Search

CN-122011136-A - Mutant of pre-fusion human metapneumovirus F protein and application thereof

CN122011136ACN 122011136 ACN122011136 ACN 122011136ACN-122011136-A

Abstract

The invention relates to the technical field of biological products, in particular to a mutant of pre-fusion human metapneumovirus F protein and application thereof. The mutant is obtained by carrying out mutation on the basis of wild type pre-fusion human metapneumovirus F protein, wherein amino acids 86-112 or 97-106 are replaced by flexible linker. The application comprises the steps of preparing a medicament for preventing or treating hMPV infection, preparing an immunogenic composition for inducing a body to generate neutralizing antibodies against hMPV, preparing a reagent or a kit for detecting the hMPV antibodies, screening or preparing anti-hMPV antibodies, and separating hMPV specific B cells. The novel pre-fusion human metapneumovirus F protein mutant is obtained through research and design, has remarkably better stability, expression level and affinity, can be used for hMPV diagnosis, antibody therapy and development of various vaccines, and has important application value.

Inventors

  • LI KELEI
  • LIU JIANKAI
  • CUI LISHA
  • LI JIN
  • Cang Tianle
  • WANG JIANLONG
  • WANG HAIXIN
  • LI MINGLI
  • LI KE
  • HOU YINGYING

Assignees

  • 北京民海生物科技有限公司
  • 深圳康泰生物制品股份有限公司

Dates

Publication Date
20260512
Application Date
20260413

Claims (15)

  1. 1. A mutant of human metapneumovirus F protein is characterized in that the mutant is obtained by carrying out mutation on the basis of wild type human metapneumovirus F protein, wherein amino acids 86-112 or 97-106 are replaced by flexible linker.
  2. 2. The mutant of claim 1 which further comprises mutating one or more of the amino acid sequences at positions 122, 125, 127, 129, 133, 134, 151, 168, 196, 258, 259, 260, 263 or 268 to C.
  3. 3. The mutant according to claim 1 or 2, further comprising at least one of the following mutations: (1) Mutation at position 131 to P; (2) Mutation at position 160 to F; (3) Mutation at position 163 to P; (4) Mutation at position 185 to P; (5) Mutation at position 459 to P; (6) Mutation at position 73 to W; (7) Mutation at position 116 to H; (8) Mutation at position 368 to N; (9) Mutation at position 453 to Q.
  4. 4. The mutant according to claim 1 or 2, further comprising at least one of the following mutations: (1) Mutation at position 123 to C and mutation at position 429 to C; (2) Mutation at position 88 to C and mutation at position 122 to C; (3) Mutation at position 127 to C and mutation at position 151 to C; (4) Mutation at position 119 to C and mutation at position 428 to C; (5) Mutation at position 115 to C and mutation at position 375 to C; (6) Mutation at position 84 to C and mutation at position 249 to C; (7) Mutation at position 140 to C and mutation at position 147 to C.
  5. 5. The mutant according to claim 1 or 2, further comprising at least one of the following mutations: (1) Mutation at position 56 to F or M; (2) Mutation at position 118 to M; (3) Mutation at position 191 to M; (4) Mutation at position 209 to E; (5) Mutation at position 231 to I; (6) Mutation at position 374 to M; (7) Mutation at position 376 to T; (8) Mutation at position 411 to M; (9) Mutation at position 430 to Q or M; (10) Mutation at position 449 to D; (11) Mutation at position 453 to P; (12) Mutation at position 404 to P; (13) Mutation at position 435 to E.
  6. 6. The mutant according to claim 1 or 2, further comprising the following mutations: truncating at any amino acid position in positions 480-489.
  7. 7. The mutant according to claim 1 or 2, further comprising the following mutations: the trimerization domain is fused at the C-terminus.
  8. 8. A mutant according to claim 1 or claim 2, wherein amino acids 86 to 112 are replaced by flexibility LINKER PGCGSGGSG or 97 to 106 are replaced by flexibility LINKER GSGSGR.
  9. 9. The mutant according to claim 1 or 2, wherein the wild-type human metapneumovirus F protein comprises an amino acid sequence as shown in SEQ ID No.1 or SEQ ID No. 2.
  10. 10. A recombinant protein multimer comprising the amino acid sequences of the mutants according to any of claims 1 to 9 at positions 26 to 85 and 113 to 485 or at positions 26 to 96 and 107 to 485.
  11. 11. A nucleic acid encoding the mutant according to any one of claims 1 to 9, or the recombinant protein multimer according to claim 10.
  12. 12. A biological material comprising the nucleic acid according to claim 11, wherein the biological material is an expression cassette, a vector, a cell or a recombinant viral particle.
  13. 13. A kit comprising the mutant of any one of claims 1-9, or the recombinant protein multimer of claim 10, or the nucleic acid of claim 11, or the biological material of claim 12.
  14. 14. Use of the mutant according to any one of claims 1 to 9, or the recombinant protein multimer according to claim 10, in any one of the following: (1) Preparing a medicament for preventing or treating hMPV infection; (2) Preparing an immunogenic composition that induces the body to produce neutralizing antibodies against hMPV; (3) Preparing a reagent or a kit for detecting the hMPV antibody; (4) Screening or preparing an anti-hMPV antibody; (5) hMPV-specific B cells were isolated.
  15. 15. Use of a nucleic acid according to claim 11 or a biomaterial according to claim 12 for the preparation of an mRNA vaccine or a DNA vaccine for the prevention or treatment of hMPV infection.

Description

Mutant of pre-fusion human metapneumovirus F protein and application thereof Technical Field The invention relates to the technical field of biological products, in particular to a mutant of pre-fusion human metapneumovirus F protein and application thereof. Background Human metapneumovirus (Human Metapneumovirus, hMPV) is one of the important pathogens causing acute respiratory infections worldwide, belonging to the family paramyxoviridae (Paramyxoviridae). Epidemiological data show that hMPV is one of the main causes of severe lower respiratory tract infection in infants, the elderly and immunocompromised individuals, and has clinical symptoms similar to Respiratory Syncytial Virus (RSV) infection, which can cause serious complications such as bronchitis and pneumonia. However, up to now, there is no preventive vaccine or specific therapeutic drug against hMPV marketed worldwide, mainly in symptomatic therapy clinically, so there is a need to develop safe and effective prevention and control strategies. Among the viral structural proteins of hMPV, fusion protein (F protein) is the main transmembrane glycoprotein located on the surface of viral envelope, belonging to the type I viral Fusion glycoprotein. The F protein plays a vital role in the virus infection process and is responsible for mediating fusion of a virus envelope and a host cell membrane so as to enable virus genetic material to enter the host cell. During biosynthesis, the F protein is initially synthesized translationally in the form of an inactive single polypeptide precursor (F0), and is subsequently cleaved by proteolytic cleavage by the host cell to form disulfide-linked F2 and F1 subunits, which are further assembled to form a biologically active Pre-fusion trimer in a metastable state. The prior study shows that the hMPV F protein has high sequence conservation among different strains and contains main neutralizing epitope, so the hMPV F protein is considered to be a vaccine target antigen with great potential. In particular, type I fusion proteins that are stable in the pre-fusion conformation are capable of inducing the production of neutralizing antibodies of high potency, which are significantly more immunogenic than the Post-fusion conformation. Although F protein is an ideal vaccine development target, the vaccine development based on wild type hMPV F protein in the prior art still faces the following significant technical bottlenecks, namely, first, poor conformational stability. Wild-type hMPV F protein has thermodynamic metastable character when in pre-fusion conformation, and is extremely unstable. The protein is highly susceptible to irreversible conformational changes during in vitro expression, purification or formulation, spontaneously transitioning from a highly immunogenic pre-fusion conformation to a less immunogenic post-fusion conformation. Studies have shown that most potent neutralizing antibody epitopes are specifically present in the pre-fusion conformation and that once conformational inversion occurs, these critical epitopes are lost or masked, resulting in a substantial decrease in the ability to induce neutralizing antibodies, thereby severely affecting the immunoprotection effect of the vaccine. Second, heterologous expression is inefficient. The wild F protein has lower expression level in the common heterologous expression systems such as CHO cells, 293F cells and the like, and high-purity and high-yield recombinant protein is difficult to obtain. This disadvantage not only increases the cost of production, but also makes it difficult to meet the demands of large-scale vaccine production and industrialization. Third, immunogenicity is to be further enhanced. Although the native F protein is capable of eliciting a certain immune response, it is often difficult to induce sufficient titers of protective neutralizing antibodies using the wild-type sequence alone due to epitope loss resulting from the conformational instability described above. In summary, how to obtain hMPV F protein mutants with high expression levels and excellent immunogenicity, which can be stably maintained in a pre-fusion conformation, by means of molecular design, is a key technical problem to be solved in the current hMPV vaccine development field. Disclosure of Invention In order to solve the problems in the prior art, the invention provides a mutant of a pre-fusion human metapneumovirus F protein and application thereof. In a first aspect, the invention provides a mutant of human metapneumovirus F protein, which is obtained by carrying out mutation on the basis of wild type human metapneumovirus F protein, wherein amino acids 86-112 or 97-106 are replaced by flexible linker. Further, the method further comprises the following steps: Mutating one or more of the amino acid sequences at positions 122, 125, 127, 129, 133, 134, 151, 168, 196, 258, 259, 260, 263 or 268 to C; Preferably, the mutation of amino acid 122 to C is also included.