Improved respiratory syncytial virus fusion f protein mutant and use thereof

Abstract

The present invention belongs to the field of biomedicines. Particularly, provided are an improved respiratory syncytial virus fusion F protein mutant and the use thereof. The provided respiratory syncytial virus fusion F protein mutant can form a trimeric structure without introducing heterologous trimerization domains. Meanwhile, by designing mutations such as the release of an electrostatic repulsion force within the protein, the deletion of furin cleavage sites, the truncation of C-terminal domains, and the introduction of interchain disulfide bonds, the mutant can be stabilized in the pre-fusion conformation, making the protein more stable. When used as a vaccine or a vaccine component, the respiratory syncytial virus F protein mutant has high immunogenicity, can induce immunized animals to produce a relatively high level of neutralizing antibodies, may be used for preparing a vaccine for preventing respiratory syncytial virus infections, and may also be used as a reagent for detecting respiratory syncytial viruses.

Description

Improved respiratory syncytial virus fusion F protein mutant and its application Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to an improved respiratory syncytial virus fusion F protein mutant and an application thereof. Background Art

[0002] Respiratory Syncytial Virus (RSV) is a common respiratory virus. Infected cells fuse together to form large cell structures resembling syncytia. The clinical manifestation of infection is upper respiratory tract disease, which can further progress to lower respiratory tract disease. RSV is a major viral pathogen that causes acute lower respiratory tract infections (ALRI) in infants, the elderly, and immunocompromised adults. In young children, RSV infection manifests as bronchiolitis, pneumonia, and tracheobronchitis. Elderly people and immunocompromised adults experience more severe manifestations of infection, including bronchiolitis, pneumonia, asthma, exacerbations of chronic obstructive pulmonary disease, or worsening of congestive heart failure.

[0003] RSV infection is primarily mediated by the transmembrane envelope glycoproteins F and G. Although both F and G proteins reside outside the viral envelope and possess multiple specific antigenic determinants, the F sequence is highly conserved across subtypes. Vaccines targeting F proteins are expected to elicit a broader spectrum of protection, making F proteins an important candidate antigen for RSV vaccines. During the fusion of the viral envelope with the host cell membrane, the F protein transitions from a metastable prefusion conformation to a stable postfusion conformation. A large amount of preclinical and clinical data show that vaccines with prefusion RSV F as antigen can stimulate the body to produce higher levels of virus neutralizing antibodies (Griffin, MR (2022). "A Challenge to Respiratory Syncytial Virus Illness in Adults." N Engl J Med 386(25):2427-2428.), but the prefusion F protein is easily converted to the postfusion conformation after physical or chemical stress or storage (McLellan, JS, et al. (2013). "Structure-based design of a fusion glycoprotein vaccine for respiratory syncytial virus." Science 342(6158):592-598.), so the F protein needs to be designed to maintain the stability of the prefusion conformation.

[0004] Currently, two preventive RSV vaccines, Arexvy and Abrysvo, have been approved for marketing internationally, using the prefusion conformation F protein as an antigen. However, no related preventive vaccine products have been approved for marketing in China. Arexvy, developed by GSK, consists of the prefusion conformation F protein conjugated to Adjuvant System 01 (AS01E), while Abrysvo, developed by Pfizer, uses the bivalent prefusion conformation F protein as an antigen. The former is intended for use in the United States for people aged 60 and older, while the latter is intended for pregnant women and those over 60. However, both Arexvy and Abrysvo vaccines suffer from suboptimal long-term protection after immunization. Reportedly, the protection rates of the two vaccines in the first epidemic season were 66.7% (LRTD2+) and 82.6% (LRTD), respectively. These rates dropped to 48.9% (LRTD2+) and 56.1% (LRTD), respectively, in the second epidemic season. Furthermore, booster vaccinations in the second epidemic season failed to improve protection rates.

[0005] To achieve soluble protein expression and maintain a trimerized conformation, the antigen designs of both Arexvy and Abrysvo vaccines utilize a heterologous trimerization domain foldon to replace the C-terminal transmembrane insoluble region. However, the use of heterologous trimerization domains can easily induce pre-existing immunity, making it difficult to provide sustained protection through repeated vaccinations if vaccine immunity is not durable. Therefore, we are committed to designing a modified prefusion conformation of the respiratory syncytial virus F protein without the heterologous trimerization domain, in order to provide sustained immune protection.

[0006] Summary of the Invention

[0007] In order to solve the above problems, the present invention provides a modified respiratory syncytial virus fusion F protein mutant, which can stably present a trimerization conformation without introducing a heterologous trimerization domain, ensuring that the protein is similar to the natural conformation, providing a new direction for clinical application, and laying the foundation for the development of new products to prevent and block RSV infection.

[0008] The present invention focuses on the research of respiratory syncytial virus fusion F protein. Based on the guidance of structural biology and bioinformatics, a specific mutant that can stably present a pre-fusion conformation is designed. Compared with the wild-type F protein, the RSV F protein mutant can form a trimeric structure without introducing a heterologous trimerization domain, and further designed mutations to maintain it in a relatively stable fusion precursor state. The target protein can be expressed in a large-scale soluble manner using mammalian cells, and the protein stability performance is excellent. When used as a vaccine or vaccine component, its immunogenicity is enhanced and it can induce immunized animals to produce higher neutralizing antibody levels. The RSV F protein mutant described in the present application can be used to prevent and / or treat RSV infection, and can also be used as a detection reagent for respiratory syncytial virus. The mutant can be used alone when used as a prevention and / or treatment of RSV infection, can be used with different adjuvants to form a vaccine composition, and can also be used with different types of vaccine products to form a multi-vaccine.

[0009] The first object of the present invention is to provide an improved respiratory syncytial virus fusion F protein mutant, wherein the mutant uses a fragment of the respiratory syncytial virus fusion F protein's own soluble expression region to replace the C-terminal transmembrane insoluble region of the respiratory syncytial virus fusion F protein.

[0010] Furthermore, a segment of the respiratory syncytial virus fusion F protein's own soluble expression region is a segment with rigid structure.

[0011] In the present invention, a structurally rigid fragment of the RSV fusion F protein's own soluble expression region is intercepted to replace its C-terminal transmembrane insoluble region, which not only achieves the soluble expression of the target protein, but also can present a trimerization conformation without introducing any heterologous trimerization domain, ensuring that the protein is similar to the natural conformation, and thus can avoid the body's pre-existing immunity when it is used as a vaccine component.

[0012] Furthermore, the amino acid sequence of the respiratory syncytial virus fusion F protein is shown in SEQ ID NO.1, and a fragment of the soluble expression region of the respiratory syncytial virus fusion F protein itself is an amino acid sequence fragment from positions 155 to 173, an amino acid sequence fragment from positions 190 to 204, or an amino acid sequence fragment from positions 255 to 275 of the amino acid sequence shown in SEQ ID NO.1.

[0013] Furthermore, the C-terminal transmembrane insoluble region of the respiratory syncytial virus fusion F protein is the amino acid fragment at position 510 and thereafter of the amino acid sequence shown in SEQ ID NO.1.

[0014] Furthermore, when performing the replacement of the amino acid fragment at position 510 and thereafter, it also includes mutating lysine at position 508 and serine at position 509 to cysteine ​​(K508C-S509C).

[0015] In the present invention, the structurally rigid fragment of the truncated self-soluble expression region is a non-trimeric conformation in its natural state. In order to further increase the interaction force between trimers, a K508C-S509C mutation is further introduced adjacent to the C-terminus. The introduction of this interchain disulfide bond can increase the interaction between the C-termini of the trimers and is included in the C-terminal design modification.

[0016] Furthermore, the mutant further comprises at least one disulfide bond mutation.

[0017] In the present invention, since there are no interchain disulfide bonds between the wild-type respiratory syncytial virus fusion F protein trimers, the mutation design of the interchain disulfide bonds can further improve the stability of the trimer.

[0018] Furthermore, the mutant is obtained by subjecting the amino acid sequence of SEQ ID NO.1 to at least one of the following mutations:

[0019] Alanine at position 74 and glutamic acid at position 218 were mutated to cysteine ​​(A74C, E218C);

[0020] The glutamine at position 279 and the alanine at position 241 were mutated to cysteine ​​(Q279C-A241C).

[0021] Furthermore, the mutant also includes mutating one or more charged amino acids in the amino acid sequence of the respiratory syncytial virus fusion F protein into polar amino acids, hydrophobic amino acids or aromatic amino acids to release the electrostatic repulsion in the RSV F protein and enhance the stability of the RSV F pre-fusion protein.

[0022] Furthermore, the mutant is obtained by subjecting the amino acid sequence of SEQ ID NO.1 to at least one of the following mutations:

[0023] The glutamic acid at position 60 was mutated to alanine (E60A), glycine (E60G), serine (E60S), threonine (E60T), leucine (E60L), methionine (E60M), or phenylalanine (E60F).

[0024] Furthermore, the mutant further comprises deleting a fragment containing a flavidin protease cleavage site in the amino acid sequence of the respiratory syncytial virus fusion F protein.

[0025] In the present invention, deletion of the fragment containing the ferin cleavage site can avoid the breakage caused by ferin cleavage in the wild-type RSV F protein, thereby maintaining the pre-fusion conformation of the F protein. The present invention does not introduce any site-directed mutagenesis of the ferin cleavage site, and does not require the introduction of a heterologous linker sequence, which can maintain a higher sequence similarity than the wild-type sequence.

[0026] Furthermore, the mutant is obtained by deleting 28 to 46 amino acids between positions 100 and 147 in the amino acid sequence shown in SEQ ID NO.1.

[0027] Furthermore, the mutant is obtained by deleting 37 amino acids between positions 105 and 143 in the amino acid sequence of SEQ ID NO. 1 (deletion of 37 amino acids between positions 105 and 143).

[0028] The second object of the present invention is to provide a nucleic acid molecule encoding the respiratory syncytial virus fusion F protein mutant.

[0029] Furthermore, the nucleic acid molecule has been codon optimized for expression in mammalian cells.

[0030] The third object of the present invention is to provide a vector comprising the nucleic acid molecule.

[0031] The fourth object of the present invention is to provide a cell, wherein the cell expresses the respiratory syncytial virus fusion F protein mutant, or contains the nucleic acid molecule, or contains the vector.

[0032] The fifth object of the present invention is to provide a vaccine comprising the respiratory syncytial virus fusion F protein mutant.

[0033] Furthermore, the vaccine also contains other active ingredients.

[0034] Furthermore, the vaccine also contains a vaccine adjuvant.

[0035] The sixth object of the present invention is to provide a pharmaceutical composition comprising the respiratory syncytial virus fusion F protein mutant, the vaccine, the nucleic acid molecule or the vector.

[0036] Furthermore, the pharmaceutical composition includes pharmaceutically acceptable excipients.

[0037] Furthermore, the pharmaceutically acceptable excipients include protective agents, stabilizers, preservatives, bactericides, inactivators, adjuvants and / or buffers.

[0038] The seventh object of the present invention is to provide the use of the respiratory syncytial virus fusion F protein mutant, the nucleic acid molecule, the vector, the cell, the vaccine or the pharmaceutical composition in the preparation of products for detecting, preventing and / or treating respiratory syncytial virus infection.

[0039] Furthermore, the application specifically includes the following steps:

[0040] When used to prepare a product for detecting respiratory syncytial virus infection, the respiratory syncytial virus fusion F protein mutant according to any one of claims 1 to 11, or the respiratory syncytial virus fusion F protein mutant produced by the nucleic acid molecule according to claim 12, the vector according to claim 13, or the cell according to claim 14, is used as a coating reagent of a detection kit, and the presence of respiratory syncytial virus infection in the sample to be tested is determined by detecting the binding antibody titer in the serum of the sample to be tested;

[0041] When used to prepare a product for preventing and / or treating respiratory syncytial virus infection, the F protein mutant described in any one of claims 1 to 11, or the respiratory syncytial virus fusion F protein mutant produced by the nucleic acid molecule described in claim 12, the vector described in claim 13, or the cell described in claim 14, is used alone or in combination with an adjuvant, and is administered via an immunization route to stimulate the body to produce neutralizing antibodies, which is used to prevent and / or treat respiratory syncytial virus infection; or the vaccine described in claim 15 or the pharmaceutical composition described in claim 16 is administered via an immunization route to stimulate the body to produce neutralizing antibodies, which is used to prevent and / or treat respiratory syncytial virus infection.

[0042] By means of the above solution, the present invention has at least the following advantages:

[0043] The present application provides a designed and improved respiratory syncytial virus fusion F protein mutant, in which the C-terminus of the RSV F protein mutant uses a fragment of the F protein's own soluble expression region to replace the C-terminal transmembrane insoluble region of the F protein, which not only achieves the ability to use mammalian cells for large-scale soluble expression of the target protein, but also can form a trimer structure without using a heterologous trimerization domain, avoiding the defect of the prior art in which the F protein produces pre-existing immunity when used as a vaccine component due to the introduction of a heterologous trimerization domain.

[0044] In addition, the present application further maintains the respiratory syncytial virus fusion F protein in a relatively stable fusion precursor state through designed mutations, and the protein exhibits excellent stability. The designed mutants have enhanced immunogenicity when used as vaccines or vaccine components, and can induce high levels of neutralizing antibodies in immunized animals. The RSV F protein mutants described herein can be used to prepare recombinant protein RSV vaccines to prevent and treat diseases and conditions caused or mediated by RSV.

[0045] The above description is only an overview of the technical solutions and partial results of the present invention. In order to more clearly understand the technical means of the present invention and implement them according to the contents of the specification, the preferred embodiments of the present invention are described below with detailed drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1. Template amino acid sequence for constructing RSV F protein mutants described in certain embodiments; the template amino acid sequence includes the heterologous signal peptide sequence "MKWVTFLLLLFISGSAFS" (underlined), the F2 polypeptide (residues 26-109), "|...|" indicating a deletion of 37 amino acids between positions 105 and 143, the F1 polypeptide (residues 143-509), and the replacement sequence (shown in bold black, residues 155-173); the amino acid positions are relative to the wild-type RSV F sequence described in SEQ ID NO. 1;

[0047] Figure 2. F protein expression levels were measured using antibodies D25 and AM14 specific for pre-fusion F protein, with SC-TM, DS-CavI, and pXCS847 serving as positive controls.

[0048] Figure 3. F protein stability was determined using antibodies D25 and AM14 that specifically recognize the pre-fusion F protein, with SC-TM, DS-CavI, and pXCS847 serving as positive controls.

[0049] FIG4 . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the purified fusion F protein mutant YK001Ag399 under reducing (R) and non-reducing (NR) conditions;

[0050] Figure 5. SEC analysis of purified F proteins of the fusion F protein mutant YK001Ag399 and the control molecules SC-TM, DS-CavI, and pXCS847;

[0051] Figure 6. Evaluation results of the immunogenicity (binding antibodies) of the vaccine composition prepared with the fusion F protein mutant YK001Ag399 as the core component. DETAILED DESCRIPTION

[0052] The present invention discloses improved respiratory syncytial virus prefusion F protein mutants and their applications. Those skilled in the art can refer to the contents of this article and appropriately improve the process parameters to achieve the desired results. It should be noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0053] the term:

[0054] In this application, the term "respiratory syncytial virus" or "RSV" belongs to the genus Pneumovirus of the family Paramyxoviridae. The virus is usually an important viral pathogen that causes acute lower respiratory tract infection (ALRI) in infants, the elderly and immunocompromised adults, and can cause interstitial pneumonia and bronchiolitis.

[0055] In this application, the term "protein mutant" generally refers to a protein whose amino acid sequence or protein structure exhibits one or more alterations compared to the wild-type protein. Such alterations may include deletion, insertion, substitution, truncation, and / or deletion of one or more amino acids, and processing or cleavage of the protein structure. In this application, protein mutants refer to respiratory syncytial virus (RSV) F protein mutants.

[0056] In this application, the term "amino acid mutation" generally refers to a modification of an amino acid in a parent amino acid sequence. For example, the modification may include the replacement, insertion, and / or deletion of one or more amino acids. In this application, the amino acid mutation may include the deletion or replacement of at least one amino acid residue at a specified position in the amino acid sequence. In certain embodiments, the amino acid mutation can optimize the conformation of the protein composed of the amino acid sequence. Amino acid mutations can be generated using genetic or chemical methods well known in the art. For example, genetic methods may include site-directed mutagenesis, PCR, and gene synthesis.

[0057] In this application, the term "codon optimization" generally refers to the replacement of one or more codons in a parent polypeptide-encoding nucleic acid with codons encoding the same amino acid residue having different relative usage frequencies to improve the expression of the nucleic acid encoding the polypeptide. In this application, as long as the amino acid mutation is the same as that in this application, all possible codons encoding the mutant amino acid are within the scope of protection of this application.

[0058] In this application, the term "trimer" generally refers to a protein structure composed of three protein subunits of the same or different types, and the protein trimer can be connected together by a special chemical structure. In this application, the protein trimer can be the F protein of respiratory syncytial virus.

[0059] In this application, the term "conformational change" generally refers to a change in the spatial structure of a protein molecule. For example, the conformational change may include a change in the chemical bonds in the protein molecule or a change in the folding mode of a polypeptide.

[0060] In this application, the term "signal peptide" generally refers to an amino acid sequence present at the N-terminus of a transmembrane protein that serves as a signal for transmembrane protein translocation. For example, the transmembrane protein may include a secretory protein or a cell membrane protein. For example, the signal peptide may be synthesized at the N-terminus of the transmembrane protein in the form of a precursor polypeptide.

[0061] In this application, the term "structural protein" generally refers to proteins that constitute components of viral particles. The structural proteins may include structural proteins of respiratory syncytial virus. The structural proteins described in this application may include F protein, G protein, and SH protein.

[0062] In the present application, term " before fusion " generally refers to the conformation presented by the structural protein of virus when membrane fusion does not occur before infected host cell. Generally speaking, before the fusion of wild-type RSV F protein, it is a kind of metastable protein, and its conformation is discontinuous after viral infection host cell and membrane fusion occurs, and progressively and irreversible conformational change is changed into the conformation (conformation after fusion) of a low-energy stable state. In the present application, RSV F protein is carried out a series of transformations so that described F protein mutant can stably be in fusion precursor state.

[0063] In this application, protein mutation sites are generally described as "amino acid + amino acid position + mutated amino acid." In this application, mutations may include, but are not limited to, additions, substitutions, deletions, and / or deletions of amino acids. For example, the term "E218C" generally refers to a mutation from glutamic acid (E) at position 218 to cysteine ​​(C).

[0064] In this application, the term "nucleic acid molecule" generally refers to nucleotides of any length in isolated form, either deoxyribonucleotides or ribonucleotides, or analogs thereof, isolated from their natural environment or artificially synthesized.

[0065] In the application, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide encoding a protein can be inserted and the protein expressed. A vector can be used to transform, transduce, or transfect host cells, allowing the genetic material elements it carries to be expressed in the host cells. For example, vectors include: plasmids; phagemids; cosmids; artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); bacteriophages such as lambda phage or M13 phage; and animal viruses. Types of animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomas (such as SV40). A vector may contain multiple elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, a vector may also contain a replication initiation site. The vector may also include components that assist it in entering cells or assisting the integration of the target element into host cells, such as viral particles, liposomes, protein coats or integrase, but not limited to these substances.

[0066] In this application, the term "pharmaceutical composition" generally refers to a composition for preventing / treating a disease or condition. The pharmaceutical composition may include RSV F protein mutants described herein, nucleic acid molecules described herein, carriers described herein and / or cells described herein, and optionally pharmaceutically acceptable carriers. In addition, the pharmaceutical composition may also include suitable formulations of one or more (pharmaceutically effective) adjuvants, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers and / or preservatives. The acceptable components of the composition are generally nontoxic to the recipient at the dosage and concentration used.

[0067] In this application, the term "pharmaceutically acceptable carrier" generally includes pharmaceutically acceptable carriers, excipients or stabilizers that are non-toxic to cells or mammals exposed thereto at the dosages and concentrations employed. Physiologically acceptable carriers may include, for example, buffers, antioxidants, low molecular weight (less than about 10 residues) polypeptides, proteins, hydrophilic polymers, amino acids, monosaccharides, disaccharides and other carbohydrates, chelating agents, sugar alcohols, salt-forming counterions, such as sodium, and / or nonionic surfactants.

[0068] In this application, the term "comprising" generally means including the features specifically stated, but not excluding other elements.

[0069] This application, based on structural biology and incorporating immunological principles, designs a novel protective vaccine with high safety, broad protection, and a reliable preparation process. The recombinant protein vaccine provided in this application exhibits significantly improved stability compared to the publicly reported DS-CavI and Pfizer's patented preferred molecule 847. The RSV F protein mutant can be stably maintained in a prefusion state and, when used alone or with a suitable adjuvant, can induce high levels of neutralizing antibodies in animal immunization studies.

[0070] In this application, the term "adjuvant" generally refers to any substance that assists or regulates the action of a drug, including but not limited to immunological adjuvants, which enhance or diversify the immune response to an antigen. In this application, the adjuvant can be used to enhance the antigenicity of the RSV F protein mutant. In certain embodiments, the adjuvant can comprise a mineral (e.g., alum, aluminum hydroxide, or phosphate) suspension. In certain embodiments, the adjuvant can comprise an oil-in-water emulsion. In certain embodiments, the adjuvant can comprise a liposome. In certain embodiments, the adjuvant can comprise an immunostimulant such as MPL, CpG, Poly:IC.

[0071] In this application, the term "vaccine" refers to a pharmaceutical composition comprising an immunogen capable of eliciting a prophylactic or therapeutic immune response in an individual. Typically, a vaccine elicits an antigen-specific immune response against a pathogen, such as a viral pathogen.

[0072] In this application, the term "prefusion-specific antibody" refers to an antibody that specifically binds to the RSV F glycoprotein in the prefusion conformation but does not bind to the RSV F protein in the postfusion conformation. Exemplary prefusion-specific antibodies include D25, AM22, 5C4, MPE8, and AM14 antibodies.

[0073] In the present application, the term "specific binding" in the context of an antibody binding to a given target molecule refers to an antibody that binds to the target molecule with a higher affinity than it binds to other substances being tested. For example, an antibody that specifically binds to the RSV F protein in its prefusion conformation is an antibody that binds to the RSV F protein in its prefusion conformation with a higher affinity than it binds to the RSV F protein in its postfusion conformation.

[0074] In the present application, the term "SC-TM" refers to a form of RSV F protein having the amino acid sequence described in the document "Krarup, A., et al. (2015). "A highly stable prefusion RSV F vaccine derived from structural analysis of the fusion mechanism." Nat Commun 6:8143".

[0075] In this application, the term "847" refers to the pXCS847 molecule in patent CN108738312A.

[0076] In the present application, the term "DS-CavI" is a molecule described in the document "McLellan, JS, et al. (2013). "Structure-based design of a fusion glycoprotein vaccine for respiratory syncytial virus." Science 342(6158):592-598".

[0077] In the present application, the term "D25" refers to the antibody described in WO 2008 / 147196 A2, which can specifically recognize the pre-fusion conformation of the F protein of respiratory syncytial virus.

[0078] In this application, the term "AM14" refers to the antibody described in the document "Wayne Harshbarger., et al. (2021) Improved epitope resolution of the prefusion trimer-specific antibody AM14 bound to the RSV F glycoprotein, mAbs, 13: 1, DOI: 10.1080 / 19420862.2021.1955812", which only binds to the trimerized and prefusion conformation of respiratory syncytial virus F protein, and does not bind to the monomeric fusion F protein.

[0079] Without intending to be bound by any theory, the following examples are merely intended to illustrate the RSV F protein mutants, preparation methods, quality characterizations, and uses of the present application, and are not intended to limit the scope of the present invention.

[0080] The present invention will be further described below in conjunction with the embodiments:

[0081] Example 1: Design and expression of RSV F protein mutants

[0082] The present invention has assessed more than 400 kinds of RSV F protein mutants in total, and the present embodiment has summarized the design and expression amount of positive control molecules (SC-TM, DS-CavI, 847) and 7 representative RSV F protein mutants (each of which is identified by a unique identifier, such as YK001Ag222, YK001Ag399 etc.).The design and expression amount of exemplary RSV F mutants are provided in Table 1.7 representative mutants are each designed and prepared based on the amino acid sequence described in SEQ ID NO.1.The method for expressing and purifying these exemplary RSV F mutants is described in Examples 2 and 4, and the method for measuring RSV F mutant expression amount is described in Example 3, and the expression amount of the RSV F mutant is the target protein content in the cell transient expression supernatant described in Example 2 of the present invention.

[0083] D25 antibody can specifically identify the F protein of respiratory syncytial virus pre-fusion conformation, and AM14 antibody can only specifically identify trimerized pre-fusion F protein.In order to ensure that the respiratory syncytial virus F protein mutant of improved design is pre-fusion conformation, and is trimerization state, the present embodiment has determined the combination of mutant and D25 and AM14, as shown in Table 1 and Figure 2, all 7 representative molecules can be specifically combined with two antibodies, and the expression amount of two antibody determinations is not much different (less than one times), illustrating that 7 representative molecules are similar to the positive control molecule conformation, and all can present trimerization conformation before fusion.Wherein YK001Ag399, YK001Ag401 and YK001Ag404 three representative molecules do not contain heterologous trimerization domain, successfully realize replacing heterologous foldon with the S155-S173 peptide segment of self homology, without affecting the trimerization of target protein.

[0084] Table 1. Sequence design and expression levels of exemplary RSV F protein mutants

[0085] Example 2: Construction of expression vectors and small-volume expression of RSV F protein mutants

[0086] The nucleic acid sequence encoding the RSV F protein mutant was codon-optimized and synthesized using CHO as the host, cloned into the YKVGS001 vector using HindIII and NotI, and the plasmid was extracted for further transfection.

[0087] Expi 293 cells, known for their rapid growth and well-developed cell morphology, were used as the transfection host for large-volume transfection. Prior to transfection, the seed cell density was adjusted to 3 million viable cells per mL. 20 mL of cell suspension was used for each transfection. For each transfection, 20 μg of plasmid and 100 μg of PEI MAX transfection reagent were diluted in 600 μL of OPM-293CD05 medium. Gently mix the diluted transfection reagent and gently add the diluted plasmid dropwise to the solution. Incubate at room temperature for 8 minutes to prepare the plasmid complex. Following incubation, the prepared plasmid complex was slowly added dropwise to the cells to be transfected. The cells were incubated at 37°C, 8% CO2, and 200 rpm in a shaker. 18-24 hours after transfection, add 10% of the culture volume of BalanCD CHO Feed 4 (0.8X) (V / V) to each flask of transfected cells and monitor the cell status daily. The transfection cycle is usually 4 days. On the 4th day, the cell culture supernatant is harvested for expression determination and transferred to the purification section for purification.

[0088] Example 3: RSV F protein mutant expression and stability determination (detected with monoclonal antibodies D25 and AM14)

[0089] The cell supernatant harvested in Example 2 of the present invention was filtered through a disposable 0.22 μm sterile filter and then packaged. The sample was directly tested for the expression of the target protein in the supernatant or placed in a 60°C constant temperature box for 1 hour to investigate the stability of the sample under high temperature conditions. The gator based on the principle of biofilm optical interferometry was used. TM The molecular interaction detector calculates the content of the target protein in the cell expression supernatant and stability test samples by testing the binding rate of RSV fusion precursor-specific recognition monoclonal antibodies D25 or AM14 with RSV F mutants.

[0090] The specific test parameters are as follows: After 50 seconds of equilibration with the protein A probe, the D25 or AM14 monoclonal antibody is captured. After 120 seconds of equilibration, the probe binds to the supernatant of cell expression of the RSV F protein mutant for 200 seconds, followed by a 120-second dissociation reaction to test the binding of the designed RSV F protein mutant to the D25 or AM14 monoclonal antibody. The target protein content is calculated based on a standard curve established with a standard sample of known concentration.

[0091] In some embodiments, the expression level determination results of the selected RSV F protein mutants are shown in Table 1 and Figure 2, and the stability test results are shown in Table 2 and Figure 3. After the improved design described in the present invention, most RSV F protein mutants can stably present a fusion precursor conformation. After being placed at 60°C for 1 hour, preferred RSV F protein mutants such as YK001Ag403, YK001Ag404, and YK001Ag399 can still maintain high binding activity with D25 monoclonal antibody and AM14.

[0092] Table 2 Stability assessment of exemplary RSV F protein mutants

[0093] Comparison of the stability data of YK001Ag400 and YK001Ag401, YK001Ag402 and YK001Ag404, and YK001Ag403 and YK001Ag399 shows that, under the premise that the designs of all mutations except the C-terminus are the same (the K508C-S509C mutation is adjacent to the C-terminus and is included in the C-terminus design to increase the interaction between the C-terminal trimers), replacing the heterologous foldon with its own homologous S155-S173 peptide segment does not affect the trimerization and stability of the target protein.

[0094] Comparing the stability data of YK001Ag400 and YK001Ag402, and YK001Ag401 and YK001Ag404, it can be seen that under the premise that the designs of other mutations are the same, the introduction of A74C-E218C mutation can greatly increase the stability of the trimer before fusion of the target protein.

[0095] Comparing the stability data of YK001Ag401 and YK001Ag399, and YK001Ag400 and YK001Ag403, it can be seen that under the premise that the designs of other mutations are the same, the introduction of Q279C-A241C mutation can also significantly increase the stability of the target protein trimer before fusion.

[0096] Comparing the expression and stability data of YK001Ag222 and YK001Ag400, it can be seen that the introduction of the E60L mutation can improve protein expression and stability to a certain extent, provided that the other mutation designs are the same.

[0097] Example 4: Purification of RSV F protein mutants

[0098] 1. Adjust the pH of the clarified harvested liquid to 7.0 with 0.5 M NaOH and adjust the conductivity to no more than 8.0 mS / cm with 20 mM PB pH 7.0 buffer;

[0099] 2: Load the pretreated sample onto a strong cation exchange chromatography column pre-equilibrated with 20 mM PB pH 7.0 buffer. After loading, perform gradient elution with 20 mM PB pH 7.0, 1 M NaCl buffer, and collect the eluate containing the target protein fraction;

[0100] 3: Adjust the pH of the strong cation exchange chromatography eluate to 7.5 using 0.5 M NaOH;

[0101] 4: After sample treatment, load the sample onto a multi-mode chromatography column pre-equilibrated with 20 mM PB pH 7.5 buffer. After loading, perform gradient elution with 20 mM PB pH 7.5, 1 M NaCl buffer, and collect the eluate containing the target protein fraction;

[0102] 5: Load the composite mode chromatography eluate onto a size exclusion chromatography column pre-equilibrated with 1xPBS pH 7.4, and collect the eluate containing the target protein component, which is the prepared high-purity protein sample.

[0103] The purification results of the selected representative RSV F mutant YK001Ag399 are shown in Figure 4. After purification, the target protein with high electrophoretic purity can be obtained, and the trimeric conformation of the F protein can be maintained. In the present invention, taking the representative RSV F mutant YK001Ag399 as an example, the fehlin protease cleavage site introduced therein is deleted in order to maintain the pre-fusion conformation of the F protein, and the introduction of other mutations is to enhance the stability of the protein; and the C-terminal design thereof uses a fragment of the soluble expression region of the F protein itself to replace the C-terminal transmembrane insoluble region of the F protein, which can play a role in maintaining the trimeric conformation of the F protein.

[0104] Example 5: Particle size distribution analysis of selected prefusion RSV F mutants

[0105] To analyze the size distribution of the designed RSV F protein mutants, this experiment used SEC-HPLC to analyze the purified samples. The specific implementation method was as follows: the chromatographic column was Agilent Bio SEC-3 (3 μm, 4.6 x 300 mm), detection wavelength at 280 nm, mobile phase 1× PBS, flow rate 0.4 ml / min, injection volume 10 μl, isocratic elution for 15 min. The test solution was prepared as follows: 50 μl of sample was placed in a 1.5 ml centrifuge tube, centrifuged at 10,000 rpm for 1 min, and the supernatant was transferred to a liquid phase vial containing an inner cannula for later use. Proteins were eluted and separated according to their molecular size.

[0106] The molecular weight of the RSV F mutant monomer of the present invention is about 53 KDa. As shown in Figure 5, the peak retention time of the selected RSV F mutant is close to that of γ-globulin with a molecular weight of 158 KDa, confirming that the RSV F mutant of the present invention is a trimer conformation.

[0107] Example 6: Structural thermal stability of selected prefusion RSV F protein mutants

[0108] To investigate the thermodynamic stability of the designed RSV F protein mutants, this experiment used the Prometheus Panta instrument to assess the particle size and thermal denaturation of the antigen. The specific method was to centrifuge 50 μl of the test sample at 15,000 g for 10 minutes at 8°C, then load it into a capillary tube (manufacturer: Nano Temper, catalog number: PR-C002) and place it in a tray. Each sample was plated in triplicate wells. The thermal unfolding module in the instrument software was selected, with a starting temperature of 25°C, an end temperature of 95°C, and a heating rate of 1°C / min for the test.

[0109] The results of the structural thermal stability investigation of representative RSV F protein mutants are shown in Table 3. The Tm value of the YK001Ag399 molecule is significantly higher than that of the SC-TM and DS-CavI positive control molecules, and is similar to that of the pXCS847 molecule, demonstrating that the improved protein has excellent structural thermal stability.

[0110] Table 3 Structural stability data of exemplary RSV protein mutants

[0111] * represents the mean, σ* represents the variance, and NA represents abnormal data that cannot be fitted.

[0112] Example 7: Immune response of improved designed RSV F protein mutants in mice

[0113] In order to study the immunogenicity of the RSV F protein mutant provided by the present invention, the inventors of the present invention prepared a vaccine composition (0.5 ml / dose) containing different adjuvants and a representative design molecule YK001Ag399 as an antigen. The group information is specifically shown in Table 4. The immunogenicity of each group of vaccine compositions was investigated in SPF female 6-8 week old BALB / c mice, with 6 mice in each group, for a total of 3 groups. The immunization method was intramuscular injection, and the injection volume was 50 μl / dose / mouse (1 / 10 of the intended human dose). Each mouse was immunized twice, with an immunization interval of 3 weeks. Two weeks after the second immunization, blood was collected from each group of mice, and the IgG antibody titer against F protein in the serum was detected by ELISA (Figure 6-A); three weeks after the second immunization, blood was collected from each group of mice, and the serum neutralizing antibody titer was detected by neutralization experiments based on wild-type A and B live viruses (Figure 6-B / C).

[0114] Table 4 Different RSV vaccine composition formulations

[0115] Compared with the vehicle control group (Group 1), high levels of binding antibodies against the F protein were detected in the sera of mice in each experimental group two weeks after the second immunization, and the sera of mice in each experimental group three weeks after the second immunization were able to effectively neutralize the replication of live wild-type A and B viruses in in vitro cells, indicating that the RSV F protein mutant provided by the present invention has excellent immunogenicity and can induce cross-protection against both A and B strains when used as a vaccine product.

[0116] The above is only a preferred embodiment of the present invention and is not intended to limit the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the technical principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. An improved respiratory syncytial virus fusion F protein mutant, characterized in that: The mutant uses a fragment of the soluble expression region of the respiratory syncytial virus fusion F protein itself to replace the C-terminal transmembrane insoluble region of the respiratory syncytial virus fusion F protein.

2. The respiratory syncytial virus fusion F protein mutant according to claim 1, characterized in that The segment of the respiratory syncytial virus fusion F protein self-soluble expression region is a segment with rigid structure.

3. The respiratory syncytial virus fusion F protein mutant according to claim 2, characterized in that: The amino acid sequence of the respiratory syncytial virus fusion F protein is shown in SEQ ID NO.1, and a fragment of the soluble expression region of the respiratory syncytial virus fusion F protein itself is an amino acid sequence fragment from positions 155 to 173, an amino acid sequence fragment from positions 190 to 204, or an amino acid sequence fragment from positions 255 to 275 of the amino acid sequence shown in SEQ ID NO.

1.

4. The respiratory syncytial virus fusion F protein mutant according to claim 3, characterized in that: The C-terminal transmembrane insoluble region of the respiratory syncytial virus fusion F protein is the amino acid fragment at position 510 and thereafter of the amino acid sequence shown in SEQ ID NO.

1.

5. The respiratory syncytial virus fusion F protein mutant according to claim 4, characterized in that: When performing the replacement of the amino acid fragment at position 510 and thereafter, it also includes mutating the lysine at position 508 and the serine at position 509 to cysteine.

6. The respiratory syncytial virus fusion F protein mutant according to any one of claims 1 to 5, characterized in that: The mutant further comprises at least one disulfide bond mutation.

7. The respiratory syncytial virus fusion F protein mutant according to claim 6, characterized in that: The mutant is obtained by subjecting the amino acid sequence of SEQ ID NO.1 to at least one of the following mutations: Mutate alanine at position 74 and glutamic acid at position 218 to cysteine; The glutamine at position 279 and the alanine at position 241 were mutated to cysteine.

8. The respiratory syncytial virus fusion F protein mutant according to any one of claims 1 to 5, characterized in that: The mutant also includes mutating one or more charged amino acids in the amino acid sequence of the respiratory syncytial virus fusion F protein into polar amino acids, hydrophobic amino acids or aromatic amino acids to release the electrostatic repulsion in the RSV F protein and enhance the stability of the RSV F pre-fusion protein.

9. The respiratory syncytial virus fusion F protein mutant according to claim 8, characterized in that: The mutant is obtained by subjecting the amino acid sequence of SEQ ID NO.1 to at least one of the following mutations: The glutamic acid at position 60 was mutated to alanine, glycine, serine, threonine, leucine, methionine or phenylalanine.

10. The respiratory syncytial virus fusion F protein mutant according to any one of claims 1 to 5, characterized in that: The mutant further comprises deleting a fragment containing a feline protease cleavage site in the amino acid sequence of the respiratory syncytial virus fusion F protein.

11. The respiratory syncytial virus fusion F protein mutant according to claim 10, characterized in that: The mutant is obtained by deleting 28 to 46 amino acids between positions 100 and 147 in the amino acid sequence shown in SEQ ID NO.

1.

12. A nucleic acid molecule encoding the respiratory syncytial virus fusion F protein mutant according to any one of claims 1 to 11.

13. A carrier, characterized in that The vector comprises the nucleic acid molecule of claim 12.

14. A cell, characterized in that The cell expresses the respiratory syncytial virus fusion F protein mutant according to any one of claims 1 to 11, or contains the nucleic acid molecule according to claim 12, or contains the vector according to claim 13.

15. A vaccine, characterized in that The vaccine comprises the respiratory syncytial virus fusion F protein mutant according to any one of claims 1 to 11.

16. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the respiratory syncytial virus fusion F protein mutant according to any one of claims 1 to 11, the vaccine according to claim 15, the nucleic acid molecule according to claim 12 or the vector according to claim 13.

17. Use of the respiratory syncytial virus fusion F protein mutant according to any one of claims 1 to 11, the nucleic acid molecule according to claim 12, the vector according to claim 13, the cell according to claim 14, the vaccine according to claim 15 or the pharmaceutical composition according to claim 16 in the preparation of a product for detecting, preventing and / or treating respiratory syncytial virus infection; in, The application specifically comprises the following steps: When used to prepare a product for detecting respiratory syncytial virus infection, the respiratory syncytial virus fusion F protein mutant described in any one of claims 1 to 11, or the respiratory syncytial virus fusion F protein mutant produced by the nucleic acid molecule described in claim 12, the vector described in claim 13, or the cell described in claim 14, is used as a coating reagent of a detection kit, and the binding antibody titer in the serum of the sample to be tested is detected to determine whether the sample to be tested is infected with respiratory syncytial virus; When used to prepare a product for preventing and / or treating respiratory syncytial virus infection, the F protein mutant described in any one of claims 1 to 11, or the respiratory syncytial virus fusion F protein mutant produced by the nucleic acid molecule described in claim 12, the vector described in claim 13, or the cell described in claim 14, is used alone or in combination with an adjuvant, and is administered via an immunization route to stimulate the body to produce neutralizing antibodies, which is used to prevent and / or treat respiratory syncytial virus infection; or the vaccine described in claim 15 or the pharmaceutical composition described in claim 16 is administered via an immunization route to stimulate the body to produce neutralizing antibodies, which is used to prevent and / or treat respiratory syncytial virus infection.

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