SARS-CoV-2 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 problem of reduced neutralizing activity of Delta mutant strain

Active Publication Date: 2022-01-14
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Studies have shown that the neutralizing activity of BNT162b2 vaccine serum against Delta mutant strains is reduced by 5.8 times

Method used

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  • SARS-CoV-2 mutant strain S protein and subunit vaccine thereof
  • SARS-CoV-2 mutant strain S protein and subunit vaccine thereof
  • SARS-CoV-2 mutant strain S protein and subunit vaccine thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0078] Example 1. Prediction of consensus sequences Due to the high mutagenicity of SARS-CoV-2, especially the key receptor binding domain (RBD) of the mutation site on the S protein, its binding ability to the receptor ACE2 may be enhanced, increasing the The transmissibility or pathogenicity of the virus; at the same time, mutations may change the key epitopes of the antigen, reducing the affinity of neutralizing antibodies that have been screened, or reducing the protective effect of vaccines and neutralizing antibodies. At present, SARS-CoV-2 has evolved Multiple types of mutations ( figure 1 ). For these mutants, we adopted four different strategies to compare the consensus sequences of mutants by software prediction.

[0079] 1. Strategy 1:

[0080] We downloaded 2674 (de-duplicated) 2019-nCoV S protein sequences from the NCBI database (as of February 28, 2021), and constructed a phylogenetic tree of these S proteins by neighbor-joining method using MEGA 7.0 software (...

Embodiment 2

[0090] Embodiment 2. Construction and expression optimization of recombinant S protein vector

[0091] 1. Construction of the S protein gene expressed in the supernatant of mammalian cells

[0092] The schematic diagram of the construction of the S protein expression gene of the present invention is shown in Image 6 and Figure 7 .

[0093] Image 6 : Consensus sequence S6 schema diagram. This sequence contains deletions of amino acids 69-70 and 144, and contains E484K, N501Y, D614G, P681H mutations. At the same time, in order to maintain the integrity of the recombinant S protein, the Furin cleavage site was mutated to GSAS, GS combination, or (GGGS)n or (GGGGS)n or (G)n, with the value of n, the length of the linker connecting S1 and S2 also change. To ensure that the recombinant S protein is secreted and expressed in the form of a native trimer, the C-terminal transmembrane domain was replaced with a T4 phage Fibritin trimer motif or a GCN4 multimer formation motif. ...

Embodiment 3

[0102] Example 3. Immunization and effect identification of recombinant S6 / S15 protein trimer vaccine

[0103] 1. The immune process of mice

[0104] The mice used in this experiment were K18-hACE female mice, 6-8 weeks, 19-25 g, purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. All animal experiments were performed in the SPF laboratory. There were 12 mice in the S6 vaccine experimental group; 5 mice in the S15 vaccine experimental group; 12 mice in the prototype strain S vaccine experimental group; and 12 mice in the control group inoculated with PBS. Orbital blood was collected from all mice 14 days after the first injection of the vaccine and 28 days after the first injection (i.e., 14 days after the second injection). The mouse serum was taken to test serum neutralizing antibody titers.

[0105] 2. The serum of mice immunized with the vaccine of the present invention contains extremely high anti-S IgG

[0106] In order to verify that the designed vaccine can...

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Abstract

The invention provides a SARS-CoV-2 mutant strain S protein and a subunit vaccine thereof. A furin cleavage site 682-RRAR-685 between the S1 subunit and the S2 subunit of the SARS-CoV-2 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, wherein n is greater than or equal to 1 and less than or equal to 3, and n is an integer; the amino acid sequence of the SARS-CoV-2 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 the SARS-CoV-2 mutant strain.

Description

[0001] Cross References to Related Applications [0002] This application claims the priority of Chinese patent application CN202110395097.0 submitted on April 13, 2021, the entire content of which is hereby incorporated by reference. technical field [0003] 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 [0004] SARS-CoV-2, which is currently in a global pandemic, is a single-stranded positive-sense RNA virus with an envelope structure, which is extremely 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 w...

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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