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CN-121991182-A - Influenza A virus H5 protein mutant and application thereof

CN121991182ACN 121991182 ACN121991182 ACN 121991182ACN-121991182-A

Abstract

The invention relates to the technical field of biology, and discloses an influenza A virus H5 protein mutant and application thereof. Aiming at the problems of weak cross immune response to epidemic strains, insufficient stability of wild HA protein, low immunogenicity and the like of the existing H5N1 vaccine, the invention constructs a stable soluble H5 protein mutant through a sequence transformation strategy. Specifically, amino acids 332-380 in the HA protein of the wild type influenza A virus H5N1 are replaced by SPGCAT, and point mutations T386C, L418P and H456P are introduced to obtain mutants. The mutant can obviously improve the structural stability of the trimeric protein, improve the expression level of the recombinant protein, widen the cross protection spectrum, strengthen the level of the immunogenic hemagglutination inhibition antibody and improve the immune response balance.

Inventors

  • LI MINGLI
  • LI KELEI
  • WANG JIANLONG
  • ZHENG HAIFA
  • ZHANG JINGFEI

Assignees

  • 北京民海生物科技有限公司

Dates

Publication Date
20260508
Application Date
20260113

Claims (10)

  1. 1. An influenza a virus H5 protein mutant, wherein the mutant is obtained by replacing amino acid residues 332-380 in the hemagglutinin protein of wild-type influenza a virus H5N1 with SPGCAT and introducing point mutations T386C, L418P and H456P.
  2. 2. The mutant according to claim 1, wherein the amino acid sequence of the mutant is shown in SEQ ID NO. 4.
  3. 3. The mutant according to claim 1 or 2, wherein the mutant further comprises at least one mutation selected from the group consisting of: (1) S219C and S233C; (2) N222C and K228C; (3) R224C and P227C; (4) T231C and H256C.
  4. 4. A mutant according to claim 3, wherein the amino acid sequence of the mutant is as shown in SEQ ID No. 5, 6, 7 or 8.
  5. 5. A nucleic acid molecule encoding the mutant according to any one of claims 1 to 4 or a biological material comprising said nucleic acid molecule; the biological material is an expression cassette, a transposon, a plasmid vector, a viral vector, engineering bacteria or a transgenic cell line.
  6. 6. Use of the mutant according to any one of claims 1 to 4 or of the nucleic acid molecule or biological material according to claim 5 for any one of the following: (1) Preparing a medicament for preventing and/or treating influenza a virus H5N1 infection; (2) Preparing an antibody of influenza A virus H5N 1; (3) Preparing a diagnostic reagent for influenza A virus H5N 1.
  7. 7. A pharmaceutical composition comprising the mutant of any one of claims 1-4 or the nucleic acid molecule or biological material of claim 5.
  8. 8. The pharmaceutical composition of claim 7, wherein the pharmaceutical composition is a vaccine; the vaccine comprises a recombinant protein vaccine, a nucleic acid vaccine, a vector vaccine or a virus-like particle vaccine.
  9. 9. The pharmaceutical composition of claim 8, wherein the nucleic acid vaccine is an mRNA-LNP vaccine, wherein the mRNA encodes the mutant of any of claims 1-4, and/or, LNP comprises cationic lipids, DSPC, cholesterol and DMG-PEG2000.
  10. 10. The pharmaceutical composition of claim 9, wherein the molar ratio of cationic lipid, DSPC, cholesterol, and DMG-PEG2000 in the mRNA-LNP vaccine is 50 (8-12): (35-40): (2-3).

Description

Influenza A virus H5 protein mutant and application thereof Technical Field The invention relates to the technical field of biology, in particular to an influenza A virus H5 protein mutant and application thereof. Background Highly pathogenic avian influenza virus H5N1 constitutes a persistent and serious threat to global public health. In recent years, 14 human H5N1 vaccines have been approved for use in different countries and regions or are included in reserve programs, including whole virus inactivated vaccines, split vaccines, subunit vaccines, attenuated live vaccines, and the like. However, the existing vaccine has weak cross immune response to the currently popular 2.3.4.4b branched virus, and development of a specific vaccine against the novel H5N1 virus is highly demanded. Besides the traditional vaccine platform, mRNA vaccine technology has obvious advantages due to the characteristics of rapid research and development, high-efficiency production, capability of inducing comprehensive immune response and the like. Influenza hemagglutinin (hemagglutinin, HA) is a key protein that mediates viral invasion into host cells and is also a major target for anti-influenza virus neutralizing antibodies. HA comprises two subunits, HA1 and HA2, distributed on the surface of the viral envelope in the form of trimeric spines. HA trimers can be structurally divided into two parts, head (head) and stem (stem). The HA head contains its receptor binding site, and the HA stem contains a hydrophobic membrane fusion peptide (fusion peptide) and a transmembrane region formed by the C-terminus of HA 2. When the virus enters the host cell, the conformation of HA changes along with the decrease of pH, and finally, the transition from the pre-membrane fusion state (pre-fusion) to the post-membrane fusion state (post-fusion) is realized, and fusion of the virus membrane and the host cell membrane is mediated. The head domain of pre-fusion HA contains multiple immunodominant sites (e.g., SA, SB, CA1, CA2, CB, etc.), which are the primary binding targets for neutralizing antibodies. After HA fusion, the structure is significantly changed, and part of the epitope may be hidden or exposed, resulting in altered immunogenicity. The stem region of fused HA may be exposed to new epitopes, but these epitopes are relatively weak in immunogenicity, and the neutralizing activity of the induced antibodies is inferior to that of antibodies directed against the head domain of HA before fusion, resulting in some limitations in vaccine design for immunogenicity of fused HA. Thus, stabilizing the trimeric conformation of HA proteins prior to fusion is critical to enhancing their immunogenicity and vaccine protection efficacy. Influenza a virus subtype H5N1 (highly pathogenic avian influenza virus) has cross-species transmission capability, can cause severe infection and death in humans, and constitutes a serious threat to public health. The existing H5N1 vaccine mainly HAs the technical bottlenecks of unstable conformation, low cross protection capability, limited cross neutralization activity on variant strains (such as 2.3.4.4b branches) due to the fact that the pre-fusion conformation of wild type HA protein is easy to be converted into the post-fusion conformation (mediated by the multi-alkaline amino acid sequences related to membrane fusion), low in immunogenicity and insufficient in induced neutralizing antibody titer, and the expression level and the solubility are low, wherein the wild type HA protein is easy to form inclusion bodies in recombinant expression, and the requirement of mass production is difficult to meet. Therefore, the development of H5 trimeric proteins that stabilize pre-fusion conformation, are highly immunogenic and cross-protected in a broad spectrum is critical to breaking through the technological bottlenecks of existing vaccines. Disclosure of Invention The invention aims to provide an influenza A virus H5 protein mutant and application thereof. To achieve the object of the present invention, in a first aspect, the present invention provides an influenza a virus H5 protein mutant obtained by replacing amino acid residues 332 to 380 in the hemagglutinin protein (HA protein) of the wild-type influenza a virus H5N1 with SPGCAT and introducing point mutations T386C, L418P and H456P. Preferably, the amino acid sequence of the mutant is shown in SEQ ID NO. 4 (i.e., mutant HA-mod 0). Further, the mutant further comprises at least one mutation selected from the group consisting of: (1) S219C and S233C; (2) N222C and K228C; (3) R224C and P227C; (4) T231C and H256C. More preferably, the amino acid sequence of the mutant is shown as SEQ ID NO. 5, 6, 7 or 8, corresponding to mutant HA-mod1, HA-mod2, HA-mod3, HA-mod4, respectively. In a second aspect, the invention provides a nucleic acid molecule encoding the mutant or a biological material comprising the nucleic acid molecule. The biological material includes, but is no