Novel coronavirus mutant strain S protein and subunit vaccine thereof

A subunit vaccine, coronavirus technology, applied in the field of new coronavirus mutant S protein and its subunit vaccine, can solve the problems of decreased neutralization effect and low effectiveness of B.1.351

Inactive Publication Date: 2021-08-10
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In addition, Moderna's data showed that the neutralization effect of the vaccine serum against the South African mutant strain was reduced by more than 7 times, and Pfizer's data also showed that the neutralization effect was significantly reduced[4]
Phase III clinical trial results of Novavax recombinant protein vaccine show that the effectiveness against B.1.351 is less than 50%
In summary, the emergence and prevalence of South African mutants and mutants with similar characteristics to South African mutants have posed a huge challenge to existing anti-epidemic strategies

Method used

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  • Novel coronavirus mutant strain S protein and subunit vaccine thereof
  • Novel coronavirus mutant strain S protein and subunit vaccine thereof
  • Novel coronavirus mutant strain S protein and subunit vaccine thereof

Examples

Experimental program
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preparation example Construction

[0062] According to another typical implementation of the embodiments of the present invention, a method for preparing the S protein of a novel coronavirus mutant strain is provided, the method comprising:

[0063] obtaining the recombinant expression vector;

[0064] Transfecting the recombinant expression vector into cells, and obtaining a cell line stably expressing the recombinant S protein through glutamine resistance screening and monoclonal screening of the cell population;

[0065] The cell line was secreted, expressed and purified to obtain a purified recombinant novel coronavirus mutant strain S protein.

[0066] According to another typical embodiment of the embodiment of the present invention, a subunit vaccine of a novel coronavirus mutant strain is provided, and the novel coronavirus mutant strain subunit vaccine comprises the recombinant S protein and a pharmaceutically acceptable adjuvant.

[0067] The adjuvant includes at least one of aluminum hydroxide, leci...

Embodiment 1

[0070] Example 1. Prediction of Consensus Sequence

[0071] Due to the high mutation of SARS-CoV-2, especially the mutation site in the key receptor-binding domain (RBD) on the S protein, it may strengthen its ability to bind to the receptor ACE2, increasing the transmission or pathogenicity of the virus At the same time, the mutation may change the key epitope of the antigen, reduce the affinity of the neutralizing antibody that has been screened, or reduce the protective effect of the vaccine and neutralizing antibody. Currently, SARS-CoV-2 has evolved various types of mutations ( figure 1 ). For these mutants, we adopted four different strategies to predict and compare the consensus sequences of the mutants by software.

[0072] 1. Strategy 1:

[0073]We downloaded 2674 (deduplicated) S protein sequences of 2019-nCoV from the NCBI database (as of February 28, 2021), and constructed a phylogenetic tree of these S proteins by the neighbor-joining method using MEGA 7.0 softw...

Embodiment 2

[0082] Through four strategies, we obtained the S protein consensus sequence (S6) with 6 typical mutations (69-HV-70 deletion, Y144 deletion, E484K, N501Y, D614G, P681H) and 15 characteristic mutations (69- HV-70 deletion, Y144 deletion, 242-LAL-244 deletion, and contains the S protein consensus sequence (S15) of A222V, K417N, L452R, E484K, N501Y, A570D, D614G, Q677H, P681H, T716I, S982A, D1118H). Example 2. Construction and expression optimization of recombinant S protein vector

[0083] 1. Construction of S protein gene expressed in mammalian cell supernatant

[0084] The schematic diagram of the construction of the S protein expression gene of the present invention is shown in Figure 6 and Figure 7 .

[0085] Figure 6 : Diagram of the consensus sequence S6 schema. The sequence contains deletions of 69-70 and 144 amino acids, and contains E484K, N501Y, D614G, P681H mutations. At the same time, in order to maintain the integrity of the recombinant S protein, the Furi...

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Abstract

The invention provides a novel coronavirus mutant strain S protein and a subunit vaccine thereof. The novel coronavirus mutant strain S protein is characterized in that a furin cleavage site 682-RRAR-685 between an S1 subunit and an S2 subunit of the novel coronavirus mutant strain S protein is replaced with a flexible protein linker; the linker is (GGCAGCGCCAGC), or (GGCGGCGGCAGC)n, or (GGCGGCGGCGGCAGC)n, or (GGC)n, or the linker is GSAS or (GGGS)n or (GGGGS)n or (G) n, n is greater than or equal to 1 and less than or equal to 3, and n is an integer; and the amino acid sequence of the novel coronavirus mutant strain S protein is as shown in SEQ ID NO: 2 or as shown in SEQ ID NO: 4. The S protein has great potential as a vaccine antigen for coping with an SARS-CoV-2 mutant strain.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a novel coronavirus mutant strain S protein and a subunit vaccine thereof. Background technique [0002] SARS-CoV-2, which is currently in a global pandemic, is a single-stranded positive-sense RNA virus with an envelope structure, which is highly prone to mutations. The recent emergence of SARS-CoV-2 mutant strains may strengthen its ability to bind to the receptor ACE2 due to the mutation site on the spike protein (S protein), especially in the key receptor binding domain (RBD), and increase the virus At the same time, the mutation may change the key epitope of the antigen, so that the affinity of the neutralizing antibody that has been screened will decrease, immune escape will occur, and the protective effect of the vaccine and neutralizing antibody will be reduced. At present, according to the new coronavirus pedigree information released by the GISAID database, there are more th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/85C12N5/10A61K39/215A61P31/14
CPCC07K14/005C12N15/85C12N5/0682A61K39/12A61P31/14C12N2770/20022C12N2770/20034C07K2319/00C07K2319/02C07K2319/03C12N2800/107C12N2800/22C12N2510/02
Inventor 徐可蓝柯
Owner WUHAN UNIV
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