RSV pre-f mutants, methods of production and uses thereof

Abstract

The application relates to a mutant of a pre-fusion F protein of RSV and preparation and application thereof. The application provides a mutant of a pre-fusion F protein of RSV, wherein the alpha 3 helix region of the F1 polypeptide has a cysteine substituent alpha 3-1, the beta 3 folding region has a cysteine substituent beta 3-1 and a cysteine substituent beta 3-2, and the beta 4 lamella has a cysteine substituent beta 4-1; the cysteine substituent beta 3-1 and the cysteine substituent beta 4-1 form a disulfide bond; the cysteine substituent alpha 3-1 and the cysteine substituent beta 3-2 form a disulfide bond. Compared with conventional technologies, the application finds another group of disulfide bond mutation positions, mainly forming disulfide bond mutations between alpha 3, beta 3 and beta 4, and the mutant formed by the disulfide bond mutations can be combined with specific monoclonal antibodies recognizing phi sites, has high expression, good stability and great potential for development of RSV vaccines.

Description

Technical Field

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

[0002] Human respiratory syncytial virus (RSV) is a common respiratory virus, a single-stranded RNA virus in the Viridae family. It is one of the main pathogens causing respiratory infections in infants and young children worldwide. RSV is most common in autumn and winter, and is most frequently transmitted among young children. RSV infection 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] Over the past few decades, many RSV vaccine candidates have been tested in clinical trials, including attenuated virus vaccines, subunit vaccines, and genetically engineered vaccines. However, due to the complex viral properties and immunological responses of RSV, the development of these vaccines faces several challenges, including the inability to achieve a long-lasting protective immune response and potential lack of efficacy in high-risk populations such as children and the elderly.

[0004] Despite this, scientists and pharmaceutical companies continue to strive for a more effective RSV vaccine. Some research teams are focusing on understanding the immunological properties of RSV and how to stimulate a durable and comprehensive immune response. At the same time, new technology platforms and strategies are being explored in vaccine development, such as micronized vaccines, nanoparticle vaccines, and the design of structure-specific antigens.

[0005] The F protein is a type I glycoprotein. It is synthesized intracellularly as an inactive precursor, F0. During maturation, F0 is cleaved by furin to form F2 (aa: 1-109), P27 (aa: 110-136), and F1 (aa: 137-574). F2 and F1 form a heterodimer, the monomer of the F protein, through disulfide bonds. The three monomers assemble to form a trimer. The F protein is expressed on the surface of the viral envelope in a metastable pre-fusion protein (pre-F). Upon contact with the host cell membrane, the secondary structural components α2, α3, β3, β4, and α4 at the N-terminus of the F1 protein undergo a series of rearrangements together with the subsequent α5 to form a larger α-helical structure. This series of processes causes the F protein to transform from a high-energy metastable pre-F structure to a stable post-fusion protein (post-F) structure. Compared to post-F, studies have shown that the vast majority of highly effective neutralizing antibodies target epitopes only in pre-F, the antigenic epitopes present at the top of the pre-F monomer. The antigenic epitope V at the top of the pre-F trimer has been shown to be extremely potent. Therefore, pre-F has become the preferred target antigen for RSV vaccines.

[0006] Current RSV vaccine research and development focuses on identifying more conformationally stable RSV pre-F mutants. Based on the principles of protein structure and stability, the DS-Cav1 mutant, designed by cavity-filling mutations (S190F and V207L) and disulfide bond replacements (S155C and S290C), stabilizes the unstable F1 protein N-terminus, resulting in a stable pre-F protein with antigenic epitopes (McLellan et al., Science, 2013). The iterative mutants designed based on structure by the National Institutes of Health (NIH) also exhibited a stable prefusion conformation (PCT / US2014 / 026714 family). The strategy for designing mutation positions for the RSV F protein mutants publicly invented by Pfizer (CN 108738312A) was also roughly similar. In addition to S155C and S290C, disulfide bond replacements included S55C and L188C, T103C and I148C, and L142C and N371C; cavity filling included substitution of I at position 190 and H at position 54; electrostatic mutations such as D486S and E487Q were added, and different combination mutations were performed at the same time. Janssen designed SC-DM (Anders Krarup et al., Nature Communications, 2015) by deleting the furin cleavage site and P27 and replacing them with GS Linker and introducing mutations N67I and S215P to maintain protein stability, and finally using the T4 phage fiber protein trimerization domain to secrete the protein and form a trimer; while GSK's Pre-F-GCN4t (Normand Blais et al., Journal of Virology, 2017) replaced the transmembrane domain and C-terminal domain of the F0 protein with the engineered Saccharomyces cerevisiae GCN4 trimerization domain; the furin cleavage site and P27 sequence were removed, and a lysine residue was retained at this position between F2 and the fusion peptide (FP); mutations L112Q, Q471G and L482K were added to increase the homogeneity and stability of the protein, and it was shown to bind Epitope-specific monoclonal antibody D25.

[0007] As can be seen from the above description, based on the RSV F protein structure and using different design strategies, multiple mutation sites have been derived. These mutations can prevent the RSV F protein from changing to the post-F protein conformation, stabilizing the RSV F protein in the prefusion conformation (pre-F). However, in order to find a more stable RSV pre-F protein conformation, current research is still insufficient, and the expression and stability of the pre-F protein still have room for improvement. Therefore, new explorations and attempts are still underway.

[0008] In 2013, the epitope was discovered by antibody D25 and named site [McLellan, Jason S., et al."Structure of RSV fusion glycoprotein trimer bound to a prefusion-specific neutralizing antibody." Science 340.6136(2013):1113-1117.], in order to solve the problem of lack of D25 antibody, site To address the instability problem, an engineered stable RSV F protein prefusion conformation (pre-F) antigen was further modified [McLellan, Jason S., et al. "Structure-based design of a fusion glycoprotein vaccine for respiratory syncytial virus." Science 342.6158(2013):592-598.], mainly involving disulfide bond mutations: DS (S155C, S290C), cavity filling: Cav1 (S190F, V207L) and the mutation combination DS-Cav1, as well as other mutants derived therefrom (PCT / US2014 / 026714 family).

[0009] In 2015, Janssen Pharmaceutical published research evidence on its construction of stabilized pre-F protein mutants. A short-chain linker was introduced between F1 and F2. After the variant bound to the fibrin domain, amino acid mutations were performed to obtain SC-DM and SC-TM [Krarup, Anders, et al. "A highly stable prefusion RSV F vaccine derived from structural analysis of the fusion mechanism." Nature communications 6.1(2015):8143.]. The amino acid mutations involved included: SC-DM (N67I, S215P), SC-TM (N67I, S215P, E487Q) (US_11338031_B2 family).

[0010] GSK conducted secondary development based on DS-Cav1 (US8563002), focusing on protecting and restricting the furin cleavage site and the C-terminal trimerization of the protein. Given its good immunogenicity and ability to induce neutralizing antibodies, GSK announced in 2022 that its RSV vaccine had achieved positive results in a Phase III clinical trial in adults over 60 years old. On May 3, 2023, it received marketing authorization (trade name: Arexvy), which is also the first vaccine against RSV virus.

[0011] Pfizer's structurally designed stabilized pre-F protein mutants (PCT / IB2016 / 057502 family) primarily involve mutation strategies including disulfide bond mutations and cavity filling. The disulfide bond mutation sites involved include: S55C / L188C, S155C / S290C, T103C / I148C, L142C / N371C, as well as 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 had achieved positive results and received marketing authorization in May 2023 (trade name: Abrysvo), becoming the second vaccine against RSV virus after Arexvy.

[0012] Currently, the main problem with stabilized RSV pre-F protein is its low expression level and instability in long-term storage. To address this issue, it is necessary to structurally engineer the RSV F protein and find a more stable mutation strategy. Summary of the Invention

[0013] Based on this, one or more embodiments of the present application provide an RSV pre-F mutant and a production method and use thereof.

[0014] One or more embodiments of the present application provide a mutant of RSV pre-F, wherein the F1 polypeptide of the mutant satisfies the following conditions (1) or (2):

[0015] (1) the β3 sheet region has a cysteine ​​substituent β3-1, and the β4 sheet region has a cysteine ​​substituent β4-1, wherein the cysteine ​​substituent β3-1 and the cysteine ​​substituent β4-1 form a disulfide bond;

[0016] (2) the α3 helical region has a cysteine ​​substituent α3-1, the β3 sheet region has a cysteine ​​substituent β3-1 and a cysteine ​​substituent β3-2, and the β4 sheet has a cysteine ​​substituent β4-1, wherein the cysteine ​​substituent β3-1 and the cysteine ​​substituent β4-1 form a disulfide bond, and the cysteine ​​substituent α3-1 and the cysteine ​​substituent β3-2 form a disulfide bond;

[0017] The mutant has one of the following cysteine ​​mutation combinations:

[0018] Combination 1 is S180C and S186C; and,

[0019] Combination 2 is K176C and S190C.

[0020] In some embodiments of the present application, the RSV pre-F mutant is derived from human or bovine species.

[0021] In some embodiments of the present application, the amino acid sequence of the F1 polypeptide of the RSV pre-F is as shown in any one of SEQ ID NO.4, SEQ ID NO.71 and SEQ ID NO.72, or has at least 80% homology to the amino acid sequence shown in any one of SEQ ID NO.4, SEQ ID NO.71 and SEQ ID NO.72;

[0022] Optionally, the F1 polypeptide satisfies one or more of the following conditions:

[0023] (1) does not contain a transmembrane domain, and

[0024] (2) does not contain an intracellular domain;

[0025] Further optionally, the F1 polypeptide has one or more of the following mutations: I379V and M447V.

[0026] In some embodiments of the present application, the mutant has one of the following cysteine ​​mutation combinations:

[0027] Combination 3 is S180C, S186C, A170C, A177C;

[0028] Combination 4 is S180C, S186C, E163C, L181C;

[0029] Combination 5 is S180C, S186C, A170C, V179C;

[0030] Combination 6 is S180C, S186C, L171C, A177C;

[0031] Combination 7 is K176C, S190C, A170C, A177C;

[0032] Combination 8 is K176C, S190C, E163C, L181C;

[0033] Combination 9 is K176C, S190C, A170C, V179C; and,

[0034] Combination 10 is K176C, S190C, L171C, and A177C.

[0035] In some embodiments of the present application, the mutant further has one or more of the following mutation sites: S55C, S155C, V207L ​​and S290C.

[0036] In some embodiments of the present application, the mutant does not contain a furin cleavage site fragment.

[0037] In some embodiments of the present application, the mutant does not contain the pep27 polypeptide, and 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;

[0038] 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.

[0039] In some embodiments of the present application, the mutant F2 polypeptide satisfies one or more of the following conditions:

[0040] 1) The C-terminus does not contain NN, and

[0041] 2) with the following mutations: P102A;

[0042] In some embodiments of the present application, a tag fragment is connected to the C-terminus of the mutant; optionally, the tag fragment includes polyhistidine; further optionally, the polyhistidine includes an 8His fragment.

[0043] In some embodiments of the present application, the mutant further comprises one or more of a structural polypeptide and a functional polypeptide; the structural polypeptide enables a plurality of monomers of the mutant to form a multimer, and the functional polypeptide enhances the biological activity of the mutant;

[0044] Optionally, the structural polypeptide comprises one or more of an aggregation motif, a GCN4 leucine zipper, and a nanoparticle conjugation fragment;

[0045] Optionally, the functional polypeptide includes an antigen-specific binding fragment;

[0046] Optionally, one or more of the structural polypeptide and the functional polypeptide.

[0047] In some embodiments of the present application, the sequence of the mutant is shown in any one of SEQ ID NO.20, SEQ ID NO.25 to SEQ ID NO.70.

[0048] One or more embodiments of the present application provide a nucleic acid molecule encoding the mutant.

[0049] One or more embodiments of the present application provide a vector comprising the nucleic acid molecule.

[0050] One or more embodiments of the present application provide an engineered cell, which expresses the mutant, or comprises the nucleic acid molecule or the vector.

[0051] One or more embodiments of the present application provide a method for producing the mutant, which comprises the following steps:

[0052] The engineered cells are cultured, and mutants are isolated from the obtained culture supernatant.

[0053] One or more embodiments of the present application provide an immune composition comprising the mutant, or the nucleic acid molecule, and an immune adjuvant;

[0054] Optionally, the immune adjuvant includes one or more of aluminum salt adjuvant, surfactant, polynucleotide, lipopolysaccharide, liposome, and oil emulsion adjuvant.

[0055] One or more embodiments of the present application provide a use of the mutant in preparing a respiratory syncytial virus antibody detection kit.

[0056] One or more embodiments of the present application provide a respiratory syncytial virus antibody detection kit, which includes the mutant.

[0057] The details of one or more embodiments of the present application are set forth in the description below, and other features, objects, and advantages of the application will become apparent from the description and from the claims thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] In order to more clearly illustrate the technical solutions in the embodiments of the present application and to more fully understand the present application and its beneficial effects, the following is a brief introduction to the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present application. Those skilled in the art can also derive other drawings based on these drawings without inventive effort.

[0059] Figure 1 An expression plasmid for constructing RSV pre-F protein;

[0060] Figure 2 Schematic diagram of the RSV F0 precursor polypeptide structure (A, B, bovine);

[0061] Figure 3 Schematic diagram of the amino acid sequence of the stabilized mutant monomeric RSV F protein;

[0062] Figure 4 Schematic diagram of the multimeric structure of the stabilized mutant RSV F protein;

[0063] Figure 5 Schematic diagram of the positions of α3, β3 and β4 (partial and overall);

[0064] Figure 6 is the distance between Cα in β3 / β4 position amino acid residue pairs;

[0065] Figure 7 is the distance between Cα in α3 / β3 position The amino acid residue pairs of

[0066] Figure 8 is the expression level of the mutant pre-F formed at positions β3 and β4;

[0067] Figure 9 The thermal stability of the mutant pre-F formed at positions β3 and β4;

[0068] Figure 10 is the expression level of the mutant pre-F formed at the α3 and β3 positions;

[0069] Figure 11 The thermal stability of the mutant pre-F formed at the α3 and β3 positions;

[0070] Figure 12 is the expression level of the double disulfide bond mutant pre-F formed at the α3 / β3 and β3 / β4 positions;

[0071] Figure 13 The thermal stability of the double disulfide bond mutant pre-F formed at the α3 / β3 and β3 / β4 positions;

[0072] Figure 14 It is the identification of RSV pre-F mutant monomer by SDS-PAGE;

[0073] Figure 15 It is the identification of RSV pre-F mutant single trimer by SDS-PAGE;

[0074] Figure 16 HPLC identification of RSV pre-F mutant monomer;

[0075] Figure 17 HPLC identification of RSV pre-F mutant trimer;

[0076] Figure 18 To identify the antigenicity of RSV pre-F mutant trimers;

[0077] Figure 19 is the RSV pre-F mutant trimer binding antibody level;

[0078] Figure 20 is the level of neutralizing antibodies against RSV pre-F mutant trimer. DETAILED DESCRIPTION

[0079] Below in conjunction with accompanying drawing, embodiment and example, the application is described in further detail.It should be understood that these embodiment and example are only used to illustrate the application and are not used to limit the scope of the application, and the purpose of providing these embodiment and example is to make the understanding of the disclosure of the application more thorough and comprehensive.It should also be understood that the application can be implemented in many different forms and is not limited to the embodiment and example described herein. Those skilled in the art can make various changes or modifications without violating the connotation of the application, and the equivalent form obtained also falls within the protection scope of the application.In addition, in the description below, a large amount of specific details are given in order to provide a more complete understanding of the application.It should be understood that the application can be implemented without one or more of these details.

[0080] 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 pertains. The terms used herein in the specification of this application are for the purpose of describing embodiments and examples only and are not intended to limit this application.

[0081] the term

[0082] Unless otherwise specified or incompatible herewith, the terms and phrases used herein shall have the following meanings:

[0083] The terms "and / or", "or / and", and "and / or" used herein include any one of two or more related listed items, and also include any and all combinations of the related listed items, wherein the arbitrary and all combinations include any combination of two related listed items, any more related listed items, or 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 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 of all being 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 four-item combination of A, B, C, and D (that is, the technical solution of all being connected by "logical AND").

[0084] In this application, "plurality", "multiple", "multiple times", "multiples", 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.

[0085] As used herein, "combination thereof", "any combination thereof", "any combination thereof" and the like include all suitable combinations of any two or more of the listed items.

[0086] 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.

[0087] Herein, "preferred", "better", "more preferred" and "suitable" are merely used to describe implementation methods or examples with better effects. It should be understood that they do not constitute limitations on the scope of protection of this application.

[0088] In this application, "further", "further", "particularly" and the like are used for descriptive purposes to indicate differences in content, but should not be understood as limiting the scope of protection of this application.

[0089] In this application, the terms "optionally," "optional," and "optional" mean optional or dispensable, i.e., they refer to either option being selected from two parallel options: "with" or "without." If a technical solution contains multiple "optional" clauses, each "optional" clause is independent unless otherwise specified and there are no contradictions or constraints.

[0090] In this application, the terms "first," "second," "third," "fourth," etc. in "the first aspect," "the second aspect," "the third aspect," "the fourth aspect," etc. are used for descriptive purposes only and should not be understood as indicating or implying relative importance or quantity, nor should they be understood as implicitly indicating the importance or quantity of the indicated technical features. Furthermore, "first," "second," "third," "fourth," etc. serve only as non-exhaustive enumeration and description, and should be understood not to constitute a closed-ended limitation on quantity.

[0091] In this 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.

[0092] In this application, when referring to a numerical interval (i.e., a numerical range), unless otherwise specified, the optional numerical distribution is considered continuous within the above numerical interval and includes the two numerical endpoints of the numerical range (i.e., the minimum and maximum values), as well as each numerical value between the two numerical endpoints. Unless otherwise specified, when a numerical interval refers only to integers within the numerical interval, it includes the two endpoint integers of the numerical range, as well as 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 merged. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges included therein.

[0093] Unless otherwise specified, the temperature parameters in this application allow for both constant temperature treatment and temperature fluctuations within a certain temperature range. It should be understood that the constant temperature treatment allows for temperature fluctuations within the accuracy range of instrument control. Fluctuations within ranges such as ±5°C, ±4°C, ±3°C, ±2°C, and ±1°C are permitted.

[0094] In this application, % (w / w) and wt% both refer to weight percentage, % (v / v) refers to volume percentage, and % (w / v) refers to mass volume percentage.

[0095] 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 in their entirety and for all purposes. When 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 cited documents are involved in this application, the examples and preferred embodiments of the cited relevant technical features may also be incorporated into this application as references, but are limited to the ability to implement 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 adaptively amended according to the description in this application.

[0096] RSV: Respiratory Syncytial Virus is a single-stranded RNA virus in the Viridae family. It is a common and contagious virus that primarily affects the respiratory tract. It belongs to the Pneumoviridae family and the genus Pneumovirus. The RSV genome is approximately 15 kilobases long and contains 10 genes encoding 11 proteins, including F, G, and SH proteins displayed on its surface. RSV usually causes mild, cold-like symptoms in most people. Infants and the elderly, or people with compromised heart, lung, or immune systems, are more likely to develop severe RSV symptoms, which may lead to pneumonia, bronchitis, and middle ear infections.

[0097] F protein: F protein (fusion protein) is a surface protein of respiratory syncytial virus (RSV) that helps the virus infect human cells. It has two states: pre-fusion and post-fusion. During the virus's infection of the host, the F protein irreversibly transforms from the metastable pre-fusion conformation to the more stable post-fusion conformation. In its natural state, the F protein is translated into the F0 precursor, which is 574 amino acids long. During the maturation process of F0, it is cleaved by two furin cleavage sites, forming the F1 polypeptide, the F2 polypeptide, and the 27-amino acid p27. The F1 polypeptide and the F2 polypeptide further form a trimer, and the mature F protein finally exists as a trimer.

[0098] Prefusion F protein: The conformation of the RSV F protein before fusion is triggered. In this conformation, the F protein is in a metastable state and can be irreversibly transformed into a more stable postfusion conformation by environmental or other stimuli. This conformation possesses epitopes distinct from those in the postfusion conformation, such as the Φ epitope and the V epitope. Specific monoclonal antibodies targeting these epitopes (such as D25, AM22, 5C4, and ADI-15568) can be used to determine whether the protein is in this prefusion conformation.

[0099] Post-fusion F protein: The RSV F protein undergoes an irreversible conformational change after being triggered by the external environment or other factors. In this conformation, the F protein does not possess the Φ epitope V and is therefore not recognized by specific monoclonal antibodies corresponding to the epitope (such as D25, AM22, 5C4, ADI-15568, etc.).

[0100] F1 polypeptide: A polypeptide fragment formed by post-translational modification and enzymatic cleavage of the RSV F0 precursor, consisting of amino acids located at positions 137 to 574 of F0. It comprises an extracellular domain (approximately located at positions 137 to 524 of F0), a transmembrane domain (approximately located at positions 524 to 550 of F0), and an intracellular domain (approximately located at positions 551 to 574 of F0). As used herein, F1 polypeptide refers to the F1 polypeptide in its native state or to a mutation or modification resulting in an amino acid sequence corresponding to a native F1 polypeptide or a portion of a native F1 polypeptide.

[0101] F2 polypeptide: A polypeptide fragment formed by post-translational modification and enzymatic cleavage of the RSV F0 precursor, comprising amino acids 26 to 109 of F0. The F1 polypeptide used herein refers to the native F1 polypeptide or a mutation or modification resulting in an amino acid sequence corresponding to a native F1 polypeptide or a portion of a native F1 polypeptide.

[0102] DS-Cav1: refers to an amino acid sequence having the same mutations as the RSV F protein described by Mcllelan et al. in Structure-Based Design of a Fusion Glycoprotein Vaccine for Respiratory Syncytial Virus, Science. 2013 November 1; 342(6158): 592–598.

[0103] Epitope: An epitope is a specific region on a protein that interacts specifically with antibodies or lymphocyte receptors. Epitopes are often associated with antigen recognition, such as the interaction between antibodies and antigens. These epitopes can be linear (along a portion of the protein sequence) or three-dimensional (within the protein's folded structure). In the F protein, epitopes include Φ, I, II, III, IV, and V.

[0104] Disulfide bond replacement: Disulfide bond replacement refers to replacing one or more pairs of amino acids with cysteines at appropriate positions in the RSV F protein, thereby forming disulfide bonds between the replaced cysteines, thereby enhancing the stability of the F protein in the pre-fusion state.

[0105] D25: D25 refers to the antibody described in WO 2008 / 147196A2, which can specifically bind to the Φ epitope of RSV F protein.

[0106] AM22: AM22 refers to the antibody described in WO 2011 / 043643A1. It can specifically bind to the Φ epitope of RSV F protein.

[0107] AM14: AM14 refers to the antibody described in WO 2008 / 147196A2, which can specifically bind to the RSV F protein in a trimeric state.

[0108] ADI-15568: ADI-15568 refers to the antibody described in US2022 / 0144922A1. It can specifically bind to the V epitope of RSV F protein.

[0109] In a first aspect of the embodiments of the present application, a mutant of RSV pre-F is provided, wherein the F1 polypeptide of the mutant satisfies the following conditions (1) or (2):

[0110] (1) the β3 sheet region has a cysteine ​​substituent β3-1, and the β4 sheet region has a cysteine ​​substituent β4-1, wherein the cysteine ​​substituent β3-1 and the cysteine ​​substituent β4-1 form a disulfide bond;

[0111] (2) the α3 helical region has a cysteine ​​substituent α3-1, the β3 sheet region has a cysteine ​​substituent β3-1 and a cysteine ​​substituent β3-2, and the β4 sheet has a cysteine ​​substituent β4-1, wherein the cysteine ​​substituent β3-1 and the cysteine ​​substituent β4-1 form a disulfide bond, and the cysteine ​​substituent α3-1 and the cysteine ​​substituent β3-2 form a disulfide bond;

[0112] The mutant has one of the following cysteine ​​mutation combinations:

[0113] Combination 1 is S180C and S186C; and,

[0114] Combination 2 is K176C and S190C.

[0115] The present application has discovered a method for producing a stable pre-fusion conformation of the RSV F protein. The method involves introducing cysteine ​​substitutions between β3 / β4 to form a disulfide bond, or simultaneously introducing cysteine ​​substitutions between β3 / β4 and between α3 / β3 to form a double disulfide bond, which can maintain the RSV F protein in the pre-fusion conformation. These mutation combinations discovered in the present application, and the mutants produced therefrom, have not been involved in the prior art. This is a new idea for producing stabilized RSV pre-F mutants. The mutants that introduce a disulfide bond between β3 / β4 and a disulfide bond at the α3 / β3 position can recognize It binds to site-specific monoclonal antibodies, has good stability and immunogenicity, can induce neutralizing antibodies that can neutralize the virus, and has great potential for developing vaccines against RSV.

[0116] Mutation, Substitution, Amino Acid Substitution: The replacement of one amino acid in an antigen by another or the deletion of an amino acid. For example, an amino acid in an antigen is replaced by an amino acid from a homologous protein.

[0117] Homologous proteins: proteins with similar structure and function, for example, proteins from two or more species or virus strains that have similar structure and function in two or more species or virus strains. For example, the RSV F protein from RSVA is a protein homologous to the RSV F protein from bovine RSV. Homologous proteins share similar protein folding features and can be considered as structural homologs. Homologous proteins generally share a high degree of sequence conservation, for example, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% or at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence conservation, and a high degree of sequence identity, for example, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity.

[0118] There are several subtypes of RSV, including human subtype A, human subtype B, and bovine subtype. Within the RSV subtypes, there are separate strains of each subtype.

[0119] In some embodiments, the amino acid sequence of the RSV pre-F F1 polypeptide is as shown in any one of SEQ ID NO.4, SEQ ID NO.72 and SEQ ID NO.73, or is at least 80% (at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) homologous to the amino acid sequence shown in any one of SEQ ID NO.4, SEQ ID NO.71 and SEQ ID NO.72;

[0120] Optionally, the F1 polypeptide satisfies one or more of the following conditions:

[0121] (1) does not contain a transmembrane domain, and

[0122] (2) does not contain an intracellular domain;

[0123] Further optionally, the F1 polypeptide has one or more of the following mutations: I379V and M447V.

[0124] In some embodiments, the mutant has one of the following combinations of cysteine ​​mutations:

[0125] Combination 3 is S180C, S186C, A170C, A177C;

[0126] Combination 4 is S180C, S186C, E163C, L181C;

[0127] Combination 5 is S180C, S186C, A170C, V179C;

[0128] Combination 6 is S180C, S186C, L171C, A177C;

[0129] Combination 7 is K176C, S190C, A170C, A177C;

[0130] Combination 8 is K176C, S190C, E163C, L181C;

[0131] Combination 9 is K176C, S190C, A170C, V179C; and,

[0132] Combination 10 is K176C, S190C, L171C, and A177C.

[0133] In some embodiments, the mutant further has one or more of the following mutation sites: S55C, S155C, V207L, and S290C.

[0134] In some embodiments, the mutant does not contain a furin cleavage site fragment.

[0135] In some embodiments, the mutant does not contain a pep27 polypeptide, and 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;

[0136] 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.

[0137] In some embodiments, the mutant F2 polypeptide satisfies one or more of the following conditions:

[0138] 1) The C-terminus does not contain NN, and

[0139] 2) with the following mutations: P102A;

[0140] In some embodiments, a tag fragment is connected to the C-terminus of the mutant; optionally, the tag fragment includes polyhistidine; further optionally, the polyhistidine includes an 8His fragment.

[0141] In some embodiments, the mutant further comprises one or more of a structural polypeptide and a functional polypeptide; the structural polypeptide enables a plurality of monomers of the mutant to form a multimer, and the functional polypeptide enhances the biological activity of the mutant;

[0142] Optionally, the structural polypeptide comprises one or more of an aggregation motif, a GCN4 leucine zipper, and a nanoparticle conjugation fragment;

[0143] Optionally, the functional polypeptide includes an antigen-specific binding fragment;

[0144] Optionally, one or more of the structural polypeptide and the functional polypeptide.

[0145] In some embodiments, the sequence of the mutant is as shown in any one of SEQ ID NO.20, SEQ ID NO.25 to SEQ ID NO.70, 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 these sequences.

[0146] The mutants provided in the examples of the present application have a stable conformation and can trigger an immune response in a subject as an antigen / immunogen to produce antibodies.

[0147] 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 laboratory animals (e.g., mice, rabbits, rats, gerbils, guinea pigs, and non-human primates).

[0148] Antibody: A polypeptide substantially encoded in nature by an immunoglobulin gene or multiple immunoglobulin genes or fragments thereof, which specifically binds and recognizes an analyte (e.g., an antigen or immunogen), such as RSV F protein or an antigenic fragment thereof. Immunoglobulin genes include kappa, lambda, alpha, gamma, beta, 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 whole antibodies and by de novo synthesis using recombinant DNA methodologies.

[0149] 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 well-known in the art and parts thereof, 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, while 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 conjugated 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.

[0150] 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 pepsin followed by reduction to obtain an intact light chain and a portion of one 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 expressed 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.

[0151] 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), which determine the functional activity of the antibody molecule: IgM, IgD, IgG, IgA, and IgE. The disclosed antibodies can be class-switched.

[0152] 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 composed 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, ie, the combined framework regions of the component light and heavy chains, serves to position and align the CDRs in three-dimensional space.

[0153] The CDRs are 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 ed., 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).

[0154] The CDRs of each chain are commonly referred to as CDR1, CDR2, and CDR3 (from N-terminus to C-terminus) and are also typically 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.

[0155] 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 the disclosed mutants of the 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 specific examples, 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 a region of an antigen that reacts with B and / or T cells.

[0156] Immunogen: A protein or portion thereof capable of inducing an immune response in a mammal, such as a mammal infected with 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, such as the PreF mutants provided in the Examples of this application.

[0157] 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.

[0158] Those 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 coded 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 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

[0159] Not all residue positions within a protein will tolerate otherwise "conservative" substitutions. For example, if an amino acid residue is essential for protein function, even otherwise conservative substitutions may destroy that activity, e.g., specific binding of an antibody to a target epitope may be destroyed by a conservative mutation in the target epitope.

[0160] Epitope: Antigenic determinant. These are specific chemical groups or peptide sequences on antigenic molecules that induce a specific immune response. 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 RSV F proteins, for example, D25 or AM22 epitopes present in the prefusion conformation of RSV F proteins. Epitopes can be formed by continuous or discontinuous amino acids juxtaposed by the tertiary folding of proteins. Epitopes formed by continuous amino acids typically remain exposed to denaturing solvents, while epitopes formed by tertiary folding are typically lost when treated with denaturing solvents. Epitopes typically include at least 3 and more typically at least 5, about 9, or about 8-10 amino acids in a unique spatial conformation. Methods for determining the spatial conformation of an epitope 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.

[0161] 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.

[0162] The amino acids in a peptide, polypeptide, or protein are typically chemically bound together via an amide bond (CONH). In addition, amino acids can be bound 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, 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, Trenton, et al., Chemistry and Biochemistry of Amino Acids, Peptides, and Proteins, ed. B. Weinstein ... 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).

[0163] Peptide modification: The peptide is stabilized in a pre-fusion conformation. The mutants of the present application examples can be modified, for example, to include amino acid substitutions compared to the native 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 other desired properties. For example, the carboxylic acid group of the protein, whether at 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 at 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 group or further converted to an amide.

[0164] The hydroxyl groups of the peptide side chains can be converted to C1-C16 alkoxy groups or C1-C16 esters using recognized techniques. The phenyl and phenol rings of the peptide side chains can be substituted with one or more halogen atoms such as F, Cl, Br or I or with 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 with any of a variety of recognized protecting groups such as acetamide groups.

[0165] In a second aspect of the embodiments of the present application, a nucleic acid molecule is provided, which encodes the mutant.

[0166] Nucleic acid molecule, nucleic acid: the polymer that is made up of the nucleotide units (ribonucleotide, deoxyribonucleotide, relevant naturally occurring structural variant and the analogue of its synthetic non-natural being) connected by phosphodiester bond, relevant naturally occurring structural variant, and the analogue of its synthetic non-natural being.Therefore, described term includes wherein nucleotide and connection therebetween include the nucleotide polymers of non-natural synthetic analogue such as and not limited to thiophosphate, phosphoramidate, methylphosphonate, chiral methylphosphonate, 2-O-methyl ribonucleotide, peptide-nucleic acid (PNA) etc. These polynucleotide can for example be synthesized using automated DNA synthesizer.Described term " oligonucleotide " generally refers to the short polynucleotide that is usually not more than about 50 nucleotide.Should be understood that when nucleotide sequence is by DNA sequence dna (that is, A, T, G, C) expression, this also includes wherein " U " replaces " T " RNA sequence (that is, A, U, G, C).

[0167] "Nucleotide" includes, but is not limited to, a monomer comprising a base linked to a sugar (e.g., a pyrimidine, a 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.

[0168] " coding " refers to the intrinsic properties of the specific sequence of nucleotides such as genes, cDNA or mRNA in polynucleotides, and the nucleotides serve as templates for synthesizing other polymers and macromolecules in biological processes, and they have the nucleotides (for example, rRNA, tRNA and mRNA) of a defined sequence or the amino acids of a defined 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. The coding strands of its nucleotide sequence and the transcription templates provided in the sequence table form and the non-coding strands used as gene or cDNA can be referred to as encoding the protein or other products of the gene or cDNA. Unless otherwise indicated, " nucleotide sequence encoding an amino acid sequence " includes all nucleotide sequences that are in degenerate form and encode the same amino acid sequence. The nucleotide sequence of coded proteins and RNA may include introns. In some instances, nucleic acid encodes the mutant of the application.

[0169] Taking into account degenerate variants and conservative variants, the present application does not impose any particular limitation on nucleic acid molecules.

[0170] Degenerate variants and conservative variants: Polynucleotides encoding polypeptides that include sequences that are 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 that is degenerate due to the genetic code. There are 20 naturally occurring 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 antigen-binding antibody encoded by the nucleotide sequence is 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 every 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 variations," which are a type of conservative variation. Each nucleic acid sequence encoding a polypeptide herein also describes every possible silent variation. Those skilled in the art will recognize that each codon in a nucleic acid (except AUG, which is generally the only codon for methionine) can be modified using 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.

[0171] In some examples, it is codon-optimized for expression in mammalian cells and is operably linked to a promoter.

[0172] Expression control sequence: a nucleic acid sequence that regulates the expression of an operably linked heterologous nucleic acid sequence. When an expression control sequence controls and regulates the transcription and, when appropriate, translation of a nucleic acid sequence, an expression control sequence is operably linked to a nucleic acid sequence. Therefore, an 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 for maintaining the gene to allow suitable translation of the mRNA, and a stop codon. The term "control sequence" is intended to minimally include components whose presence can affect expression, and may also include other components whose presence is advantageous, such as a leader sequence and a fusion partner sequence. An expression control sequence may include a promoter.

[0173] A promoter is a minimal sequence sufficient to direct transcription. Also included are promoter elements sufficient to allow promoter-independent gene expression to be cell type-specific, tissue-specific, 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), etc., may be used. In one embodiment, when cloning in a mammalian cell system, promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or promoters derived from the genome of mammalian viruses (e.g., retroviral long terminal repeats; adenovirus late promoter; vaccinia virus 7.5K promoter) may be used. Promoters produced by recombinant DNA or synthetic techniques may also be used to provide transcription of nucleic acid sequences.

[0174] 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 typically contains an origin of replication, a promoter, and specific nucleic acid sequences that allow phenotypic selection of transformed cells.

[0175] 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)).

[0176] In a third aspect of the embodiments of the present application, a vector is provided, which includes the nucleic acid molecule.

[0177] In a fourth aspect of the embodiments of the present application, an engineered cell is provided, which expresses the mutant, or comprises the nucleic acid molecule or the vector.

[0178] 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.

[0179] Engineered cell, host cell: A cell in which a vector can reproduce 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, as mutations may occur during replication. However, these progeny are included when the term "host cell" is used.

[0180] A fifth aspect of the embodiments of the present application provides a method for producing the mutant, comprising the following steps:

[0181] The engineered cells are cultured, and mutants are isolated from the obtained culture supernatant.

[0182] In a sixth aspect of the embodiments of the present application, an immune composition is provided, which includes the mutant or the nucleic acid molecule, and an immune adjuvant.

[0183] Immune 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 (such as those including CpG motifs) can also be used as adjuvants. Adjuvants include biological molecules ("biological adjuvants"), such as costimulatory 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 antigens. Optionally, the immunoadjuvants in the examples of this application include one or more of aluminum salt adjuvants, surfactants, polynucleotides, lipopolysaccharides, liposomes, and oil emulsion adjuvants. Examples include Alum, CpG, Alum + CpG, MF59, AS04, and AS01E. MF59 is a water-mixable adjuvant composed primarily of three components: an oil phase, an emulsifier, and an adjuvant. 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 evenly; and the adjuvants primarily include humectants such as glycerol, threitol, and ATP, as well as buffers. AS04 adjuvant is a mixture of AS03 adjuvant and MPL adjuvant. AS03 adjuvant is a mixture of liposomes, TWEEN 80, and SORBITAN surfactants. AS01E is a nano-sized liposome solution prepared from DOPC, Chol, MPL, and QS-21. The main component of MPL adjuvant is lipopolysaccharide.

[0184] 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 (such as 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 suppressing 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 the RSV F protein, or induces a measurable B cell response (e.g., antibody production) to the RSV F protein. It further refers to an isolated nucleic acid encoding an antigen, eg, a nucleic acid that can be used to express the antigen (and thereby to induce an immune response against such a polypeptide).

[0185] 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 readily tested for its ability to induce a CTL or B cell response by recognized assays. The immunogenic composition may include adjuvants well known to those skilled in the art.

[0186] Immunological reaction conditions: include 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 that of 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 primary solvent, and exists at a temperature above 0°C and below 50°C. The molar osmotic pressure concentration is within the range that supports cell viability and proliferation.

[0187] In a seventh aspect of the embodiments of the present application, a use of the mutant in preparing a respiratory syncytial virus antibody detection kit is provided.

[0188] In the embodiments of the present application, the respiratory syncytial virus antibody detection kit can be used to detect corresponding antibodies. The definition of antibodies refers to the first aspect and can be neutralizing antibodies or binding antibodies.

[0189] In an eighth aspect of the present invention, a respiratory syncytial virus antibody detection kit is provided, comprising the mutant. The definition of the respiratory syncytial virus antibody detection kit is as described in the seventh aspect.

[0190] In a ninth aspect of the embodiments of the present application, the 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.

[0191] 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 the subject.

[0192] 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 signs or symptoms of a condition or disease, such as RSV infection. For example, this may be the amount required to inhibit viral replication or measurably alter the outward symptoms 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 will generally be used that will achieve target tissue concentrations (e.g., in respiratory tissues) that have been demonstrated to achieve in vitro inhibition of viral replication. 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 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.

[0193] To prevent, treat, or cure means, for example, to inhibit the full development of a disease or condition in a subject at risk of 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 "improve" with respect to a disease or pathological condition refers to any observable beneficial therapeutic effect. A beneficial effect can be demonstrated, for example, by a 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 subject's overall health or well-being, or other parameters well known in the art that are specific to a particular disease. A "prophylactic" treatment is a treatment administered to a subject who does not exhibit signs of the disease or only exhibits early signs in order to reduce the risk of developing the disease. The term "reduce" is a relative term, such that an agent reduces a reaction or condition if the reaction or condition is quantitatively reduced after administration of the agent, or if it is reduced after administration of the agent compared to a reference agent. Similarly, the term "prevent" does not necessarily mean that the agent completely eliminates the reaction or condition, as long as at least one characteristic of the reaction or condition is eliminated. Thus, an immunogenic composition that reduces or prevents an infection or response (e.g., a pathological response, e.g., a vaccine-enhanced viral disease) can, but need not necessarily, completely eliminate such an infection or response, so long as the infection or response is measurably reduced, e.g., 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), compared to the infection or response in the absence of the agent or compared to a reference agent.

[0194] In a tenth aspect of the embodiments of the present application, a method for detecting or isolating RSV F binding antibodies in a sample is provided, comprising the following steps:

[0195] contacting the mutant described in the first aspect with an RSV F binding antibody in a sample to form an immune complex; and,

[0196] The immune complex is detected, thereby detecting or isolating RSV F binding antibodies in the sample.

[0197] The embodiments of the present application will be described in detail below with reference to the examples. It should be understood that these examples are intended to illustrate the present application only 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 manuals or conventional conditions in this area, or according to the conditions recommended by the manufacturer, or with reference to experimental methods known in the art.

[0198] In the following specific examples, 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 testing accuracy or operational accuracy are allowed.

[0199] (1) Construction of RSV pre-F mutant vector

[0200] A mutagenesis strategy was used to determine the amino acid sequence of the RSV pre-F mutant. The corresponding nucleic acid sequence was codon-optimized for expression in Chinese hamster ovary (CHO) cells. The resulting sequence was then ligated into the pcDNA3.1 vector using the BamHI and XhoⅠ restriction sites.

[0201] The ClonExpress II Recombination Cloning Kit (Novagen Biotech Co., Ltd.) was used to perform substitutions, insertions, and deletions. Polymerase chain reaction (PCR) was used to amplify the fragments at either end of the mutation site using the high-fidelity enzyme Phanta Max (Novagen Biotech Co., Ltd.). After gel recovery, the two fragments were fused by PCR. The recovered fusion fragments were homologously recombined into the pcDNA3.1 vector, which had been double-digested with BamHI and XhoⅠ. The constructed point mutation vector was sequenced and confirmed by Beijing Qingke Biotech Co., Ltd. Figure 1 It is an expression plasmid used to construct RSV pre-F mutant.

[0202] Inoculate the cloning vector into 300 mL of LB(Amp+) medium and incubate at 37°C and 180 rpm for 16 hours. Extract the plasmid using a large plasmid extraction kit and store in 1 mL of sterile TE buffer. All commercial kits and reagents were used according to the manufacturer's instructions.

[0203] Figure 2 This diagram depicts the amino acid structure of the pre-F polypeptide of the wild-type RSV F protein. The amino acid sequence indicated in this diagram can be derived from either subtype A or subtype B of the human respiratory syncytial virus F protein. Ribosomal translation of the natural F protein forms the F0 precursor polypeptide, which consists of approximately 574 amino acids, including a signal peptide (1aa-25aa), an F2 polypeptide (26aa-109aa), a pep27 polypeptide (110aa-136aa), and an F1 polypeptide (137aa-574aa). The F1 polypeptide includes a transmembrane domain (514aa-550aa) and an intracellular domain (551aa-574aa). For the natural full-length precursor polypeptides of human RSV A2 and human RSV B, the natural full-length bovine RSV B pre-F polypeptide is 572 aa in length, and the F1 polypeptide corresponds to aa137-572 aa, containing the furin cleavage sites RAKR and KKRKRR.

[0204] The RSV pre-F signal peptide is cleaved during endoplasmic reticulum translocation. Two furin cleavage sites (RARR and KKRKRR) within the polypeptide are processed by intracellular furin-like proteases to produce three peptide fragments: the F1 polypeptide, the F2 polypeptide, and the Pep27 polypeptide. During F protein maturation, the Pep27 polypeptide is deleted, resulting in a protein consisting of the N-terminal F2 polypeptide and the C-terminal F1 polypeptide, linked to the F2 polypeptide by two disulfide bonds. The mature F protein forms a trimer and is anchored to the cell membrane by the F1 polypeptide via its transmembrane domain.

[0205] Figure 3 The structural diagram of the constructed stabilized RSV pre-F mutant monomer is described (SEQ ID NO.11). Unlike the wild-type RSV pre-F monomer, 1) the p27 peptide is replaced by GS, which is not affected by furin, and the mature monomer F protein will be composed of a single-chain polypeptide; 2) the transmembrane domain (TM, 514aa-550aa) and the intracellular domain (CT, 551aa-574aa) are deleted at the same time, and the final monomer protein is expressed in a secretory form; 3) and a His-Tag is added to the C-terminus of the protein to facilitate purification. Unless otherwise specified, the other monomer RSV F protein mutations involved in the examples of this application are designed on this basis, but it is understandable that, based on the design concept of the examples of this application, the design starting point polypeptide of the RSV pre-F mutant monomer is not limited to SEQ ID NO: 10 that meets the above conditions.

[0206] Figure 4 The structural schematic diagram of the monomers contained in the constructed stabilized RSV pre-F trimer is described (SEQ ID NO.12). On the basis of the stabilized RSV pre-F mutant monomer, the trimerization motif (T4foldon) of the bacteriophage T4 fiber protein (foldon) is fused to its C-terminus, so that the mature F protein formed finally forms a trimer to simulate the natural trimeric state of the F protein, and a purification tag is added to this C-terminus. Unless otherwise specified, the other trimeric RSV pre-F protein mutations involved in the embodiments of the present application are designed on this basis. Involving other polymers constructed based on mutants, such as dimers formed based on antibody Fc fragments, or nanoparticles formed based on iron nanoparticles (ferritin), T4foldon can be replaced in the structure shown in the legend, replaced with the corresponding antibody Fc fragment, etc.

[0207] Figure 5Described are a three-dimensional structural schematic diagram and a local enlarged schematic diagram (marked in green) of the mutation locations that can stabilize the RSV pre-F mutant. The mutation locations include the α3 helix, β3 sheet, and β4 sheet. The schematic diagram shows that the α3 helix and β3 sheet, as well as the β3 sheet and β4 sheet are parallel and close to each other. The RSV pre-F conformation is metastable and forms an irreversible rearrangement when triggered by the environment, thereby forming a more stable non-functional post-F conformation. Among them, the α2, α3, β3, β4, α4 and α5 in the sequence are conformationally transformed to form a long helical bundle, and the long helix formed with the C-terminus forms a long handle consisting of 6 helices in the case of a trimer, which is a typical feature of post-F. In the examples of the present application, we found that by introducing a cysteine ​​substitution between the β3 sheet and the β4 sheet, the β3 sheet and the β4 sheet can be fixed to prevent their conformational changes, so that the RSV F protein can be maintained in the pre-fusion conformation. When β3 and β4 as well as α3 and β3 are simultaneously fixed through the action of disulfide bridges, the RSV F protein can be placed in a more stable pre-fusion conformation.

[0208] Figure 6 The amino acids involved in the β3 / β4 position and the distance between the two amino acids that are prone to forming a disulfide bond are described in the examples of this application. The amino acids that form disulfide bonds include: 181L and 185V 180S and 186S 179V and 187V 178V and 188L 177A and 189T 176K and 190S

[0209] Figure 7 The amino acids involved in the α3 / β3 position and the distance between the two amino acids that are prone to forming a disulfide bond are described in the examples of this application. The amino acids that form disulfide bonds include: 163E and 181L 166K and 179V 167I and 179V 170A and 179V 170A and 177A 171L and 189T 171L and 177A 191K and 175N

[0210] Table 1. Amino acids contained in the α3 helix, β3 sheet, and β4 sheet of RSV F0 protein

[0211]

[0212] Table 2. Amino acid pairs that are prone to disulfide bond formation after cysteine ​​mutation

[0213] β4(aa:185V-aa:194D) β3(aa:176K-aa:181L) α3(aa:163E-aa:173S) 190S 176K 189T 177A 170A, 171L 188L 178V 187V 179V 166K, 167I, 170A 186S 180S 185V 181L 163E

[0214] Table 3. RSV pre-F mutant sites

[0215]

[0216]

[0217] Table 4

[0218] serial number Sequence number mutation site F2 position F1 position Linker JW-38 SEQ ID NO.27 S180C, S186C, A170C, V179C 103 145 GS JW-L10 SEQ ID NO.62 S180C, S186C, A170C, V179C 109 137 / JW-L11 SEQ ID NO.63 S180C, S186C, A170C, V179C 105 145 GS JW-L12 SEQ ID NO.64 S180C, S186C, A170C, V179C 103 145 SS JW-L13 SEQ ID NO.65 S180C, S186C, A170C, V179C 103 145 PG JW-L14 SEQ ID NO.66 S180C, S186C, A170C, V179C 103 145 GPG JW-L15 SEQ ID NO.67 S180C, S186C, A170C, V179C 103 145 GSGS JW-L16 SEQ ID NO.68 S180C, S186C 103 145 PG JW-L17 SEQ ID NO.69 S180C, S186C, L171C, A177C 103 145 GSGS JW-L18 SEQ ID NO.70 S180C, S186C, A170C, A177C, S190F, V207L 105 145 GS

[0219] (2) Expression of RSV pre-F mutant protein

[0220] Mutant proteins were generated by ExpiCHO TM Expression was performed using the ExpiCHO-S expression system (Thermofisher). Transient expression was performed according to the manufacturer's standard protocol. Briefly, one day before transfection (day –1), the ExpiCHO-S TM The final density of the culture was 3 × 10 6 ~4×10 6 viable cells / mL. The next day (day 0), measure the viable cell density and survival rate. The cell density should reach approximately 7×10 6 ~10×10 6 viable cells / mL. The viability should be 95% to 99% before continuing with transfection. Use fresh ExpiCHO preheated to 37°C. TM Dilute the cells to a final density of 6 × 10 6 Gently shake the culture flask to mix the cells and discard the remaining cells. Prepare ExpiFectamine using cold reagent (4°C) according to the instructions in the reagent manual. TM CHO / plasmid DNA complex. After inversion, add ExpiFectamine to the shake flask. T M CHO / DNA complex, the cells were cultured on a shaker (8% CO2, 37°C, 120 rpm). The day after transfection (day 1, 18-22 hours after transfection), ExpiFectamine TM CHO Enhancer and ExpiCHO TM According to the standard experimental protocol, the culture was terminated on the 8th day after transfection and samples were collected for testing.

[0221] The expression level was determined by referring to “(7) Detection of RSV pre-F mutant content by double antibody sandwich method”.

[0222] Thermal stability assay: Thermal stability is assessed by comparing the loss of prefusion conformation of the mutant protein before and after heat treatment. Briefly, the mutant protein is heat treated at 50°C for 3 h. The concentration of the prefusion conformation protein before and after heat treatment is determined by referring to "(7) Double antibody sandwich assay for RSV pre-F mutant content." The remaining percentage is calculated by dividing the post-heat treatment prefusion conformation protein concentration by the pre-heat treatment prefusion conformation protein concentration. This percentage is then used to assess the thermal stability of the mutant.

[0223] Table 5. Expression and thermal stability of mutants

[0224]

[0225]

[0226] Figure 8 The expression levels of pre-F mutants at positions β3 and β4 were described. JW-31, JW-34, and JW-35 were significantly expressed in CHO cells, with JW-31 and JW-35 showing significant increases compared to WT (SEQ ID NO. 11) and F0-GS (SEQ ID NO. 12). JW-32 and JW-33 showed almost no expression, demonstrating that some mutations at positions β3 and β4 can produce stable pre-F proteins.

[0227] Figure 9 The thermal stability of pre-F mutants at positions β3 and β4 was characterized. The results showed that mutants with high pre-F protein expression exhibited improved thermal stability compared to WT and F0-GS. However, WT and F0-GS without the cysteine ​​mutations were significantly unstable after treatment at 50°C.

[0228] Figure 10 The expression levels of pre-F mutants at positions α3 and β3 are described. With the exception of JW-03, which showed a relatively low expression level, all other mutations introduced at these positions significantly increased expression levels compared to WT and F0-GS. This demonstrates that some mutations at positions α3 and β3 can also produce stable pre-F proteins.

[0229] Figure 11 The thermal stability of pre-F mutants at positions α3 and β3 was described. Mutations at different positions in α3 and β3 had significant effects on pre-F stability. JW-04 and JW-06 exhibited good thermal stability.

[0230] Figure 12The expression levels of the pre-F mutants with double disulfide bonds formed at the α3 / β3 and β3 / β4 positions are described.

[0231] Figure 13 The thermal stability of the pre-F mutant with double disulfide bonds at the α3 / β3 and β3 / β4 positions was investigated. Compared with the single disulfide bond mutant, the double disulfide bond mutant significantly improved the thermal stability of the mutant, allowing >60% of the pre-F protein to remain after treatment at 50°C for 3 hours.

[0232] (3) Purification of recombinant mutant protein

[0233] Protein purification was performed in two steps using affinity chromatography and ion exchange chromatography. The expressed product was centrifuged (8000 rpm, 20 min), and the culture supernatant was harvested to remove cells and cell debris. The culture supernatant was filtered through a 0.45 μm filter to remove impurities. The treated supernatant was applied to a Ni-Sepharose 6FF column (Cytiva) equilibrated with 25 mM Tris-HCl, 0.15 M NaCl (Buffer A; pH 8.0). The column was then further washed with 25 mM Tris-HCl (150 mM NaCl, pH 8.0) until the A280 reached baseline. The column was then linearly eluted using 0-100% elution buffer (Buffer B: 25 mM Tris-HCl, 150 mM NaCl, 500 mM imidazole, pH 8.0). The eluate was collected and replaced with the equilibration buffer to remove the imidazole.

[0234] After the solution change, the sample was diluted with PBS buffer to a conductivity of less than 4 ms / cm and the pH was adjusted to 6.0. The protein was loaded onto Capto SPImpres packing (Cytiva) equilibrated with 20 mM phosphate buffer, pH 6.0. After loading, the sample was eluted with 20 mM phosphate buffer, pH 6.0, until the A280 reached the baseline level. The eluate was linearly eluted with 0-100% B elution buffer (20 mM phosphate buffer, 1 M NaCl, pH 6.0), and the eluate was collected. The pH was adjusted to 7.4-8.0 with 0.2 M Na2HPO4, and insoluble particles were further removed by filtration through a 0.45 μm filter.

[0235] (4) SDS-PAGE identification of mutant proteins

[0236] The purified recombinant protein was analyzed by SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis). The purified test sample was added to a sample buffer containing SDS and a reducing agent (such as dimercaptoethanol or thioglycolic acid), treated in a boiling water bath, and then loaded. After electrophoresis, the gel was stained with Coomassie blue and the gel image was acquired using a transparency scanner or protein imaging system.

[0237] Figure 14 The results of SDS-PAGE characterization of RSV pre-F mutant monomers are described. The different mutants exhibited only individual amino acid mutations, resulting in minimal changes in molecular weight. The SDS-PAGE electrophoretic bands were uniform and slightly larger than the theoretical molecular weight (50.55 kDa), likely due to band shifts caused by glycosylation modifications.

[0238] Figure 15 The results of SDS-PAGE characterization of RSV pre-F mutant trimers are described. The different mutants exhibited only individual amino acid mutations, resulting in minor changes in molecular weight. The SDS-PAGE electrophoretic bands were uniform and slightly larger than the theoretical molecular weight (53.99 kDa), likely due to band shifts caused by glycosylation modifications.

[0239] (5) HPLC detection of the purity and homogeneity of mutant proteins

[0240] The purity of the mutant protein was analyzed by HPLC (High Performance Liquid Chromatography) using a Thermo U3000 high-performance liquid chromatograph and a Thermo TSKgel UP-SW2000 size exclusion column (TOSOH). The mobile phase consisted of 20 mM PBS buffer at a flow rate of 1 mL / min, and the elution composition was measured at 280 nm. HPLC was used to determine the purity and homogeneity of the target protein.

[0241] Figure 16 The purity and homogeneity of the mutant monomer JW038 were determined by HPLC. A specific peak appeared at 18.713 minutes. The target peak was single, indicating good homogeneity.

[0242] Figure 17 The purity and homogeneity of the mutant trimer JW038-T4 were determined by HPLC. A specific peak for the mutant trimer appeared at 15.958 min. The mutant trimer exhibited good homogeneity.

[0243] (6) Differential scanning calorimetry to detect the thermal stability of mutant proteins

[0244] Differential Scanning Calorimetry (DSC) was performed using a DSC Q2000 (TA Instruments) system. The temperature range of the device was 0°C to 400°C, and the thermal expansion rate was 10°C / min in a nitrogen flow of 8 mL / min. Measurements were performed under a nitrogen atmosphere.

[0245] Table 6. Detection of Tm values ​​of mutants

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[0247]

[0248] According to Table 6, the Tm values ​​of the mutant proteins of different mutants were all >60°C when tested using differential scanning calorimetry, indicating that the mutant proteins had good thermal stability, which is also consistent with the good stability of the mutant proteins in the treatment at 50°C.

[0249] (7) Detection of RSV pre-F mutant content by double antibody sandwich method

[0250] The double antibody sandwich ELISA method was used to quantify RSV pre-F mutants. Palivizumab, which can simultaneously recognize pre-F, post-F and F proteins, was used as the coating antibody to specifically recognize RSV pre-F. The specific monoclonal antibody D25 labeled with horseradish peroxidase (D25-HRP) was used as the detection antibody for detection. Purified pre-F monomeric and trimeric proteins were used as calibrants to establish standard curves. The content of the test sample was determined by measuring its absorbance at OD450 and converting the dilution factor using the standard curve. This method can be used to measure the expression level of mutant pre-F proteins in transient expression and the residual content of pre-F mutant proteins in mutant thermal stability tests. The sample testing process is as follows: the monoclonal antibody is diluted to 1 μg / L in carbonate buffer and coated on an ELISA plate (Corning 9018). 100 μL was added to each well, and the plate was placed at 37°C for 1 hour, and then placed at 2-8°C overnight; the liquid in the 96-well plate was discarded, and the plate was washed three times with 20mM PBS. Then, 200 μL of blocking solution (2% bovine serum albumin, component V) was added to each well and blocked at room temperature for 60 minutes; the blocking solution in the well was aspirated, and the plate was washed three times with 20mM PBS-T solution. A series of 3-fold diluted mutant pre-fusion conformation F proteins were added to the first column of wells of the 96-well ELISA plate, and the negative control was PBS. The plate was reacted at 37°C for 60 minutes, the blocking solution in the well was aspirated, and the plate was washed three times with 20mM PBS-T solution; the anti-HIS-HRP conjugate was diluted 1:2000 with enzyme conjugate diluent, and then added to the 96-well ELISA plate, 100 μL per well, and the plate was reacted at 37°C for 10 minutes; the secondary antibody in the well was aspirated and diluted with 20mM The cells were washed three times with PBS-T solution, 100 μL of TMB colorimetric solution was added to each well, and 50 μL of stop solution was added to terminate the reaction after 10 min. The A450 and absorbance values ​​were then measured using a microplate reader.

[0251] Figure 8 , Figure 10 and Figure 12The expression level of monomeric RSV pre-F mutant protein in cell culture after transient transfection of Expi-CHO cells is shown. In this example, palivizumab, which binds independently of conformational changes in the F antigen, was used as the capture antibody, and horseradish peroxidase-labeled D25 antibody, which specifically detects the epitope Φ, was used as the detection antibody. A standard curve was established using purified DS-Cav1 monomeric protein or trimer (consistent with the expression form of the target substance being detected) as a reference. The expression level of pre-F protein in the culture supernatant was calibrated and converted based on the standard curve. The above procedures yielded the following results: Compared to the unmutated WT and F0-GS (p27 peptide replaced by GS), the mutant containing cysteine ​​point mutations at α3 and β3 to form a disulfide bond showed significantly improved conservation of the epitope Φ, and pre-F was efficiently expressed in Expi-CHO cells. In terms of thermal stability, the stability of the protein was evaluated by assessing the proportion of protein remaining bound to D25 monoclonal antibody after treatment at 50°C (1h, 2h, 3h). The purified protein sample was diluted to 0.1 mg / mL with PBS (pH 7.4) and detected by double antibody sandwich ELSIFA method after heat treatment. Figure 9 , Figure 11 and Figure 13 shown.

[0252] (8) Antigenicity detection of RSV pre-F mutants

[0253] Each purified mutant protein was diluted with carbonate buffer (pH 9.6) at a starting concentration of 10 μg / mL, serially diluted 3-fold, and then coated on a 96-well ELISA plate (Corning 9018) at 100 μL / well. After placing at 37°C for 1 hour, place it at 2-8°C overnight; discard the liquid in the 96-well plate, wash three times with 20mM PBS, then add 200μL of blocking solution (2% bovine serum albumin, component V) to each well, and block at room temperature for 60 minutes; aspirate the blocking solution in the well, wash three times with 20mM PBS-T solution, take anti-D25-HRP, AM22-HRP, AM14-HRP, ADI-15568-HRP and other conjugates, dilute them 1:2000 with enzyme conjugate diluent, and then add them to the 96-well ELISA plate, 100μL per well, and react at 37°C for 10 minutes; aspirate the excess antibody in the well, wash three times with 20mM PBS-T solution, add 100μL of TMB colorimetric solution to each well, add 50μL of stop solution after 10 minutes to terminate the reaction, and then measure the A450 and A630 absorbance values ​​with a microplate reader.

[0254] Figure 18 The results of antigenic characterization of each mutant are described. Figure 18Figures A and B show that the purified mutants can be The mutant was recognized by epitope-specific monoclonal antibodies D25 and AM22, confirming that the mutant could maintain the pre-fusion conformation (pre-F) of RSV F protein. At the same time, the mutant could also be recognized by ADI-15568 monoclonal antibody that recognizes epitope V ( Figure 18 However, a trimer-specific antibody (AM14) can recognize the mutant trimer but not the mutant monomer, and mutations at different positions have a significant impact on the stability of the trimer.

[0255] (9) Evaluation of the immunogenicity of RSV pre-F protein

[0256] To evaluate the immunogenicity of RSV F in different prefusion conformations, mice were immunized with different mutant proteins. Female Balb / c mice weighing 14-16 g were immunized with 5 μg of vaccine antigen adjuvanted with aluminum hydroxide. The mice were injected intramuscularly at week 0 and week 4 (28 days). Serum was collected 2 weeks after the booster immunization (42 days) to determine total IgG antibody titers and neutralizing antibody titers. The immunization groups are shown in the table below:

[0257] Table 7

[0258]

[0259] (10) Antigen-specific antibody level detection

[0260] Total IgG antibodies were detected by indirect ELISA. Pre-F protein was pre-coated in a 96-well plate. After blocking, the test serum was added and serially diluted, starting at 800-fold and then diluted threefold. After washing to remove unbound serum, HRP-labeled goat anti-mouse secondary antibody was added for incubation. After washing to remove unbound secondary antibody, substrate was added for color development. The mouse serum titer was determined by measuring the absorbance at 450nm and 630nm. The dilution titer was calculated based on the dilution factor of the serum in the last well diluted to an absorbance greater than the cut-off value. Different mutant trimeric proteins were added with aluminum hydroxide adjuvant to immunize BalB / c mice. The levels of antigen-specific IgG antibodies in mouse serum were detected using the pre-F mutant trimer as the coating antigen.

[0261] Figure 19 The levels of RSV pre-F-specific binding antibodies induced by different mutants in BalB / c mice are described. Both mutant monomers and mutant trimers showed good immunogenicity and induced high levels of humoral immune responses in mice. Among them, JW-38-T4 and JW-48-T4 induced higher levels of binding antibodies than mutant monomers (JW-36), F0 protein, and post-F protein, with statistically significant differences.

[0262] (11) Neutralizing antibody level detection

[0263] The neutralizing antibody titer was detected using a high-throughput microplate method. Specifically, the density of Hep-2 cells was adjusted, inoculated into a 96-well plate, and placed in a cell culture incubator (37°C, 5% CO2) for overnight culture. The test was started after ensuring that the cell confluence was about 90% on the next day. The serum was then inactivated at 56°C in a water bath for 30 minutes to prepare inactivated serum. 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 neutralized in a 37°C, 5% CO2 incubator for about 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 fluorescently labeled detection antibodies were added. The plate was read using a CTL instrument. The neutralizing antibody titer was set as the serum dilution factor that resulted in a 50% reduction in infectious units (NT50).

[0264] Figure 20 The neutralizing antibody levels induced by different mutants in mice were described. The results showed that sera from mice immunized with the mutants were able to neutralize RSV:A2. Neutralizing antibodies induced by the different mutants varied, but all were superior to those induced by the F0 and post-F proteins. The stabilized pre-F protein retained key neutralizing epitopes and induced higher levels of neutralizing antibodies than the post-F protein. Furthermore, the F0 protein also induced higher levels of neutralizing antibodies than the post-F protein.

[0265] The embodiments of the present application provide a mutation strategy for producing a stabilized RSV pre-F protein, which prevents the conformational change of the F protein by forming a disulfide bond between β3 and β4, or simultaneously forming a disulfide bond between α3 and β3, and between β3 and β4. This mutation strategy is different from the current mainstream mutation strategy. It is an innovative mutation that enriches the structure-based antigen design for RSV F protein. The introduction of disulfide bonds produces a stable RSV F protein pre-fusion conformation (pre-F) with a high expression level. The expression level in CHO cell transient expression reaches 300 mg / L, and the mutant has very high thermal stability and can retain more than 85% of the pre-F protein after being treated at 50°C for 3 hours. The mutant has a site that can be recognized by the D25 antibody The mutant retains the antigenic epitopes recognized by other currently identified antibodies specific for RSV F protein. Immunization with this mutant can induce high levels of neutralizing antibodies in animals. This mutant has great potential as a vaccine.

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[0277] The various technical features of the above-mentioned implementation modes and examples can be combined in any appropriate manner. In order to make the description concise, not all possible combinations of the various 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 the description in this specification.

[0278] The above-described embodiments only express several implementation methods of the present application, which facilitate a specific and detailed understanding of the technical solutions of the present application, but cannot be understood as limiting 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 variations and improvements can be made, which all fall within the scope of protection of the present application. In addition, it should be understood that after reading the above-mentioned teaching content of the present application, those skilled in the art can make various changes or modifications to the present application, and the equivalent forms obtained also fall within the scope of protection of the present application. It should also be understood that the technical solutions obtained by those skilled in the art through logical analysis, reasoning or limited experiments on the basis of the technical solutions provided in the present application are all within the scope of protection of the claims attached to the present application. Therefore, the scope of protection of the patent application of the present application shall be based on the content of the attached claims, and the description and drawings can be used to interpret the content of the claims.

Claims

1. A mutant of RSV pre-F, characterized in that The sequence of the mutant is shown in any one of SEQ ID NO.20, SEQ ID NO.25 to SEQ ID NO.

70.

2. A nucleic acid molecule, characterized in that It encodes the mutant according to claim 1.

3. A carrier, characterized in that It comprises the nucleic acid molecule according to claim 2.

4. An engineered cell, characterized in that It expresses the mutant according to claim 1, or it comprises the nucleic acid molecule according to claim 2 or the vector according to claim 3.

5. The method for producing a mutant according to claim 1, characterized in that: It includes the following steps: Cultivating the engineered cell according to claim 4, and isolating the mutant from the obtained culture supernatant.

6. An immune composition comprising the mutant according to claim 1 or the nucleic acid molecule according to claim 2, and an immune adjuvant.

7. The immune composition according to claim 6, characterized in that The immune adjuvant includes one or more of aluminum salt adjuvant, surfactant, polynucleotide, lipopolysaccharide, liposome, and oil emulsion adjuvant.

8. Use of the mutant according to claim 1 in preparing a respiratory syncytial virus antibody detection kit.

9. A respiratory syncytial virus antibody detection kit comprising the mutant according to claim 1.

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