Mutants of RSV prefusion F protein, their preparation and applications

Abstract

This application belongs to the field of biotechnology and relates to a mutant of the RSV prefusion F protein, its preparation and application. This application provides a mutant of the RSV prefusion F protein, in which the α3 helix region of the F1 polypeptide has a cysteine substituent 1, and the β3 sheet region has a cysteine substituent 2, and the cysteine substituent 1 and the cysteine substituent 2 form a disulfide bond. Compared with the traditional technology, this application discovers another disulfide bond mutation position, mainly forming a disulfide bond mutation between α3 and β3. The corresponding mutant can bind to a specific monoclonal antibody that recognizes the φ site, has a high expression level, has good stability, and has great potential for developing a vaccine against RSV.

Description

Technical Field

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

[0002] Respiratory syncytial virus (RSV) is a highly contagious respiratory pathogen and one of the leading causes of hospitalization and death in infants under 6 months of age worldwide. It can cause severe lower respiratory tract infections in infants, the elderly, and immunocompromised patients. WHO studies on the etiology of acute respiratory infections in children show that the number of acute respiratory infections caused by RSV accounts for more than 60% of the total number of respiratory diseases in children. In 2019, 33 million cases of acute lower respiratory tract infections (ALRI) caused by RSV infection occurred in children aged 0-5 years worldwide, 3.6 million children were hospitalized for this reason, and a total of 101,000 children in the hospitalized group eventually died, of which about 46% were infants aged 0-6 months, which imposed a huge economic burden on the public health resources of various countries.

[0003] RSV belongs to the Paramyxoviridae family. The viral genome consists of 15,200 nucleotides, including 10 genes encoding 11 proteins. Among them, the structural proteins include three transmembrane surface glycoproteins: attachment protein G (Glycoprotein, G), fusion protein F (Fusion protein, F) and small hydrophobic protein SH (small hydrophobic protein, SH). Fusion protein F and attachment protein G, two transmembrane surface glycoproteins, are key targets for neutralizing antibodies. However, the highly variable conformational changes of glycosylated attachment protein G and fusion protein F pose challenges to vaccine development.

[0004] Fusion protein F is a type I membrane fusion protein. The virus-encoded F0 protein forms a trimer in the endoplasmic reticulum and contains two Furin protease cleavage sites. After Furin protease cleavage, it can form two proteins, F1 and F2, and a Pep27 polypeptide fragment. The F1 protein contains a hydrophobic fusion peptide at its N-terminus and two heptad repeat regions (HRA and HRB). Under physiological conditions, the pre-F protein (pre-Fusion conformation, pre-F) is in a metastable state and can easily transform into a stable post-fusion conformation (Postfusion conformation, post-F), losing the main neutralizing antigen epitope. However, in the sera of patients with natural RSV infection, most neutralizing antibodies are directed against the prefusion conformation.

[0005] At present, the crystal structures of two conformations of the F protein have been resolved, and based on its molecular structure, several mutants with relatively stable pre-F conformations have been obtained through point mutations. For example, the DS-Cav1 mutants (S155C, S290C, S190F, V207L) described in CN105473604B, US10017543B2, US11130785B2; the pre-F stable mutants (N67I, S215P, E487Q) containing SC-DM and SC-TM described in US20150320854A1, WO2014174018A1, AU2014259474B2, CN105408348B; the pre-F mutants (S55C, L188C, L142C, N371C) described in US20210023200A1, CN108738312A; the pre-F mutant containing mutations at positions L141C, L142C, and L373C described in CN114929877A. Traditional mutation strategies mainly include disulfide bond mutation, cavity filling, or charge balance, aiming to maintain the stabilized conformation of pre-F or improve the expression level of pre-F. However, the stabilization of the pre-type F protein is still insufficient, and structure-based optimization is under continuous research.

[0006] In view of this, the present application is specifically proposed. Summary of the Invention

[0007] The main object of the embodiments of the present application is a mutant of the RSV prefusion F protein, its preparation, and application. The technical solutions include:

[0008] In the first aspect of the embodiments of the present application, a mutant of the RSV prefusion F protein is provided, wherein the α3 helix region of the F1 polypeptide has a cysteine substituent 1, and the β3 sheet region has a cysteine substituent 2, and the cysteine substituent 1 and the cysteine substituent 2 form a disulfide bond.

[0009] In some embodiments of the present application, the species source of the RSV prefusion F protein is human or bovine.

[0010] In some embodiments of the present application, the F1 polypeptide of the RSV prefusion F protein has the amino acid sequence shown in SEQ ID NO.51 or has at least 80% homology with the amino acid sequence shown in SEQ ID NO.51.

[0011] In some embodiments of the present application, the F1 polypeptide of the pre-fusion F protein of RSV has the amino acid sequence shown in SEQ ID NO.51, SEQ ID NO.74 or SEQ ID NO.75. In some embodiments of the present application, the F1 polypeptide of the pre-fusion F protein of RSV satisfies one or more of the following conditions:

[0012] (1) It does not contain a transmembrane domain, and

[0013] (2) It does not contain an intracellular domain.

[0014] In some embodiments of the present application, the F1 polypeptide of the pre-fusion F protein of RSV has one or more of the following mutations: I379V and M447V.

[0015] In some embodiments of the present application, the F1 polypeptide of the pre-fusion F protein of RSV has the amino acid sequence shown in SEQ ID NO.52.

[0016] In some embodiments of the present application, the distance between the cysteine substituent 1 and the cysteine substituent 2 is

[0017] In some embodiments of the present application, the α3 helix region of the F1 polypeptide of the mutant has one or more of the following mutations: E163C, K166C, I167C, A170C, and L171C.

[0018] In some embodiments of the present application, the β3 sheet region of the F1 polypeptide of the mutant has one or more of the following mutations: A177C, V179C, and L181C.

[0019] In some embodiments of the present application, the F1 polypeptide of the mutant has one of the following mutation combinations:

[0020] Group 1 is E163C and L181C,

[0021] Group 2 is K166C and V179C,

[0022] Group 3 is I167C and V179C,

[0023] Group 4 is A170C and V179C,

[0024] Group 5 is A170C and A177C, and

[0025] Group 6 is L171C and A177C.

[0026] In some embodiments of the present application, the mutant also has one or more of the following mutation sites: S55C, S155C, L188C, S190F, V207L, and S290C.

[0027] In some embodiments of the present application, the mutant has one of the following combinations of mutation sites:

[0028] Group 1) S155C and S290C,

[0029] Group 2) S55C and L188C, and

[0030] Group 3) S190F and V207L.

[0031] In some embodiments of the present application, the mutant does not contain a furin cleavage site fragment.

[0032] 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 through a flexible short peptide.

[0033] In some embodiments of the present application, 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.

[0034] In some embodiments of the present application, the C-terminus of the F1 polypeptide of the mutant is connected to a tag fragment and / or an aggregation motif.

[0035] In some embodiments of the present application, the tag fragment includes a 6His polypeptide.

[0036] In some embodiments of the present application, the aggregation motif is as shown in SEQ ID NO. 48.

[0037] In some embodiments of the present application, the F2 polypeptide of the mutant has the amino acid sequence shown in SEQ ID NO. 49 or has at least 80% homology with the amino acid sequence shown in SEQ ID NO. 49.

[0038] In some embodiments of the present application, the F2 polypeptide of the mutant has the amino acid sequence shown in SEQ ID NO. 49, SEQ ID NO. 76 or SEQ ID NO. 77.

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

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

[0041] 2) It has the following mutation: P102A.

[0042] In some embodiments of the present application, the F2 polypeptide of the mutant has the amino acid sequence shown in SEQ ID NO.50.

[0043] In some embodiments of the present application, the amino acid sequence of the mutant is as shown in any one of SEQ ID NO.12 to SEQ ID NO.35, SEQ ID NO.37, SEQ ID NO.39, SEQ ID NO.42 to SEQ ID NO.47, and SEQ ID NO.53 to SEQ ID NO.73.

[0044] In the second aspect of the embodiments of the present application, there is provided a nucleic acid molecule encoding the mutant of the RSV prefusion F protein described in the first aspect.

[0045] In the third aspect of the embodiments of the present application, there is provided a vector comprising the nucleic acid molecule described in the second aspect.

[0046] In the fourth aspect of the embodiments of the present application, there is provided a genetically engineered cell that expresses the mutant of the RSV prefusion F protein described in the first aspect, or that comprises the nucleic acid molecule described in the second aspect, or the vector described in the third aspect.

[0047] In the fifth aspect of the embodiments of the present application, there is provided a method for producing the mutant of the RSV prefusion F protein described above, which comprises the following steps:

[0048] Culturing the genetically engineered cell described in the fourth aspect, and isolating the mutant of the RSV prefusion F protein from the resulting culture supernatant.

[0049] In the sixth aspect of the embodiments of the present application, there is provided an immunocomposition comprising the mutant of the RSV prefusion F protein described in the first aspect or the nucleic acid molecule described in the second aspect, and an immunoadjuvant.

[0050] In some embodiments of the present application, the immunoadjuvant comprises one or more of an aluminum salt adjuvant, a surfactant, a polynucleotide, a lipopolysaccharide, a liposome, and an oil-in-water adjuvant.

[0051] In the seventh aspect of the embodiments of the present application, there is provided an application of the mutant of the RSV prefusion F protein described in the first aspect in the preparation of a kit for detecting respiratory syncytial virus antibodies.

[0052] In the eighth aspect of the embodiments of the present application, a respiratory syncytial virus antibody detection kit is provided, which includes the mutant of the RSV prefusion F protein described in the first aspect.

[0053] In the ninth aspect of the embodiments of the present application, a method for preventing and treating lower respiratory tract infections caused by respiratory syncytial virus is provided, which includes the following steps: administering to a subject a therapeutically effective amount of the immunocomposition described in the sixth aspect.

[0054] In the tenth aspect of the embodiments of the present application, a method for detecting or isolating RSV F-binding antibodies in a sample is provided, which includes the following steps:

[0055] Contacting the mutant of the RSV prefusion F protein described in the first aspect with the RSV F-binding antibodies in the sample to form an immune complex; and,

[0056] Detecting the immune complex, thereby achieving the detection or isolation of RSV F-binding antibodies in the sample.

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

[0058] 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 drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and those skilled in the art can obtain other drawings based on these drawings without creative efforts.

[0059] Figure 1 Schematic diagram of the RSV F0 protein structure (A, B, bovine);

[0060] Figure 2 Schematic diagram of the structure of the stabilized mutant RSV F protein polypeptide monomer;

[0061] Figure 3 Schematic diagram of the structure of the stabilized mutant RSV F protein polypeptide trimer;

[0062] Figure 4 Identification diagram of a total of 7 pairs of disulfide bonds in the native RSV F0 precursor protein or mature F protein;

[0063] Figure 5 Schematic diagram of the three-dimensional structure of the RSV F protein monomer (left) and a partially enlarged schematic diagram of the α3 and β3 sheets (right);

[0064] Figure 6 To identify the amino acids involved in the α3 helix and β3 sheet positions in this application and the distance between two amino acid atoms that are prone to form disulfide bonds

[0065] Figure 7 Results of SDS-PAGE analysis of the mutant under reducing conditions; among them, JW-05 is the mutant monomer, and JW-05-T4 is the mutant trimer;

[0066] Figure 8 Results of high performance liquid chromatography (HPLC) analysis of the RSV pre-F mutant; among them, Figure 8 a is the mutant monomer, Figure 8 b is the mutant trimer;

[0067] Figure 9 Statistical chart of the expression level of the RSV pre-F monomer formed by mutations at the α3 / β3 position;

[0068] Figure 10 Statistical chart of the thermal stability of the RSV pre-F monomer formed by mutations at the α3 / β3 position under treatment at 50°C;

[0069] Figure 11 Statistical chart of the expression level of the RSV pre-F mutant trimer;

[0070] Figure 12 Statistical chart of the thermal stability of the RSV pre-F mutant trimer under treatment at 50°C;

[0071] Figure 13 Binding specificity of the RSV pre-F mutant trimer with the D25 monoclonal antibody ( epitope);

[0072] Figure 14 Binding specificity of the RSV pre-F mutant trimer with the AM22 monoclonal antibody ( epitope);

[0073] Figure 15 Binding specificity of the RSV pre-F mutant trimer with the AM14 monoclonal antibody (trimer epitope);

[0074] Figure 16 Results of detection of the level of antigen-specific antibodies of the RSV pre-F mutant;

[0075] Figure 17 Results of detection of neutralizing antibodies of the RSV pre-F mutant;

[0076] Figure 18 The adsorption rate of the RSV pre-F mutant to neutralizing antibodies in serum. Detailed implementation manners

[0077] The present application will be further described in detail below in conjunction with the accompanying drawings, implementation manners and examples. It should be understood that these implementation manners and examples are only used to illustrate the present application and not to limit the scope of the present application. The purpose of providing these implementation manners and examples is to make the understanding of the disclosed content of the present application more thorough and comprehensive. It should also be understood that the present application can be implemented in many different forms and is not limited to the implementation manners and examples described herein. Those skilled in the art can make various changes or modifications without departing from the connotation of the present application, and the equivalent forms obtained also fall within the protection scope of the present application. In addition, in the following description, a large number of specific details are given to provide a more thorough understanding of the present application. It should be understood that the present application can be implemented without one or more of these details.

[0078] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which this application belongs. The terms used in the description of the present application herein are only for the purpose of describing the implementation manners and examples and are not intended to limit the present application.

[0079] Term

[0080] Unless otherwise stated or there is a contradiction, the terms or phrases used herein have the following meanings:

[0081] The term "and / or", "or / and", "and / or" used herein includes any one of two or more related listed items, and also includes any and all combinations of the related listed items. The said any and all combinations include any two related listed items, any more related listed items, or the combination of all related listed items. It should be noted that when at least two conjunctions selected from "and / or", "or / and", "and / or" are used to connect at least three items, it should be understood that in the present application, this technical solution undoubtedly includes the technical solution connected by "logical AND", and also undoubtedly includes the technical solution connected by "logical OR". For example, "A and / or B" includes three parallel solutions: A, B, and A + B. Another example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, D (that is, the technical solution connected by "logical OR"), and also includes any and all combinations of A, B, C, D, that is, it includes the combination of any two or any three of A, B, C, D, and also includes the four-item combination of A, B, C, D (that is, the technical solution connected by "logical AND").

[0082] In this application, terms such as "multiple", "diverse", "multiple times", "pluralistic", etc., unless otherwise specifically defined, mean greater than 2 or equal to 2 in quantity. For example, "one or more" means one or greater than or equal to two.

[0083] As used herein, "its combination", "any combination thereof", "any combination mode thereof", etc. include all suitable combination modes of any two or more of the listed items.

[0084] In this article, the "suitable" in "suitable combination mode", "suitable mode", "any suitable mode", etc. is subject to being able to implement the technical solution of this application, solve the technical problems of this application, and achieve the expected technical effects of this application.

[0085] In this article, "preferred", "better", "more preferable", "it is advisable" are only used to describe the implementation modes or embodiments with better effects, and it should be understood that they do not constitute a limitation on the protection scope of this application.

[0086] In this application, "further", "even further", "especially", etc. are used for descriptive purposes, indicating differences in content, but should not be understood as a limitation on the protection scope of this application.

[0087] In this application, "optionally", "optional", "optional" mean that it can be either present or absent, that is, it refers to any one of the two parallel options of "present" or "absent". If "optional" appears multiple times in a technical solution, unless otherwise specified and there are no contradictions or mutual restrictions, each "optional" is independent of each other.

[0088] In this application, in "the first aspect", "the second aspect", "the third aspect", "the fourth aspect", etc., the terms "first", "second", "third", "fourth", etc. are only used for descriptive purposes, and cannot be understood as indicating or implying relative importance or quantity, nor can it be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first", "second", "third", "fourth", etc. only serve the purpose of non-exhaustive listing and description, and it should be understood that they do not constitute a closed limitation on quantity.

[0089] In this application, for the technical features described in an open-ended manner, it includes the closed technical solutions composed of the listed features, and also includes the open-ended technical solutions containing the listed features.

[0090] In this application, when it comes to numerical intervals (i.e., numerical ranges), unless otherwise specified, the selectable numerical values are considered continuous within the above numerical intervals, and include the two numerical endpoints of the numerical range (i.e., the minimum value and the maximum value), as well as each numerical value between these two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to the integers within the numerical interval, it includes the two endpoint integers of the numerical range, as well as each integer between the two endpoints. In this article, it is equivalent to directly listing each integer. For example, if t is an integer selected from 1 to 10, it means that t is any integer selected from the integer group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. In addition, when multiple ranges are provided to describe features or characteristics, these ranges can be combined. In other words, unless otherwise specified, the ranges disclosed in this article should be understood to include any and all sub-ranges subsumed therein.

[0091] The temperature parameter in this application, unless otherwise specified, allows both constant temperature treatment and variation within a certain temperature range. It should be understood that the constant temperature treatment allows the temperature to fluctuate within the accuracy range controlled by the instrument. Fluctuation within ranges such as ±5°C, ±4°C, ±3°C, ±2°C, ±1°C is allowed.

[0092] In this application, %(w / w) and wt% both represent weight percentage, %(v / v) refers to volume percentage, and %(w / v) refers to mass-volume percentage.

[0093] All documents mentioned in this application are cited in this application as references, just as if each document was cited separately as a reference. Unless it conflicts with the application purpose and / or technical solution of this application, the cited documents involved in this application are cited for all their contents and all their purposes. When this application involves cited documents, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited. When this application involves cited documents, the examples and preferred methods of the relevant technical features cited can also be incorporated into this application as references, but only to the extent that this application can be implemented. It should be understood that when the cited content conflicts with the description in this application, this application shall prevail or be amended adaptively according to the description in this application.

[0094] Unless otherwise indicated, technical terms are used according to conventional usage. Definitions of common terms in molecular biology can be found in Benjamin Lewin, Genes VII, published by Oxford University Press, 1999; Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994; and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995; and other similar references.

[0095] The present application provides a method for stabilizing RSV F0 in the pre-F state by amino acid mutation, and provides a disulfide bond mutation position not involved in the prior art or existing patents. This position involves introducing a disulfide bond mutation between α3 / β3 of the RSV F protein, and the mutation combination based on this mutation can bind to a specific monoclonal antibody recognizing the site, and has good stability. The multimer of the mutant constructed thereby not only significantly increases the expression level of the pre-F protein, but also has good immunogenicity, can induce a high level of neutralizing antibodies, and has great potential for developing vaccines.

[0096] Compared with the traditional technology, the present application discovers another disulfide bond mutation position, mainly the disulfide bond mutation between α3 and β3, which is not involved in the prior art or existing patents. The single disulfide bond mutation at this position or the mutant combined with mutations at other positions can bind to a specific monoclonal antibody recognizing the site, has a high expression level, has good stability, and has great potential for developing an RSV vaccine. The solutions of the examples of the present application include:

[0097] The first aspect of the embodiments of the present application , The examples of the present application provide a mutant of the RSV prefusion F protein, wherein the α3 helix region of the F1 polypeptide has a cysteine substituent 1, and the β3 sheet region has a cysteine substituent 2, and the cysteine substituent 1 and the cysteine substituent 2 form a disulfide bond.

[0098] Substituent, amino acid substitution: One amino acid in an antigen is replaced by another amino acid or an amino acid is deleted. For example, an amino acid in an antigen is replaced by an amino acid from a homologous protein.

[0099] Homologous proteins: Proteins having similar structures and functions. For example, proteins from two or more species or viral strains having similar structures and functions in two or more species or viral 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 characteristics and can be regarded as structural homologs. Homologous proteins generally share a high degree of sequence conservation, such as 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, such as 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.

[0100] There are several subtypes of RSV, including human subtype A, human subtype B, and bovine subtype. Within the RSV subtypes, there are individual strains of each subtype. In the examples of this application, among the mutants of the RSV prefusion F protein, except that the F1 polypeptide forms a disulfide bond through cysteine substituent 1 and cysteine substituent 2, the amino acid sequences of the remaining fragments can be wild-type sequences or sequences with other mutations through artificial or natural means on the basis of the wild-type sequences, and the introduction of other mutations can still make the mutants stable.

[0101] In some embodiments of this application, the F1 polypeptide of the RSV prefusion F protein has the amino acid sequence shown in SEQ ID NO.51 or has at least 80% (at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) homology with the amino acid sequence shown in SEQ ID NO.51. Optionally, the F1 polypeptide of the RSV prefusion F protein has the amino acid sequence shown in SEQ ID NO.51, SEQ ID NO.74 or SEQ ID NO.75. Optionally,

[0102] The F1 polypeptide of the RSV prefusion F protein described above satisfies one or more of the following conditions:

[0103] (1) Does not contain a transmembrane domain, and

[0104] (2) Does not contain an intracellular domain.

[0105] Further optionally, the F1 polypeptide of the prefusion F protein of RSV has one or more of the following mutations: I379V and M447V. For example, the F1 polypeptide of the prefusion F protein of RSV has the amino acid sequence shown in SEQ ID NO. 52.

[0106] In some embodiments of the present application, the distance between the cysteine substituent 1 and the cysteine substituent 2 is (for example

[0107] In some embodiments of the present application, the α3 helix region of the F1 polypeptide of the mutant has one or more of the following mutations: E163C, K166C, I167C, A170C, and L171C.

[0108] In some embodiments of the present application, the β3 sheet region of the F1 polypeptide of the mutant has one or more of the following mutations: A177C, V179C, and L181C.

[0109] In some embodiments of the present application, the F1 polypeptide of the mutant has one of the following mutation combinations:

[0110] Group 1 is E163C and L181C,

[0111] Group 2 is K166C and V179C,

[0112] Group 3 is I167C and V179C,

[0113] Group 4 is A170C and V179C,

[0114] Group 5 is A170C and A177C, and

[0115] Group 6 is L171C and A177C.

[0116] In some embodiments of the present application, the mutant also has one or more of the following mutation sites: S55C, S155C, L188C, S190F, V207L, and S290C. S55C is located in the F2 polypeptide. S155C, S180C, S186C, L188C, S190F, V207L, and S290C are located in the F1 polypeptide.

[0117] In some embodiments of the present application, the mutant has one of the following mutation site combinations:

[0118] Group 1) S155C and S290C,

[0119] Group 2) S55C and L188C, and

[0120] Group 3) S190F and V207L.

[0121] In some embodiments of the present application, the mutant does not contain a furin cleavage site fragment.

[0122] 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 through a flexible short peptide.

[0123] The term "connection" may refer to making two molecules into a continuous molecule; for example, connecting two other polypeptides into a continuous polypeptide, or covalently connecting a vector molecule or other molecule to an immunogenic polypeptide, such as the mutant disclosed herein. The connection may be by chemical or recombinant means.

[0124] In some embodiments of the present application, 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.

[0125] In some embodiments of the present application, the C-terminus of the F1 polypeptide of the mutant is connected to a tag fragment and / or an aggregation motif.

[0126] In some embodiments of the present application, the tag fragment includes a 6His polypeptide.

[0127] In some embodiments of the present application, the aggregation motif is as shown in SEQ ID NO. 48.

[0128] In some embodiments of the present application, the F2 polypeptide of the mutant has the amino acid sequence shown in SEQ ID NO. 49 or has at least 80% (at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) homology with the amino acid sequence shown in SEQ ID NO. 49. Optionally, the F2 polypeptide of the mutant has the amino acid sequence shown in SEQ ID NO. 49, SEQ ID NO. 76 or SEQ ID NO. 77. Further optionally, the F2 polypeptide of the mutant satisfies one or more of the following conditions: 1) The C-terminus does not contain NN, and 2) has the following mutation: P102A. For example, the F2 polypeptide of the mutant has the amino acid sequence shown in SEQ ID NO. 50.

[0129] In some embodiments of the present application, the amino acid sequence of the mutant is as shown in any one of SEQ ID NO.12 to SEQ ID NO.35, SEQ ID NO.37, SEQ ID NO.39, SEQ ID NO.42 to SEQ ID NO.47, and SEQ ID NO.53 to SEQ ID NO.73.

[0130] The mutant of the RSV prefusion F protein provided by the embodiments of the present application has a stable conformation and can trigger an immune response in a subject as an antigen / immunogen to produce antibodies.

[0131] The subject is an animal. An animal is a living multicellular vertebrate or invertebrate organism, and the categories include, for example, mammals. The term mammal includes human mammals and non-human mammals. Similarly, the term "subject" includes human and veterinary subjects, such as non-human primates. Thus, administration to a subject can include administration to a human subject. Non-limiting examples of veterinary subjects include domestic animals (such as cats and dogs), livestock (such as cattle, horses, pigs, sheep, and goats), and laboratory animals (such as mice, rabbits, rats, gerbils, guinea pigs, and non-human primates).

[0132] Antibody: A polypeptide that is substantially encoded by one immunoglobulin gene or multiple immunoglobulin genes or fragments thereof in nature and specifically binds to and recognizes an analyte (such as an antigen or immunogen), such as the RSV F protein or its antigenic fragment. Immunoglobulin genes include κ, λ, α, γ, 6, ε, and μ constant region genes, as well as numerous immunoglobulin variable region genes. As used herein, the term "antibody" includes, for example, antibody fragments produced by modification of intact antibodies and de novo synthesis using recombinant DNA methods.

[0133] Antibodies exist, for example, in the form of intact immunoglobulins and in a variety of well-characterized antibody fragments. For example, Fab, Fv, and single-chain Fv (scFv) that bind to the RSVF protein would be RSVF protein-specific binding agents. This includes intact immunoglobulins and variants and portions thereof well known in the art, such as Fab′ fragments, F(ab)′2 fragments, single-chain Fv proteins (“scFv”), and disulfide-stabilized Fv proteins (“dsFv”). An scFv protein is a fusion protein in which the variable region of the light chain of an immunoglobulin is joined to the variable region of the heavy chain of the immunoglobulin by a linker, and in a dsFv, the chains have been mutated to introduce a disulfide bond to stabilize the association of the chains. The term also includes genetically engineered forms such as chimeric antibodies (e.g., humanized murine antibodies), heteroconjugate antibodies (e.g., bispecific antibodies). See also Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, IL); Kuby, J., Immunology, 3rd ed., W.H. Freeman & Co., New York, 1997.

[0134] Antibody fragments are defined as follows: (1) Fab, a fragment of 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 the 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 in which two Fab′ fragments are held together by two disulfide bonds; (5) Fv, a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain, represented as two chains; and (6) single-chain antibody (“SCA”), a genetically engineered molecule in the form of a single-chain molecule of genetic fusion containing the variable region of the light chain and the variable region of the heavy chain joined by a suitable polypeptide linker.

[0135] Typically, naturally occurring immunoglobulins have heavy (H) and light (L) chains that are linked to each other by disulfide bonds. There are two types of light chains, λ and κ. There are five main heavy chain classes (or isotypes) that determine the functional activity of the antibody molecule: IgM, IgD, IgG, IgA, and IgE. The disclosed antibodies can be class switched.

[0136] Each heavy and light chain contains a constant region and a variable region (the regions are also referred to as "domains"). In some embodiments, the heavy and light chain variable domains combine to specifically bind an antigen. In other embodiments, only the heavy chain variable domain is required. For example, naturally occurring camel antibodies consisting only of heavy chains are functional and stable in the absence of light chains (see, e.g., Hamers-Casterman et al., Nature, 363:446-448, 1993; Sheriff et al., Nat. Struct. Biol., 3:733-736, 1996). The light and heavy chain variable domains contain "framework" regions interrupted by three hypervariable regions (also referred to as "complementary determining regions" or "CDRs") (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, U.S. 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 regions of an antibody, i.e., the combined framework regions of the component light and heavy chains, are used to position and align the CDRs in three-dimensional space.

[0137] The CDRs are primarily responsible for binding to the epitope of an 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; the "Kabat" numbering scheme), Al-Lazikani et al. (JMB 273, 927-948, 1997; the "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; the "IMGT" numbering scheme).

[0138] The CDRs of each chain are commonly referred to as CDR1, CDR2, and CDR3 (from the N-terminus to the C-terminus), and are also typically identified by the chain in which a particular CDR is located. Thus, VH CDR3 is located in the variable domain of the heavy chain of the antibody in which it is present, while VL CDR1 is the CDR1 from 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.

[0139] Antigen: A compound, composition, or substance that can stimulate the production of an antibody or T cell response in an animal, including compositions that are injected or absorbed into the animal. An antigen reacts with the products of specific humoral or cellular immunity, including those induced by heterologous antigens, such as mutants of the RSV prefusion F protein disclosed herein. Examples of antigens include (but are not limited to) polypeptides, peptides, lipids, polysaccharides, combinations thereof (such as glycopeptides), and nucleic acids containing antigenic determinants, such as those recognized by immune cells. In some examples, the antigen includes a peptide derived from a pathogen of interest, such as RSV. In a specific example, the antigen is derived from RSV, such as an antigen comprising a modified RSV F protein stabilized in the prefusion conformation. An "epitope" or "antigenic determinant" refers to the region of an antigen that reacts with B and / or T cells.

[0140] Immunogen: A protein or a portion thereof that is capable of inducing an immune response in a mammal, such as a mammal infected with a pathogen or at risk of being infected 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 mutant provided in the examples of the present application.

[0141] 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 ("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.

[0142] One of ordinary skill in the art will recognize that individual substitutions, deletions or additions which alter, add or delete a single amino acid or a small percentage of amino acids in an encoding sequence (e.g., less than 20%, 15, 10, 5%, etc.) are conservative variations where the alteration results in substitution of an amino acid with a chemically similar amino acid. Conservative amino acid substitutions which provide functionally similar amino acids are well known in the art. The following six groups each contain amino acids which are conservative substitutions for one another: 1) alanine (A), serine (S), threonine (T); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); and

[0182] 6) phenylalanine (F), tyrosine (Y), tryptophan (W).

[0143] Not all residue positions within a protein will tolerate an originally "conservative" substitution. For example, if an amino acid residue is essential for protein function, then even an originally conservative substitution may disrupt the activity, e.g., the specific binding of an antibody to a target epitope may be disrupted by a conservative mutation in the target epitope.

[0144] Epitope: Antigenic determinant. These are specific chemical groups or peptide sequences on an antigenic molecule such that they induce a specific immune response, e.g., an epitope is an antigenic region that reacts with B and / or T cells. Antibodies bind to specific antigenic epitopes, e.g., epitopes of the RSVF protein, e.g., the D25 or AM22 epitopes present on the prefusion conformation of the RSVF protein. Epitopes can be formed by contiguous amino acids or non-contiguous amino acids juxtaposed by the tertiary folding of a protein. Epitopes formed by contiguous amino acids generally remain exposed to denaturing solvents, while epitopes formed by tertiary folding generally are lost upon treatment with denaturing solvents. Epitopes generally include at least 3 and more usually 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 can also include post-translational modifications of amino acids, such as N-linked glycosylation.

[0145] A "target epitope" is a specific epitope on an antigen that specifically binds an antibody of interest, such as a monoclonal antibody. In some instances, 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.

[0146] The amino acids in a peptide, polypeptide, or protein are usually chemically bonded together via amide bonds (CONH). Additionally, amino acids can be bonded 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, edited by B. Weinstein, Marcel Dekker, New York, page 267 (1983); Spatola, A.F., Vega Data (March 1983), Volume 1, Issue 3, Peptide Backbone Modifications (general review); Morley, Trends Pharm Sci pages 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. 1 307-314, 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).

[0147] Peptide modification: Peptides such as the pre-fusion conformation-stabilized mutants of this application's examples can be modified, for example, to include amino acid substitutions compared to the native RSV protein sequence, or by various chemical techniques to produce derivatives having substantially the same activity and conformation as the unmodified peptide and optionally having other desired properties. For example, the carboxylic acid groups of a protein, whether at the carboxyl terminus or side chain, can be provided in the form of a salt with a pharmaceutically acceptable cation or esterified to form a C1-C16 ester, or converted to an amide of the formula NR1R2, where R1 and R2 are each independently H or a C1-C16 alkyl, or combined to form a heterocycle, such as a 5- or 6-membered ring. The amino groups of a peptide, whether at the amino terminus or side chain, can be in the form of a pharmaceutically acceptable acid addition salt such as HCl, HBr, acetate, benzoate, tosylate, maleate, tartrate, and other organic salts, or can be modified to a C1-C16 alkyl or dialkylamino or further converted to an amide.

[0148] The hydroxyl groups of the peptide side chains can be converted to C1-C16 alkoxy groups or C1-C16 esters using well-known 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. The thiols can be protected with any of a variety of well-known protecting groups such as acetamide groups.

[0149] The second aspect of the embodiments of the present application , An embodiment of the present application provides a nucleic acid molecule that encodes a mutant of the prefusion F protein of RSV described in the first aspect.

[0150] Nucleic acid molecule, nucleic acid: A polymer composed of nucleotide units (ribonucleotides, deoxyribonucleotides, related naturally occurring structural variants, and their synthetic non-naturally occurring analogs) linked by phosphodiester bonds, related naturally occurring structural variants, and their synthetic non-naturally occurring analogs. Thus, the term includes nucleotide polymers in which the nucleotides and the linkages between them include non-naturally occurring synthetic analogs such as, and not limited to, phosphorothioates, phosphoroamidates, methylphosphonates, chiral methylphosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs), etc. These polynucleotides can be synthesized, for example, using an automated DNA synthesizer. The term "oligonucleotide" generally refers to short polynucleotides that are usually no greater than about 50 nucleotides. It should be understood that when a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), this also includes an RNA sequence in which "U" replaces "T" (i.e., A, U, G, C).

[0151] "Nucleotide" includes (but is not limited to) monomers that include a base linked to a sugar (e.g., pyrimidine, purine, or their synthetic analogs) or monomers that include a base linked to an amino acid (such as in peptide-nucleic acid (PNA)). A nucleotide is a monomer in a polynucleotide. A nucleotide sequence refers to the sequence of bases in a polynucleotide.

[0152] "Encoding" refers to the inherent property of a specific sequence of nucleotides in a polynucleotide, such as a gene, cDNA, or mRNA, which serves as a template for the synthesis of other polymers and macromolecules in biological processes, having nucleotides of a defined sequence (e.g., rRNA, tRNA, and mRNA) or amino acids of a defined sequence and the resulting biological properties. Thus, if the transcription and translation of mRNA produced by a gene result in a protein in a cell or other biological system, the gene encodes the protein. The coding strand, whose nucleotide sequence is identical to the mRNA sequence and is usually provided in the form of a sequence listing, and the non-coding strand that serves as the transcription template for the gene or cDNA can be said to encode the protein or other products of the gene or cDNA. Unless otherwise stated, "nucleotide sequences encoding an amino acid sequence" includes all nucleotide sequences that are degenerate to each other and encode the same amino acid sequence. Nucleotide sequences encoding proteins and RNAs may include introns. In some examples, the nucleic acid encodes mutants of the present application.

[0153] Considering degenerate variants and conservative variants, the present application does not make specific limitations on nucleic acid molecules.

[0154] Degenerate variants and conservative variants: Polynucleotides encoding polypeptides including sequences that are degenerate due to the genetic code. For example, polynucleotides encoding the disclosed antigens or antibodies that specifically bind to the disclosed antigens, including sequences that are degenerate due to the genetic code. There are 20 natural amino acids, most of which are specified by more than one codon. Thus, all degenerate nucleotide sequences are included, as long as the amino acid sequence of the antigen or antibody that binds the antigen encoded by the nucleotide sequence remains unchanged. Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given polypeptide. For example, the codons CGU, CGC, CGA, CGG, AGA, and AGG all encode the amino acid arginine. Thus, at each position specifying arginine within a protein-coding sequence, the codon can be changed to any one of the corresponding codons without changing 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 each possible silent variation. Those skilled in the art will recognize that each codon in a nucleic acid (except AUG, which is usually the only codon for methionine) can be modified by standard techniques to produce a functionally identical molecule. Thus, each "silent variation" of a nucleic acid encoding a polypeptide is implicit in each such sequence.

[0155] In some examples, it is codon-optimized for expression in mammalian cells and is operably linked to a promoter.

[0156] Expression control sequence: A nucleic acid sequence that regulates the expression of an operably linked heterologous nucleic acid sequence. An expression control sequence is operably linked to a nucleic acid sequence when it controls and regulates the transcription and, where appropriate, translation of the nucleic acid sequence. Thus, an expression control sequence can include a suitable promoter, enhancer, transcription terminator, start codon (ATG) preceding a protein-coding gene, splicing signals for introns, maintaining the proper reading frame of the gene to allow proper translation of the mRNA, and a stop codon. The term "control sequence" is intended to minimally include components whose presence can affect expression and can also include other components whose presence is advantageous, such as, leader sequences and fusion partner sequences. An expression control sequence can include a promoter.

[0157] A promoter is the minimal sequence sufficient to direct transcription. Also included are those promoter elements sufficient to render promoter-independent gene expression controllable for cell-type specificity, tissue-specificity or inducible by external signals or agents; these elements can be located in the 5' or 3' regions of the gene. Constitutive and inducible promoters are included (see, for example, Bitter et al., Methods in Enzymology 153:516-544, 1987). For example, when cloning in a bacterial system, inducible promoters such as pL of phage λ, plac, ptrp, ptac (ptrp-lac hybrid promoter), etc. can 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 repeat; adenovirus late promoter; vaccinia virus 7.5K promoter) can be used. Promoters generated by recombinant DNA or synthetic techniques can also be used to provide transcription of the nucleic acid sequence.

[0158] A 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.

[0159] 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 are known in the art (see, for example, Tan et al., PLOS one, 7: e51439, 2011; Sambrook et al., Molecular Cloning, a Laboratory Manual, 2nd Edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989); Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, New York, N.Y. (1994)).

[0160] The third aspect of the embodiments of the present application , embodiments of the present application provide a vector, which comprises the nucleic acid molecule described in the second aspect.

[0161] The fourth aspect of the embodiments of the present application , embodiments of the present application provide a genetically engineered cell that expresses a mutant of the prefusion F protein of RSV described in the first aspect, or that comprises the nucleic acid molecule described in the second aspect, or the vector described in the third aspect.

[0162] Expression: the translation of nucleic acid into protein. The protein can be expressed and retained within the cell, become a component of the cell surface membrane, or be secreted into the extracellular matrix or culture medium.

[0163] Genetically engineered cell, host cell: a cell in which a vector can replicate 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 parental cell because mutations may occur during replication. However, such progeny are included when the term "host cell" is used.

[0164] The fifth aspect of the embodiments of the present application , embodiments of the present application provide a method for producing the mutant of the prefusion F protein of RSV described above, which comprises the following steps:

[0165] culturing the genetically engineered cell described in the fourth aspect, and isolating the mutant of the prefusion F protein of RSV from the resulting culture supernatant.

[0166] The sixth aspect of the embodiments of the present application , embodiments of the present application provide an immunocomposition, which comprises the mutant of the prefusion F protein of RSV described in the first aspect or the nucleic acid molecule described in the second aspect, and an immunoadjuvant.

[0167] Immunoadjuvant: A mediator used to enhance antigenicity. Adjuvants include suspensions of minerals (alum, aluminum hydroxide or phosphate) adsorbed with antigens; or water-in-oil emulsions, for example, in which an antigen solution is emulsified in mineral oil (Freund's incomplete adjuvant), sometimes including heat-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 biomolecules ("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. Those of ordinary skill in the art are familiar with adjuvants (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 embodiments of the present application are one or more of aluminum salt adjuvants, surfactants, polynucleotides, lipopolysaccharides, liposomes, and oil emulsion adjuvants. For example, Alum, CpG, Alum + CpG, MF59, AS04, AS01E, etc. MF59 is a water-miscible adjuvant mainly composed of three parts: 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 can uniformly mix the oil phase and water; the adjuvant mainly includes humectants and buffers such as glycerol, threitol, and ATP. The AS04 adjuvant is a mixture composed of the AS03 adjuvant and the MPL adjuvant. The AS03 adjuvant is a mixture composed of liposomes, TWEEN80, and sorbitan, three surfactants. The AS01E is a nanoscale liposome solution prepared from DOPC, Chol, MPL, and QS-21. The main component of the MPL adjuvant is lipopolysaccharide.

[0168] Immune composition (immunogenic composition): A composition comprising an antigen that induces an immune response, such as a measurable CTL response against a virus expressing the antigen or a measurable B cell response against the antigen (e.g., antibody production). Thus, an immunogenic composition comprises one or more antigens (e.g., polypeptide antigens) or epitopes. The immunogenic composition may further comprise one or more additional components capable of inducing or enhancing an immune response, such as excipients, carriers, and / or adjuvants. In certain cases, an immunogenic composition is administered to induce an immune response that protects a subject from symptoms or conditions induced by a pathogen. In some cases, symptoms or diseases caused by a pathogen are prevented (or reduced or ameliorated) by inhibiting replication of the pathogen after the subject is exposed to the pathogen (e.g., RSV). In one example, an "immunogenic composition" comprises a recombinant RSV F protein stabilized in the prefusion conformation that induces a measurable CTL response against a virus expressing the RSV F protein or induces a measurable B cell response against the RSV F protein (e.g., antibody production). It further refers to an isolated nucleic acid encoding an antigen, such as a nucleic acid that can be used to express the antigen (and thus to induce an immune response against such a polypeptide).

[0169] For in vitro use, the immunogenic composition may comprise an antigen or a nucleic acid encoding an antigen. For in vivo use, the immunogenic composition will generally comprise a protein, immunogenic peptide, or nucleic acid in a pharmaceutically acceptable carrier and / or other agents. The ability of any particular peptide, such as the disclosed RSV F protein stabilized in the prefusion conformation or a nucleic acid encoding the disclosed RSV F protein stabilized in the prefusion conformation, to induce a CTL or B cell response can be readily tested by well-recognized assays. The immunogenic composition may comprise adjuvants well known to those skilled in the art.

[0170] Immunological reaction conditions: Include conditions that allow antibodies produced against a particular epitope to bind to the epitope and bind to a detectably greater extent compared to substantially all other epitopes and / or substantially exclude binding to substantially all other epitopes. Immunological reaction conditions depend on the form of the antibody-binding reaction and are generally those used in immunoassay protocols or those encountered in vivo. The immunological reaction conditions used in the methods are "physiological conditions", which include conditions typical inside a living mammal or mammalian cell (e.g., temperature, osmolality, pH). While it is recognized that some organs are subject to extreme conditions, the in vivo and intracellular environments are generally about pH 7 (e.g., pH 6.0 to pH 8.0, more typically pH 6.5 to 7.5), contain water as the major solvent, and exist at temperatures above 0°C and below 50°C. The osmolality is within the range that supports cell viability and proliferation.

[0171] The seventh aspect of the embodiments of the present application, an embodiment of the present application provides an application of the mutant of the RSV prefusion F protein described in the first aspect in the preparation of a respiratory syncytial virus antibody detection kit.

[0172] In an embodiment of the present application, the respiratory syncytial virus antibody detection kit can be used to detect corresponding antibodies. The definition of the antibody refers to the first aspect and can be a neutralizing antibody or a binding antibody.

[0173] The eighth aspect of the embodiments of the present application , an embodiment of the present application provides a respiratory syncytial virus antibody detection kit, which includes the mutant of the RSV prefusion F protein described in the first aspect.

[0174] The definition of the respiratory syncytial virus antibody detection kit refers to the seventh aspect.

[0175] The ninth aspect of the embodiments of the present application , an embodiment of the present application provides a method for preventing and treating lower respiratory tract infections caused by respiratory syncytial virus, which includes the following steps: administering to a subject a therapeutically effective amount of the immunocomposition described in the sixth aspect.

[0176] Administration: introducing the composition into the subject by the selected route. Administration can be local or systemic. For example, if the selected route is intravenous, the composition is administered by introducing the composition into the vein of the subject.

[0177] Effective amount: an amount of an agent such as a PreF antigen, a nucleic acid encoding a PreF antigen, or other agent that is sufficient to produce a desired response, such as an immune response against the RSV F protein, or to reduce or eliminate the 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 external 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 that will achieve a target tissue concentration (e.g., in respiratory tissues) that has been shown to achieve in vitro inhibition of viral replication will typically be used. In some instances, an "effective amount" is an amount that treats (including preventing) one or more symptoms and / or underlying causes of any condition or disease, such as an amount that treats RSV infection. In one instance, the effective amount is a therapeutically effective amount. In one instance, the effective amount is an amount that prevents the development of one or more signs or symptoms (e.g., one or more signs or symptoms associated with RSV infection) of a particular disease or condition.

[0178] Prevention and treatment mean prophylaxis and therapy. For example, in a subject at risk of a disease (such as RSV infection), the full development of the disease or condition is inhibited. "Treatment" refers to a therapeutic intervention that improves the signs or symptoms of a disease or pathological condition after it has begun to develop. The term "improvement" with respect to a disease or pathological condition refers to any observable beneficial therapeutic effect. The beneficial effect can be demonstrated, for example, by: the delayed onset of the clinical symptoms of the disease in a susceptible subject, the reduction in the severity of some or all of the clinical symptoms of the disease, the slower progression of the disease, the improvement of the overall health or health status of the subject, or other parameters specific to a particular disease well known in the art. "Prophylactic" treatment is administered to a subject who does not exhibit signs of the disease or only exhibits early signs for the purpose of reducing the risk of developing the lesion. The term "reduce" is a relative term such that if the response or condition is quantitatively attenuated after administration of the agent, or if it is attenuated compared to a reference agent after administration of the agent, the agent reduces the response or condition. Similarly, the term "prevent" does not necessarily mean that the agent completely eliminates the response or condition, as long as at least one characteristic of the response or condition is eliminated. Thus, an immunogenic composition that reduces or prevents the immunogenicity of an infection or response (such as a pathological response, such as a vaccine-enhanced viral disease) can, but does not necessarily, completely eliminate such infection or response, as long as the infection or response is measurably attenuated compared to the infection or response in the absence of the agent or compared to a reference agent, for example, by at least about 50%, such as at least about 70%, or about 80%, or even about 90% (i.e., reduced to 10% or less).

[0179] In the tenth aspect of the embodiments of the present application , embodiments of the present application provide a method for detecting or isolating RSV F-binding antibodies in a sample, which comprises the following steps:

[0180] Contacting the mutant of the prefusion F protein of RSV described in the first aspect with RSV F-binding antibodies in the sample to form an immune complex; and,

[0181] Detecting the immune complex, thereby achieving the detection or isolation of RSV F-binding antibodies in the sample.

[0182] The embodiments of the present application will be described in detail below in conjunction with the examples. It should be understood that these examples are only used to illustrate the present application and not to limit the scope of the present application. For the experimental methods without specific conditions noted in the following examples, the guidance given in the present application is preferably referred to, and it can also be carried out according to the experimental manuals or conventional conditions in the art, or according to the conditions recommended by the manufacturers, or by referring to the experimental methods known in the art.

[0183] In the following specific embodiments, regarding the measurement parameters of raw material components, if there is no special instruction, there may be slight deviations within the weighing accuracy range. Regarding temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

[0184] Example 1

[0185] 1. Design of RSV pre-F mutant

[0186] This example relates to the respiratory syncytial virus F0 protein, and its structural characteristics are as Figure 1 shown. Figure 1 Described is the structure of the wild-type RSV virus F protein precursor polypeptide, corresponding to the amino acid sequences SEQ ID NO.1 (human wild-type RSV A, STRAIN A2, 574aa), SEQ ID NO.2 (human wild-type RSV B, STRAIN 18537, 574aa), and SEQ ID NO.3 (bovine wild-type RSV B, STRAIN A51908, 572aa).

[0187] For the native full-length human RSV A2, the precursor polypeptide includes a signal peptide (aa1-25, as shown in SEQ ID NO.7), an F2 polypeptide (aa26-109, as shown in SEQ ID NO.5), a pep27 polypeptide (aa110-136, as shown in SEQ ID NO.6), an Fl polypeptide (aa137-574, as shown in SEQ ID NO.4), and the furin cleavage sites are RARR and KKRKRR. The structural division of the native full-length human RSV B is consistent with that of the native full-length human RSV A2.

[0188] Relative to the precursor polypeptides of the native full-length human RSV A2 and human RSV B, the precursor polypeptide of the native full-length bovine RSV B is 572aa in length, and the Fl polypeptide corresponds to aa137-572aa, containing the furin cleavage sites RAKR and KKRKRR.

[0189] The α3 helices in the native full-length human RSV A2 precursor polypeptide and the human RSV B precursor polypeptide are as shown in SEQ ID NO.8 and SEQ ID NO.53 respectively, and the α3 helix in the native full-length bovine RSV B precursor polypeptide is also as shown in SEQ ID NO.53, and the β3 sheets of the three native full-length precursor polypeptides are all as shown in SEQ ID NO.9.

[0190] Table 1

[0191] Type α3 helix β3 sheet Human RSV A2 <![CDATA[SEQ ID NO.8: EVNKIK S ALLS]]> SEQ ID NO.9: KAVVSL Human RSV B <![CDATA[SEQ ID NO.78: EVNKIK N ALLS]]> SEQ ID NO.9: KAVVSL Bovine RSV B <![CDATA[SEQ ID NO.78: EVNKIK N ALLS]]> SEQ ID NO.9: KAVVSL

[0192] The native RSV F protein is first expressed as the F0 polypeptide (precursor). After the endoplasmic reticulum translocation signal peptide of the F0 polypeptide is cleaved, it is processed by an intracellular furin-like protease at two sites, the pep27 polypeptide is excised, and the mature F protein contains the F2 polypeptide at the N-terminus and the F1 polypeptide at the C-terminal part. The F1 polypeptide is linked to the F2 polypeptide by two disulfide bonds. The mature F protein forms a trimer and is anchored to the cell membrane via the F1 polypeptide through a transmembrane domain.

[0193] Taking the native full-length human RSV precursor polypeptide as an example, the pre-F mutants of the RSV F protein are designed as shown in Figure 2 and Figure 3 follows:

[0194] (1) Delete its P27 polypeptide and use GS as a linker to connect the F1 and F2 polypeptides;

[0195] (2) Delete the transmembrane domain and intracellular domain of the protein to enable soluble expression of the mutant;

[0196] (3) Introduce a trimerization motif at the C-terminus of the monomeric protein of the mutant, which can enable the mutant to be recombinantly secreted and expressed in the form of a trimer;

[0197] (4) Add a His tag at the C-terminus to facilitate the purification and preparation of the mutant.

[0198] There is an allosteric process of the RSV F protein from the pre-F conformation to the PostF conformation. During the allosteric process, the N-terminus and C-terminus of the F1 polypeptide allosterically become upward long α-helices. By analyzing its structure, it is found that introducing a disulfide bond between its α3 helix and β3 sheet can obtain a stabilized pre-F conformation.

[0199] Table 2

[0200]

[0201] Figure 2Describes the structural schematic diagram of the stabilized mutant RSV F protein polypeptide monomer for construction (amino acid sequence is SEQ ID NO.10). This schematic diagram corresponds to the natural mutant of RSV / A2 subtype (amino acid sequence is SEQ ID NO.1), where the positions of three natural mutations are P102A, I379V, and M447V respectively. Unless otherwise specified, other mutations involved in this application are designed based on this. In the implementation cases of this application, the mutants involved are based on this for amino acid mutations. Different from the wild-type F protein, the pep27 polypeptide is replaced by GS, not affected by furin protease, and at the same time the transmembrane domain is deleted, and a His-Tag is added to the C-terminus of the protein to facilitate purification. Such mutants exist in the form of soluble monomers in the cell culture supernatant after expression.

[0202] Figure 3 Describes the structural schematic diagram of the stabilized mutant RSV F protein polypeptide trimer for construction (amino acid sequence is SEQ ID NO.11). Different from the wild-type, the pep27 peptide is replaced by GS and is not affected by furin protease. Preferably, the mutant RSV F protein of this application forms a trimer to mimic the natural trimer state of the F protein. Therefore, the trimerization motif (T4foldon) of phage T4 fibritin is fused to its C-terminus, and at the same time a purification tag is added to this C-terminus. Such mutants exist in the form of soluble trimers in the cell culture supernatant after expression.

[0203] Figure 4 It is described that there are a total of 7 pairs of disulfide bonds in the natural RSV F0 precursor protein or mature F protein, which play an important role in stabilizing the structural characteristics of the RSV F protein. Among them, the F2 polypeptide and the F1 polypeptide are linked together by two pairs of disulfide bonds (C37 / C439; C69 / C212), which play a key role in stabilizing the epitope at the RSV pre-F apex. The remaining 5 pairs of disulfide bonds are distributed in the F1 polypeptide to facilitate the stabilization of the unique structural characteristics of the F protein. This application makes mutations at other positions on the basis of maintaining the natural disulfide bonds of the RSV F protein, so that it maintains the pre-fusion conformation (pre-F) without changing the basic structure of the RSV F protein.

[0204] Table 3. Disulfide bond pairing naturally present in wild-type RSV F0 protein

[0205] Serial number Disulfide bond pairing disulf1 C37-C439 disulf2 C69-C212 disulf3 C313-C343 disulf4 C322-C333 disulf5 C358-C367 disulf6 C382-C393 disulf7 C416-C422

[0206] Figure 5Describes the three-dimensional structural schematic diagram of the RSV F protein monomer (left) and the partial enlarged schematic diagram located in the α3 and β3 sheets (right). The schematic diagram shows that the α3 and β3 sheets are antiparallel and close in distance. The RSV pre-F conformation is metastable and undergoes irreversible rearrangement to mediate membrane fusion or the non-functional post-F conformation when spontaneously triggered. Among them, the allosteric formation of a long coiled-coil bundle by α2, α3, β3, β4, α4, and α5 in the sequence is a typical feature of forming post-F. Therefore, preventing its allostery through amino acid mutations is an important means to maintain the stabilized pre-fusion conformation. In this application, we found that a disulfide bond formed by appropriate amino acid mutations between the α3 helix and the β3 sheet can produce a stabilized pre-F conformation, and the mutant combination formed based on this can further improve the stability of the pre-F mutant.

[0207] Table 4. Amino acids contained in the α3 helix and β3 sheet of the RSV F0 protein

[0208] Position Amino acid residue α3(163aa-173aa) 163E / 164V / 165N / 166K / 167I / 168K / 169S / 170A / 171L / 172L / 173S β3(176aa-181aa) 176K / 177A / 178V / 179V / 180S / 181L

[0209] Figure 6 Describes the amino acids involved in the α3 and β3 positions in this application and the distance between two amino acid atoms that are prone to form a disulfide bond

[0210] Table 5. Mutation sites in the α3 helix, β3 sheet, and other regions of each mutant

[0211]

[0212]

[0213] In the table, " / " indicates that the mutations S55C, S155C, S180C, S186C, L188C, S190F, V207L, and S290C are not present.

[0214] 2. Vector construction of the RSV pre-F mutant

[0215] According to the mutation strategy, the amino acid sequence of the mutant is determined, and the corresponding nucleic acid sequence is codon-optimized. The optimized codons are beneficial for expression in Chinese hamster ovary cells Cricetulus griseus (CHO cells). Finally, it is ligated to the pcDNA3.1 vector through the BamHⅠ and XhoⅠ restriction enzyme sites.

[0216] The vector substitution, insertion, deletion, etc. were carried out using the ClonExpress II Recombinant Cloning Kit (Novoprotein Scientific Inc.). The two fragments at both ends of the mutation site were amplified by polymerase chain reaction (PCR) with the high-fidelity enzyme Phanta Max (Novoprotein Scientific Inc.). After the amplification products were recovered by gel extraction, the two fragments were subjected to fusion PCR. The recovered fusion fragment was homologously recombined onto the pcDNA3.1 vector digested with BamHⅠ and XhoⅠ. The constructed point mutation vector was sequenced by Tsingke Biotechnology Co., Ltd. Beijing and the sequence was confirmed to be correct.

[0217] The cloning vector was inoculated into 300 mL of LB (Amp+) medium and cultured at 37 °C and 180 rpm for 16 h. The plasmid was extracted using a large / large plasmid extraction kit; finally, it was stored in 1 mL of sterile TE Buffer. For all commercial kits or reagents, the manufacturer's instructions were followed.

[0218] 3. Expression of RSV pre-F mutant

[0219] The mutant protein was expressed using the ExpiCHO TM expression system (Thermofisher). Transient transfection and expression were carried out completely according to the manufacturer's standard protocol. Briefly, one day before transfection (day –1), the ExpiCHO-S TM culture was seeded, and the final density was 3×10 6 ~4×10 6 viable cells / mL. The next day (day 0), the viable cell density and the percentage of viability were measured. The cell density should reach approximately 7×10 6 –10×10 6 viable cells / mL. The viability should be 95–99% to proceed with transfection. The cells were diluted to a final density of 6×10 TM viable cells / mL using fresh pre-warmed (37 °C) ExpiCHO 6 expression medium. The culture flask was gently shaken to mix the cells, and the remaining cells were discarded. The ExpiFectamine TM CHO / plasmid DNA complex was prepared according to the operating sequence of the reagent instructions using cold reagents (4 °C). After inverting and mixing, the ExpiFectamineTM CHO / DNA complex was added to the shake flask, and the cells were placed on a shaker (8% CO2, 37 °C, 120 rpm). One day after transfection (day 1, 18–22 hours after transfection), ExpiFectamine TM CHO enhancer and ExpiCHO TM supplements were added. The culture was terminated on day 8 after transfection according to the standard test protocol, and samples were taken for detection.

[0220] 4. Purification of RSV pre-F Mutant

[0221] Protein purification was completed in two steps using affinity chromatography and ion exchange chromatography. Generally, the expression product was centrifuged (8000 rpm, 20 min) to harvest the culture supernatant and remove cells and cell debris. The culture supernatant was filtered through a 0.45 μm filter membrane to remove impurities. The treated supernatant was added to a Ni-Sepharose 6FF column (Cytiva) equilibrated with a buffer (Buffer A; pH 8.0) containing 25 mM Tris-HCl and 0.15 M NaCl. Then, it was further rinsed with 25 mM Tris-HCl, 150 mM NaCl, pH 8.0 until the A280 reached the baseline level, and eluted linearly with an elution buffer (Buffer B: 25 mM Tris-HCl, 150 mM NaCl, 500 mM imidazole, pH 8.0) from 0 to 100%. The eluate was collected and the imidazole was removed using the equilibration buffer as a replacement solution.

[0222] After the buffer replacement was completed, the sample was diluted with PBS buffer to make its conductivity less than 4 ms / cm and the pH was adjusted to 6.0. The protein was loaded onto a Capto SP Impres packing material (Cytiva) equilibrated with 20 mM phosphate buffer pH 6.0. After loading, it was eluted with 20 mM phosphate buffer pH 6.0 until the A280 reached the baseline level, and then eluted linearly with an elution buffer (20 mM phosphate buffer, 1 M NaCl pH 6.0) from 0 to 100% of Buffer B. The eluate was collected. The pH was adjusted to 7.4 - 8.0 with 0.2 M Na2HPO4, and further insoluble particles were removed by filtration through a 0.45 μm filter membrane.

[0223] 5. SDS-PAGE Identification of RSV pre-F Mutant

[0224] The purified recombinant protein was detected 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 dithiothreitol or mercaptoacetic acid), loaded after boiling water bath treatment. After electrophoresis, the gel was stained for proteins using the Coomassie blue staining method, and the gel image was obtained using a transparent scanner or a protein imaging system.

[0225] Figure 7Describes the SDS-PAGE identification results of mutant monomers and trimers based on mutations at the α3 / β3 position. Since the mutants only differ from the wild-type F protein by amino acid substitutions and the molecular weights of different mutant proteins are basically the same and show the same pattern on SDS-PAGE, only JW-05 is used to represent the mutant protein monomer. Similarly, JW-05-T4 is used to represent the mutant trimer. Judging from the results, the monomers and trimers are slightly larger than the theoretical molecular weights (51KDa / 54KDa), probably due to glycosylation of the mutants.

[0226] 6. HPLC detection of the purity and homogeneity of the mutants

[0227] 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 was 20 mM PBS buffer with a flow rate of 1 mL / min, and the elution composition was detected at 280 nm. HPLC was used to detect the purity and homogeneity of the target protein.

[0228] Figure 8 Describes the HPLC detection results of mutant monomers and trimers. Among them, Figure 8 a shows that the JW-05 monomer protein has a characteristic peak at 18.731 min. Figure 8 b shows that the trimer JW-05-T4 has a characteristic peak at 15.958 min, and the target protein is single without protein aggregation.

[0229] 7. Detection of the content of RSV pre-F mutants by the double antibody sandwich method

[0230] The RSV pre-F mutants were quantified by the double antibody sandwich ELISA method. Palivizumab, which can recognize pre-F, post-F, and F proteins simultaneously, was used as the coating antibody, and a specific antibody was used to recognize RSV pre-F Detection was performed using the site-specific monoclonal antibody D25 labeled with horseradish peroxidase (D25-HRP) as the detection antibody. At the same time, the purified pre-F monomer protein and trimer protein were used as calibration products to establish a standard curve, and the content of the sample to be detected was obtained by measuring its absorbance at OD450 and dilution factor through the standard curve conversion. By this method, the expression level of the mutant pre-F protein under transient transfection and the residual content of the pre-F mutant protein in the mutant thermal stability test can be detected. The sample detection process is as follows: The monoclonal antibody was diluted to 1 μg / L using carbonate buffer and coated on an enzyme-linked immunosorbent assay (ELISA) plate (Corning 9018). 100 μL was added to each well and incubated at 37 °C for 1 h, then at 2-8 °C overnight. The liquid in the 96-well plate was discarded and washed 3 times with 20 mM 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 min. The blocking solution in the wells was aspirated, and the wells were washed 3 times with 20 mM PBS-T solution. The mutated pre-fusion conformation F protein after a series of 3-fold dilutions was added to the wells in the first column of the 96-well ELISA plate. The negative control was PBS, and the reaction was carried out at 37 °C for 60 min. The blocking solution in the wells was aspirated, and the wells were washed 3 times with 20 mM PBS-T solution. The anti-HIS-HRP conjugate was diluted 1:2000 with the enzyme conjugate diluent and then added to the 96-well ELISA plate, 100 μL per well, and the reaction was carried out at 37 °C for 10 min. The secondary antibody in the wells was aspirated, and the wells were washed 3 times with 20 mM PBS-T solution. 100 μL of TMB chromogenic solution was added to each well, and after 10 min, 50 μL of the stop solution was added to terminate the reaction. Then, the absorbance at A450 was measured using an ELISA reader.

[0231] Figure 9 Shows the expression level of the pre-F protein in the cell culture supernatant after transient transfection of the monomeric RSV pre-F mutant protein in Expi-CHO cells. In this example, the results showed that a relatively low level of pre-F protein could also be detected in the culture supernatant of transient transfection of the native F protein (WT). When the p27 peptide was deleted and replaced with GS (F0-GS, SEQ ID NO: 10), the content of the pre-F protein increased. When a cysteine substitution was introduced at the α3 / β3 position of the F0 protein, the mutant significantly increased the content of the pre-F protein under the action of the formed disulfide bond bridge, indicating that the disulfide bond formed at the α3 / β3 position prevented the conformational change of the F protein to the post-F conformation, keeping the F protein in the pre-fusion conformation.

[0232] Figure 10The remaining percentages of the pre-F protein after heat treatment at 50 °C for 1 h, 2 h, and 3 h were shown when the concentration of the purified monomer mutant protein was 0.1 mg / mL in a pH 7.4 buffer system containing 20 mM PB and 150 mM NaCl. The results showed that although the pre-F protein could be detected in WT and F0-GS, its stability was very poor. Compared with WT and F0-GS, the mutants formed at the α3 / β3 position significantly improved the stability of the pre-F protein.

[0233] Figure 11 The results in showed that disulfide bonds formed at the α3 / β3 position, in combination with mutations at other positions, could also form stable pre-F proteins, although different combinations led to significant differences in the content of the pre-F protein. When trimerized with the T4 foldon motif, the expression level of the pre-F protein decreased compared with the monomer expression level.

[0234] Figure 12 The results in showed that the mutant trimers generated based on the mutations at the α3 / β3 position had higher thermal stability than the monomers. In particular, the mutant combinations based on the mutations at the α3 / β3 position had better thermal stability than the single disulfide bond mutation at the α3 / β3 position, indicating that mutations at the α3 / β3 position provided a very promising possibility for generating stabilized pre-F.

[0235] Figure 13 and Figure 14 The results in showed that the purified pre-F mutants could be recognized by the specific monoclonal antibodies D25 and AM22 against the site in the pre-F protein.

[0236] Figure 15 The results in showed that although different mutants could form pre-F trimer proteins, significant differences were found in the trimerization of pre-F protein mutants formed by mutations at different positions when detected by the pre-F protein trimer-specific monoclonal antibody AM14. The reason was that the mutations at different positions caused subtle structural differences, resulting in conformational changes in the protein after trimerization and inability to be recognized by the AM14 monoclonal antibody. The AM14 monoclonal antibody could only recognize the trimeric pre-F protein, and this result confirmed that the trimeric pre-F mutants could be correctly assembled.

[0237] 8. Immunogenicity evaluation of RSV pre-F protein

[0238] To evaluate the immunogenicity of RSV F in different pre-fusion conformations, female Balb / c mice weighing 14 - 16 g were immunized with different mutant proteins. 5 μg of vaccine antigen was added with aluminum hydroxide as an adjuvant to immunize the female Balb / c mice. The inactivated RSV vaccine (FI-RSV) group was added as a control. Intramuscular injection was performed at week 0 and week 4 (28 d). Serum was collected 2 weeks (42 d) after the second immunization to measure the total IgG antibody titer and neutralizing antibody titer. The immunization groups are shown in the following table:

[0239] Table 6

[0240]

[0241] 9. Detection of antigen-specific antibody levels

[0242] The total IgG antibody was detected by the indirect ELISA method. The Pre F protein was pre-coated in a 96-well plate. After blocking, the serum to be tested was added. The serum to be tested was serially diluted, with the starting dilution factor of 800-fold, and then diluted three-fold. After washing to remove the unbound serum, HRP-labeled goat anti-mouse secondary antibody was added for incubation. After the incubation, the unbound secondary antibody was removed by washing. After washing, the substrate was added for color development. The serum titer of the mice was judged by detecting the absorbance at 450 nm and 630 nm. The dilution titer was calculated according to the serum dilution factor of the last well with the absorbance greater than the CUT-OFF value after dilution.

[0243] BalB / c mice were immunized with different mutant trimeric proteins added with aluminum hydroxide adjuvant, and the inactivated vaccine was used as a control at the same time. The antigen-specific IgG antibody level in the mouse serum was detected using the pre-F mutant trimer as the coating antigen.

[0244] Figure 16 The results showed that the five pre-F mutant trimers had good immunogenicity and could induce a high level of antibody response. In particular, the mutant trimer generated by the mutation combination at the α3 / β3 position induced a higher antibody level than the single mutation. The reason was that the difference in the position of the mutant caused differences in the antigen structure, which in turn led to changes in protein stability. The final result was that the immunogenicity of the protein was affected. Although there were almost no antigenic epitopes against the site in the inactivated vaccine, there were still antibodies against other antigenic epitopes in the immune serum of the inactivated vaccine.

[0245] 10. Detection of neutralizing antibody levels

[0246] The neutralizing antibody titer was detected by the plaque method. First, prepare a 24-well plate filled with a monolayer of Hep2 cells; heat inactivate the serum in a 56 °C water bath for 30 min; after inactivation, cool the serum to room temperature and dilute the serum with EMEM serum-free medium in proportion (1:32, 1:64, 1:128, 1:256, 1:512, 1:1024, 1:2048); take 100 μL of virus solution with a titer of 50 pfu and mix it evenly with the serum dilution, and incubate it in a 37 °C incubator for 1 h; remove the culture medium in the ppC VBNM well plate, wash it 2 times with sterile PBS, take out the remaining serum, add the incubated serum-virus mixture into the well, and continue to incubate it in the incubator for 1 h, shaking it once every 15 min during this period; discard the serum-virus mixture, cover the cells with 2 mL of 0.5% low melting point agar containing 2% serum, and culture it in a 37 °C 5% CO2 incubator for 9 d. After 9 d, remove the overlay, add PBS and gently wash off the remaining overlay, add about 0.5 mL of cold methanol to fix it for 10 min. Discard the methanol, stain the cells with crystal violet staining solution for 10 min, discard the crystal violet staining solution, and wash it 2 times with deionized water. Count, and the neutralizing antibody titer is set as the serum dilution multiple (NT50) when the infectious unit is reduced by 50%.

[0247] Figure 17 The neutralizing antibody detection results of the mutant protein and the RSV inactivated vaccine (FI-RSV) are shown. It shows that the mutant trimer has good immunogenicity and can induce a high level of neutralizing antibodies against RSV A2 type in mice, and the neutralizing antibody level is significantly higher than that of the inactivated vaccine group.

[0248] To verify the existence of neutralizing epitopes in the mutant, 3 healthy adult sera were screened and verified as positive sera by the neutralization test of the RSV / A2 strain. For the 3 healthy adult sera, BeaverBeads TMHis-tag ProteinPurification magnetic beads (BEAVERBIO) were used to adsorb the mutant monomer antigens. First, the magnetic beads were washed twice with PBS (pH 7.4) and aliquoted into 50 μL portions. 50 μg of monomeric proteins JW-04-T4, JW-05-T4, JW-25-T4, and JW-29-T4 were mixed with the magnetic beads respectively, then blocked with bovine serum albumin. Next, 100 μL of each healthy adult serum was diluted 10-fold with PBS (pH 7.4). The magnetic beads adsorbed with mutant antigens were added to the serum diluent, and incubated at 4 °C for 1 h under stirring conditions. After centrifugation, the supernatant was taken to remove the antibodies bound to the antigen. The adsorbed human serum diluent was subjected to a virus-infected cell experiment according to the operation procedure of neutralizing antibody detection. The human serum diluent without antigen absorption was used as the positive control for the virus neutralization test. The positive control should show no cytopathic effect 3 - 4 days after virus cell infection, and the cell state should be consistent with the blank control cell wells, indicating that the positive serum can neutralize virus infection. The virus without serum can normally infect cells and show typical cytopathic effects at 3 - 4 d, indicating that the test is valid. The antigen was evaluated by the neutralizing ability of the serum adsorbed with the antigen against the virus.

[0249] Figure 18 The adsorption rate of neutralizing antibodies in the serum. The results showed that the serum without antigen adsorption could completely neutralize the virus and no cytopathic effect occurred in the cells. After adsorption with each mutant antigen, the neutralizing antibodies in the serum were completely removed, and the serum could not prevent the virus from infecting cells, showing typical cytopathic effects. These results indicate that each mutant has the same neutralizing epitope and can be recognized by the serum antibodies of natural infection, which is also the basis for its development as a RSV pre-F candidate vaccine.

[0250] Example 2

[0251] Referring to the designs of JW-01 to JW-06 in Example 1, starting from the F protein precursor polypeptides of human RSV STRAIN 18537 and bovine RSV STRAIN A51908 respectively, the corresponding mutants JW-01-18537 to JW-06-18537, JW-01-51908 to JW-06-51908 were designed, as shown in the following table.

[0252] Meanwhile, referring to the double antibody sandwich method in Example 1, the content of the mutants (denoted as V) was detected. Further, the samples with determined content were divided into two groups for treatment (one group was placed at 4 °C for 4 weeks, and one group was placed at 50 °C for 3 h), and then the content of the mutants in the samples was detected again (denoted as V'), and V' / V×100% was calculated. The results are shown in the following table.

[0253] Table 7

[0254]

[0255] Note: "N / A" means no detection was performed; "not detected" means detection was performed but no target was detected.

[0256] Table 7 describes the effects of introducing disulfide bonds in the α3 helix and β3 sheet of the F protein of different subtypes of RSV virus on the expression level and stability of mutants. The results show that because the F proteins of different subtypes of RSV virus are relatively conserved, introducing disulfide bonds in the α3 helix and β3 sheet has similar effects on the expression level and stability of the F proteins of different subtypes of RSV virus, and preF protein can be expressed to varying degrees.

[0257] Example 3

[0258] Referring to the design of JW-05 in Example 1, mutants JW-L01 to JW-L09 were designed in this example, as shown in Table 8 below.

[0259] Meanwhile, the content of the mutants (denoted as V) was detected by the double antibody sandwich method referring to Example 1. Further, the samples with measured content were divided into two groups for treatment (one group was placed at 4 °C for 4 weeks, and one group was placed at 50 °C for 3 h), and then the content of the mutants in the samples was detected again (denoted as V'), and V' / V×100% was calculated. The results are shown in Table 9 below.

[0260] Table 8. Different flexible peptide mutants of F protein p27 polypeptide

[0261] Number Sequence number Mutation site F2 position F1 position Linker JW-05 SEQ ID NO.16 A170C, A177C 103 145 GS JW-L01 SEQ ID NO.65 A170C, A177C 109 137 / JW-L02 SEQ ID NO.66 A170C, A177C 105 145 GS JW-L03 SEQ ID NO.67 A170C, A177C 103 145 SS JW-L04 SEQ ID NO.68 A170C, A177C 103 145 PG JW-L05 SEQ ID NO.69 A170C, A177C 103 145 GPG JW-L06 SEQ ID NO.70 A170C, A177C 103 145 GSGS JW-L07 SEQ ID NO.71 A170C, V179C 103 145 PG JW-L08 SEQ ID NO.72 L171C, A177C 103 145 GSGS JW-L09 SEQ ID NO.73 E163C, L181C 105 145 GS

[0262] Table 9. Expression level and thermal stability of different flexible peptide mutants of F protein p27 polypeptide

[0263] Number Expression level (mg / mL) Stability at 4°C (% After 4W) Stability at 50°C (% After 3h) JW-05 103 82 33 JW-L01 73 40 20 JW-L02 89 77 40 JW-L03 143 59 29 JW-L04 90 72 45 JW-L05 120 65 35 JW-L06 77 69 56 JW-L07 104 70 36 JW-L08 73 57 23 JW-L09 82 65 34

[0264] Table 9 describes the expression level of the mutant proteins obtained by connecting different flexible peptides at the p27 position of the mutant F protein in the α3 helix and β3 sheet and the thermal stability of the proteins. The results show that there are slight differences in the thermal stability of the mutant proteins obtained with different lengths of flexible peptides and different connection positions, and all can be expressed normally.

[0265] Table 10

[0266]

[0267]

[0268]

[0269]

[0270]

[0271]

[0272]

[0273]

[0274]

[0275]

[0276] The technical features of the above-described embodiments and examples can be combined in any suitable manner. For the sake of brevity of description, not all possible combinations of the various technical features in the above-described embodiments and examples are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered to be within the scope described in this specification.

[0277] The above-described examples only represent several embodiments of the present application, which are convenient for understanding the technical solutions of the present application specifically and in detail, but should not be construed as a limitation on the scope of patent protection of the application. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present application, several modifications and improvements can still be made, and these all belong to the protection scope of the present application. In addition, it should be understood that after reading the above teachings 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 protection scope 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 based on the technical solutions provided by the present application are all within the protection scope of the appended claims of the present application. Therefore, the protection scope of the patent of the present application shall be subject to the content of the appended claims, and the specification and drawings can be used to explain the content of the claims.

Claims

1. A mutant of RSV pre-fusion F protein, wherein the α3 helical region of the F1 polypeptide has a cysteine ​​substituent 1, and the β3 sheet region has a cysteine ​​substituent 2, and the cysteine ​​substituent 1 and the cysteine ​​substituent 2 form a disulfide bond; The F1 polypeptide of the RSV pre-fusion F protein satisfies the following conditions (1) and (2): (1) does not contain a transmembrane domain, and (2) does not contain an intracellular domain; The mutant does not contain a furin cleavage site fragment; The mutant does not contain the pep27 polypeptide; The mutant F2 polypeptide meets the following conditions: the C-terminus does not contain NN; The amino acid sequence of the mutant is shown in any one of SEQ ID NOs. 12-17, 21, 22, 24, 28, 30, 36, 40, 42, 46, 53, 54, 56-60, 62-73.

2. A nucleic acid molecule encoding a mutant of the RSV pre-fusion F protein according to claim 1.

3. A vector comprising the nucleic acid molecule of claim 2.

4. An engineered cell expressing a mutant of the RSV pre-fusion F protein according to claim 1, or comprising the nucleic acid molecule according to claim 2 or the vector according to claim 3.

5. The engineered cell according to claim 4, wherein The engineered cells are CHO cells.

6. A method for producing a mutant of the RSV pre-fusion F protein according to claim 1, comprising the steps of: Cultivate the engineered cell according to any one of claims 4 to 5, and isolate the mutant of RSV pre-fusion F protein from the obtained culture supernatant.

7. An immune composition comprising a mutant of the RSV pre-fusion F protein according to claim 1 or a nucleic acid molecule according to claim 2, and an immune adjuvant.

8. The immunogenic composition according to claim 7, wherein The immune adjuvant is one or more of aluminum salt adjuvant, surfactant, polynucleotide, lipopolysaccharide, liposome, and oil emulsion adjuvant.

9. Use of the mutant of RSV pre-fusion F protein according to claim 1 in preparing a respiratory syncytial virus antibody detection kit.

10. A respiratory syncytial virus antibody detection kit comprising a mutant of the RSV pre-fusion F protein according to claim 1.

Images