen-specific binding antibodies was evaluated, and this method was used to evaluate the antibody quality. Fig.12 .

[0196] The antigen design strategy of this application effectively improves the pre-fusion conformation stability, trimer stability and expression level of the trimerized RSV pre-F protein through a series of mutation strategies. The exogenous trimer domain is removed, and the trimeric F protein without T4 has good stability and good immunogenicity.

[0197] The present application embodiment constructs 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, so that the RSV F protein is stabilized in a pre-fusion conformation and exists in a trimer form. During the stabilization process, two pairs of intrachain disulfide bonds (A170C / V179C, S180C / S186C) are respectively connected to the (α3 / β3 and β3 / β4) in the monomer to stabilize the RSV F protein in a pre-fusion conformation, providing a basis for constructing a trimerized RSV pre-F protein. During the stabilization process, two pairs of intrachain disulfide bonds and two pairs of interchain disulfide bonds (A241C / Q279C, D486C / A490C) connected the three monomers of the trimer in pairs in the middle and tail of the F protein through interchain disulfide bonds, providing a basis for the stable existence of the trimer and ensuring that the mutant F protein can still have good trimer stability in the absence of an exogenous trimer domain. During the stabilization process, the expression level of the trimerized RSV pre-F protein was effectively increased by introducing L305I present in the wild-type B subtype.

[0198]

[0199]

[0200]

[0201] The technical features of the above-mentioned implementation modes and examples can be combined in any appropriate manner. To make the description concise, not all possible combinations of the technical features in the above-mentioned implementation modes 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 above-described embodiments only express several implementation methods of the present application, which is convenient for understanding the technical solution of the present application in detail, but it cannot be understood as a limitation on the scope of protection of the patent application. It should be pointed out that for ordinary technicians in this field, without departing from the concept of the present application, several deformations and improvements can be made, which all belong to the protection scope of the present application. In addition, it should be understood that after reading the above-mentioned teaching content of the present application, the technicians in this field can make various changes or modifications to the present application, and the equivalent forms obtained also fall within the protection scope of the present application. It should also be understood that the technical solutions obtained by the technicians in this field through logical analysis, reasoning or limited experiments on the basis of the technical solutions provided in the present application are all within the protection scope of the claims attached to the present application. Therefore, the protection scope of the patent of the present application shall be based on the content of the attached claims, and the description and drawings can be used to explain the content of the claims.

Claims

1. A RSV pre-F mutant, characterized in that Relative to the wild-type F0 polypeptide, the RSV pre-F mutant meets the following conditions: (1) Not containing the furin cleavage site fragment, P27 polypeptide, transmembrane domain and intracellular domain; (2) having mutation combination 1, mutation combination 2, mutation combination 3 and mutation combination 4, wherein: Mutation combination 1 is S180C and S186C, Mutation combination 2 is A170C and V179C, Mutation combination 3 is A241C and Q279C, Mutation combination 4 is D486C and A490C, or D486C and D489C; and, (3) the C-terminus of the F1 polypeptide is not connected or is connected to an aggregation motif; Optionally, the RSV pre-F mutant further satisfies the following conditions: (4) The 305th position is I.

2. The RSV pre-F mutant according to claim 1, characterized in that The RSV pre-F mutant also has one or more of the following mutations: P102A, I167C, I379V and M447V.

3. The RSV pre-F mutant according to claim 1, characterized in that The RSV pre-F mutant does not have one or more of the following mutations: P102A, I167C, I379V and M447V.

4. The RSV pre-F mutant according to any one of claims 1 to 3, characterized in that In the RSV pre-F mutant, the C-terminus of the F2 polypeptide does not contain NN.

5. The RSV pre-F mutant according to any one of claims 1 to 3, characterized in that In the RSV pre-F mutant, the C-terminus of the F2 polypeptide and the N-terminus of the F1 polypeptide are directly connected by an amide bond or indirectly connected by a flexible short peptide; Optionally, the flexible short peptide is GS, G, S, GS, SG, SS, GG, PG, GGG, GGS, SSS, GSG, SGS, GPG, GSGS, GGGS, GPGS, GGGG, GSGG, GGSG, SGGG, GSSG, SGSG, GSSG, GGPGG or GGGGS.

6. The RSV pre-F mutant according to any one of claims 1 to 3, characterized in that The C-terminus of the RSV pre-F mutant is connected to a tag fragment.

7. The RSV pre-F mutant according to any one of claims 1 to 3, characterized in that The aggregation motif has the amino acid sequence shown in positions 475 to 505 of SEQ ID NO:7; Optionally, the C-terminus of the F1 polypeptide and the N-terminus of the aggregation motif are directly connected via an amide bond or indirectly connected via a flexible short peptide; Optionally, the flexible short peptide is GS, G, S, GS, SG, SS, GG, PG, GGG, GGS, SSS, GSG, SGS, GPG, GSGS, GGGS, GPGS, GGGG, GSGG, GGSG, SGGG, GSSG, SGSG, GSSG, GGPGG or GGGGS.

8. The RSV pre-F mutant according to any one of claims 1 to 3, characterized in that The RSV pre-F mutant has the amino acid sequence shown at positions 1 to 474 in SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:13 and SEQ ID NO:

14.

9. A nucleic acid molecule encoding the RSV pre-F mutant of any one of claims 1 to 8.

10. A vector comprising the nucleic acid molecule of claim 9.

11. An engineered cell expressing the RSV pre-F mutant according to any one of claims 1 to 8, or comprising the nucleic acid molecule according to claim 9 or the vector according to claim 10.

12. A method for producing the RSV pre-F mutant according to any one of claims 1 to 8, comprising the steps of: Cultivating the engineered cell according to claim 11, and isolating the RSV pre-F mutant from the obtained culture supernatant.

13. An immune composition comprising the RSV pre-F mutant according to any one of claims 1 to 8 or the nucleic acid molecule according to claim 15, and an immune adjuvant.

14. Use of the RSV pre-F mutant according to any one of claims 1 to 8 in preparing a respiratory syncytial virus antibody detection kit.

15. A respiratory syncytial virus antibody detection kit comprising the RSV pre-F mutant according to any one of claims 1 to 8.

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