Coronavirus vaccine based on controllable secretory expression of attenuated salmonella, preparation method therefor, and application thereof
View Patent ↗A coronavirus vaccine based on controllable secretory expression of attenuated Salmonella , a preparation method therefor, and use thereof. The method includes constructing controllable and stable expression plasmids for secretory expression of different antigenic structural domain proteins of the new coronaviruses and their attenuated Salmonella expression strains, and then mixing various attenuated Salmonella antigen-presenting strains that can achieve controllable intracellular secretory expression in antigen-presenting cells. With the aid of a unique secretion system, a variety of different antigenic proteins can be secretory-expressed efficiently in antigen-presenting cells after oral gavaging. The secretory-expressed antigenic proteins can be efficiently processed and presented by the antigen-presenting cells, and finally activate/regulate the immune system to produce more potent antibodies to make the vaccine work.
1 . A SARS-CoV-2 coronavirus vaccine based on secretory expression of attenuated Salmonella , comprising:
an antigen expression vector and an antigen presentation system for the SARS-CoV-2 coronavirus vaccine,
wherein the attenuated Salmonella is htrA gene-deficient attenuated Salmonella VNP20009 (Ah-1);
wherein the antigen expression vector is an oral vaccine presentation system capable of secretory expression through a Salmonella type III secretory expression system induced by an intracellular environment of antigen-presenting cells, the Salmonella type III antigen secretory expression system comprises a type III secretion system promoter and a signal peptide sequence and a plasmid anti-loss element;
wherein the type III secretion system promoter comprises a sifB promotor having a gene sequence of the nucleotide sequence shown in SEQ ID No. 4, the signal peptide sequence comprises a SseJ signal peptide having a gene sequence of the nucleotide sequence shown in SEQ ID No. 5; and the plasmid anti-loss element is an AT element having a gene sequence of the nucleotide sequence shown in SEQ ID No. 10; and
wherein the SARS-CoV-2 vaccine comprises a recombinant VNP20009 (Ah-1) strain that expresses a SARS-CoV-2 spike protein (S protein) RBD structural domain located at positions 319-541 of full amino acid sequence of the S protein, and the RBD structural domain has a gene sequence of the nucleotide sequence shown in SEQ ID No. 6.
2 . A method of preparing the coronavirus vaccine based on secretory expression of attenuated Salmonella according to claim 1 , comprising the steps of: mixing different attenuated Salmonella antigen-presenting strains that can express different antigenic structural domain proteins of the coronavirus by intracellular secretion to prepare the coronavirus vaccine.
3 . A method of preparing the coronavirus vaccine based on secretory expression of attenuated Salmonella according to claim 1 , comprising the steps of: constructing attenuated Salmonella antigen-presenting strains with controllable, stable and efficient intracellular secretory expression of different antigenic structural domain proteins of the coronavirus; using an intracellular inducible promoter to regulate a bacterial secretion signal to secrete and express the antigen, using the Salmonella secretory expression system to obtain secretion of the antigen, and adding a plasmid anti-loss element to improve plasmid stability of the expression vector in Salmonella cells, to obtain an efficient, stable and antigen-presenting cells intracellularly regulated secretory expression of different antigen structural domains in attenuated Salmonella cells.
4 . A method of preparing the coronavirus vaccine based on secretory expression of attenuated Salmonella according to claim 1 , comprising a combination of selected coronavirus antigenic epitope structural domains that can induce an optimal immune response.
5 . The method of preparing the coronavirus vaccine based on secretory expression of attenuated Salmonella according to claim 4 , wherein the combination of coronavirus antigenic epitope structural domains that can induce an optimal immune response is the SARS-CoV-2 spike protein (S protein) RBD structural domain located at positions 319-541 of full amino acid sequence of the S protein, and the RBD structural domain has a gene sequence of the nucleotide sequence shown in SEQ ID No. 6; or the combination of coronavirus antigenic epitope structural domains that can induce an optimal immune response is the SARS-CoV-2 spike protein (S protein) NTD structural domain located at positions 13-303 of the full amino acid sequence of the S protein, and the NTD structural domain of the ARS-CoV-2 spike protein has a gene sequence of the nucleotide sequence shown in SEQ ID No. 7; or the combination of coronavirus antigenic epitope structural domains that can induce an optimal immune response is part of the sequence of the SARS-CoV-2 spike protein (S protein) S2 structural domain located at positions 886-1077 of the full amino acid sequence of the S protein, and the S2 structural domain has a gene sequence of the nucleotide sequence shown in SEQ ID No. 8.
6 . The method of preparing the coronavirus vaccine based on secretory expression of attenuated Salmonella according to claim 4 , wherein the intracellular inducible promoter in the antigen presenting cells is Salmonella SifB promoter having a gene sequence of the nucleotide sequence shown in SEQ ID No. 4; the plasmid anti-loss element is AT element sequence having a gene sequence of the nucleotide sequence shown in SEQ ID No. 10; the secretory expression system containing bacterial secretion signal is the Salmonella type III secretory expression system, the Salmonella type III secretion signal is Salmonella virulence island 2 (SPI-2) effector protein SseJ signal peptide having a gene sequence of the nucleotide sequence shown in SEQ ID No. 5; expression of multiple vaccine antigens is achieved by attenuated Salmonella , and the attenuated Salmonella is VNP20009 or htrA gene-deficient attenuated Salmonella VNP20009 (Ah-1).
7 . A method of preparing the coronavirus vaccine based on secretory expression of attenuated Salmonella according to claim 1 , comprising the steps of: preparing a vaccine of a strain mixture of multiple recombinant attenuated Salmonella , the strain mixture comprises two recombinant attenuated Salmonella strains loaded with Ah-BJ-RBD and Ah-BJ-NTD expression plasmids, respectively, and the strain mixture comprises at least two of the abovementioned strains.
8 . The SARS-CoV-2 coronavirus vaccine based on secretory expression of attenuated Salmonella according to claim 1 , wherein the vaccine further comprises a recombinant VNP20009 (Ah-1) strain that expresses a SARS-CoV-2 spike protein (S protein) NTD structural domain located at positions 13-303 of the full amino acid sequence of the S protein, and the NTD structural domain of the ARS-CoV-2 spike protein has a gene sequence of the nucleotide sequence shown in SEQ ID No. 7.
9 . The SARS-CoV-2 coronavirus vaccine based on secretory expression of attenuated Salmonella according to claim 1 , wherein the vaccine further comprises a recombinant VNP20009 (Ah-1) strain that expresses part of the sequence of the SARS-CoV-2 spike protein (S protein) S2 structural domain located at positions 886-1077 of the full amino acid sequence of the S protein shown in SEQ ID No. 8.
10 . A SARS-CoV-2 coronavirus preventive drug comprising the SARS-CoV-2 coronavirus vaccine according claim 1 .