A nano-delivery system based on attenuated Salmonella EN-VNP outer membrane vesicles and its preparation method and application

Abstract

The present invention discloses a nano-delivery system based on attenuated Salmonella EN-VNP outer membrane vesicles, a preparation method and an application thereof, wherein the nano-delivery system based on attenuated Salmonella EN-VNP outer membrane vesicles comprises an OMV vesicle carrier EMV of EN-VNP, a protein antigen and a linker; the protein antigen is any one of a bacterial antigen, a viral antigen or a tumor antigen; and the linker is a biotin-streptavidin coupling technology to achieve antigen coupling. The present invention provides a safe, efficient and stable antigen delivery system, which has broad application prospects, especially in the fields of vaccine development and tumor treatment.

Description

Technical Field

[0001] The present invention relates to the fields of biogenetic engineering technology and vaccine manufacturing, and in particular to an attenuated Salmonella EN-VNP outer membrane vesicle (EMV) nano-delivery system and a preparation method and application thereof. Background Art

[0002] OMVs are outer membrane vesicles released by Gram-negative bacteria, mainly containing bacterial outer membrane and periplasmic components. The outer membrane is composed of phospholipids and lipopolysaccharides, with lipopolysaccharides located on the outside of the membrane and interspersed with membrane proteins. The lumen of the vesicle may contain a variety of substances derived from the bacterial periplasm or cytoplasm, such as proteins, RNA or DNA, and peptidoglycan. Since OMVs have been found to induce T cell immune responses, many studies have been conducted to study the potential immunity of OMV components in vivo. OMVs rely on receptor-mediated endocytosis to enter cells and are able to present the antigens they carry to APC cells, making OMVs both adjuvants and very ideal vaccine carriers.

[0003] In addition, OMV itself can also be used as a vaccine. So far, the first generation of vaccines containing OMV components, Bexsero (Novartis), has been approved for marketing to prevent epidemic meningitis group B bacterial infection. There are also several OMV-based vaccine products focusing on infectious diseases, and vaccine development is in the clinical stage, including pneumonia, meningitis, whooping cough, etc. OMVs have demonstrated a series of unique abilities in inducing host cell immune responses, including the production of protective humoral and cellular immune responses. Therefore, OMVs can provide a platform for vaccine development. Using OMVs derived from Escherichia coli and Salmonella as vaccine antigen delivery systems to induce effective immune cell responses is one of the current research hotspots, and has been discussed in depth in many literatures.

[0004] Compared with traditional whole pathogen vaccines, subunit vaccines based on single antigens provide more precise targeting and excellent safety. However, this also determines that they have weak immunogenicity, and adjuvants need to be added to improve the immunogenicity of antigens and activate immune responses. Unfortunately, there are few clinically applicable adjuvants with high efficiency and low toxicity, and new safe and potent adjuvants are urgently needed. OMV has good biocompatibility and can be taken up by APC (antigen presenting cells). It has the potential to be used as an adjuvant and antigen delivery carrier, but OMV contains a variety of pathogen-associated molecular patterns (PAMP) components, such as lipopolysaccharide (LPS), flagellin, lipoprotein, DNA, RNA, and peptidoglycan, which can promote the release of inflammatory cytokines, causing fever, programmed cell death, and sepsis, etc., which limits the clinical use of OMV. Therefore, the construction of a safe and effective outer membrane vesicle delivery system is of great significance in vaccine research.

[0005] When it comes to antigen delivery, there are many customized solutions, but there is no one-size-fits-all solution, and different solutions are suitable for different antigen or pathogen delivery.

[0006] For example, the most successful case so far is GSK's AS delivery system, which mainly includes MPLA (monophosphoryl lipid A derived from Salmonella typhimurium), QS-21 (plant saponin components extracted from soap trees), liposomes and aluminum adjuvants, etc. Different combinations are formulated for different pathogen antigens to achieve the goal of activating humoral immune responses and cellular immune responses. The products on the market include herpes zoster vaccine, human papillomavirus vaccine HPV, etc. When reviewing the data on AS adjuvant-delivered vaccines, it was found that the adverse reactions of AS were high, especially for VZV vaccines, which had a poor vaccination experience and poor compliance, seriously affecting the application of vaccines. In addition, MPLA is derived from LPS, an extract of the outer membrane of Salmonella, and is prepared through a hydrolysis process. This hydrolysis process determines that MPLA has heterogeneity, large batch differences, and difficult quality control.

[0007] LNP is also a very important delivery system at present. The surface of LNP has a positive charge and is mainly used for the delivery of mRNA nucleic acid vaccines. It is not suitable for the delivery of protein antigens.

[0008] Tuberculosis caused by infection with Mycobacterium tuberculosis is an ancient disease that seriously endangers human health. There are multiple vaccine development routes being promoted simultaneously, including live attenuated vaccine BCG, subunit vaccine, mRNA vaccine, DNA vaccine, etc. The combination of the two antigens ESAT-6 and CFP-10 is a research hotspot in this field. ESAT-6 and CFP-10 are used to develop diagnostic reagents on the one hand and vaccines on the other. Specific diagnostic reagents have been successfully used in large-scale population tuberculosis screening, and there has been no breakthrough in vaccine research. A suitable delivery system is needed to present antigens to human cells and activate appropriate humoral and cellular immune responses. Summary of the invention

[0009] In view of the deficiencies in the above-mentioned prior art, the present application provides a Salmonella EN-VNP attenuated based on flagellin and LPS mutations, and its outer membrane vesicle OMV is used as an antigen delivery system, referred to as EMV. The EMV of the attenuated Salmonella EN-VNP has high safety and T cell adjuvant effect as an antigen delivery nanocarrier. The EMV delivery system of the present invention is suitable for various types of protein antigens and is a universal platform. The present invention selects tuberculosis-specific antigens as model antigens and describes the preparation and application of the delivery system. Therefore, the EMV of the present invention can also deliver protein antigens of other viral or bacterial origin.

[0010] To achieve the above application purpose, the technical solution adopted in this application is as follows:

[0011] In a first aspect, the present application provides an outer membrane vesicle nano-delivery system based on attenuated Salmonella EN-VNP.

[0012] In a second aspect, the present application provides a method for preparing an outer membrane vesicle nanodelivery system based on attenuated Salmonella EN-VNP.

[0013] In a third aspect, the present application provides a vaccine.

[0014] In a fourth aspect, the present application provides the use of an attenuated Salmonella EN-VNP outer membrane vesicle nanodelivery system as a vaccine carrier.

[0015] The first aspect of the present application provides a nano-delivery system based on outer membrane vesicles of attenuated Salmonella EN-VNP. The nano-delivery system based on outer membrane vesicles of attenuated Salmonella EN-VNP includes outer membrane vesicles EMV, protein antigens and linkers; the protein antigen is any one of bacterial antigens, viral antigens or tumor antigens; the linker is a biotin-streptavidin coupling technology to achieve antigen coupling.

[0016] Furthermore, the outer membrane vesicle EMV is derived from the outer membrane vesicle formed by the modified attenuated Salmonella typhimurium EN-VNP strain, does not contain flagella and LPS toxic components; the outer membrane vesicle has a double-layer membrane structure, and the diameter of the outer membrane vesicle is 20-200nm.

[0017] Furthermore, a modified attenuated Salmonella typhimurium EN-VNP strain was prepared: based on the Salmonella VNP20009 strain, the flagellin genes fliC, fljB, the phosphoethanolamine transferase gene eptA, the 4-amino-4-deoxy-L-arabinose transferase gene arnT, the phosphotransferase yeiU were knocked out or silenced in sequence, and the phosphotransferase gene lpxE was knocked in or overexpressed to form a strain to obtain the attenuated Salmonella typhimurium EN-VNP strain.

[0018] Furthermore, the nucleotide sequence of the gene fliC is shown in SEQ ID NO.1, and the amino acid sequence of the protein encoded by the gene fliC is shown in SEQ ID NO.2; the nucleotide sequence of the gene fljB is shown in SEQ ID NO.3, and the amino acid sequence of the protein encoded by the gene fljB is shown in SEQ ID NO.4. The phosphoethanolamine transferase gene The nucleotide sequence of eptA is shown in SEQ ID NO.5,The amino acid sequence of the protein encoded by the phosphoethanolamine transferase gene eptA is shown in SEQ ID NO.6; the nucleotide sequence of the 4-amino-4-deoxy-L-arabinose transferase gene arnT is shown in SEQ ID NO.7, and the amino acid sequence of the protein encoded by the 4-amino-4-deoxy-L-arabinose transferase gene arnT is shown in SEQ ID NO.8; the nucleotide sequence of the phosphotransferase gene yeiU is shown in SEQ ID NO.9, and the amino acid sequence of the protein encoded by the phosphotransferase gene yeiU is shown in SEQ ID NO.10; the nucleotide sequence of the phosphotransferase gene lpxE is shown in SEQ ID NO.11, and the amino acid sequence of the protein encoded by the phosphotransferase gene lpxE is shown in SEQ ID NO.12; the nucleotide sequence of the upstream homologous arm of the knockout flagellin gene fliC is shown in SEQ ID NO.13, and the nucleotide sequence of the downstream homologous arm of the knockout flagellin gene fliC is shown in SEQ ID NO. IDNO.14; the nucleotide sequence of the upstream homologous arm of the knockout flagellin gene fljB is shown in SEQ ID NO.15, and the nucleotide sequence of the downstream homologous arm of the knockout flagellin gene fljB is shown in SEQ ID NO.16; the nucleotide sequence of the upstream homologous arm of the knockout phosphoethanolamine transferase gene eptA is shown in SEQ ID NO.17, and the nucleotide sequence of the downstream homologous arm of the knockout phosphoethanolamine transferase gene eptA is shown in SEQ ID NO.18.

[0019] Furthermore, the nucleotide sequence of the upstream homologous arm of the knocked-out 4-amino-4-deoxy-L-arabinose transferase gene arnT is shown as SEQ ID NO.19, and the nucleotide sequence of the downstream homologous arm of the knocked-out 4-amino-4-deoxy-L-arabinose transferase gene arnT is shown as SEQ ID NO.20; the nucleotide sequence of the upstream homologous arm of the knocked-out phosphotransferase yeiU is shown as SEQ ID NO.21, and the nucleotide sequence of the downstream homologous arm of the knocked-out phosphotransferase yeiU is shown as SEQ ID NO.22; the nucleotide sequence of the upstream homologous arm of the knocked-in phosphotransferase gene lpxE is shown as SEQ ID NO.23, and the nucleotide sequence of the downstream homologous arm of the knocked-in phosphotransferase gene lpxE is shown as SEQ ID NO.24.

[0020] Furthermore, the LPS component is a non-toxic LPS modified by gene editing; the coupling of biotin to EMV vesicles is a chemical coupling of biotinylated EMV, and the connection of streptavidin to the target protein is fusion expressed on a peptide chain, with streptavidin at the N-terminus of the peptide chain.

[0021] The second aspect of the present application provides a method for preparing an attenuated Salmonella EN-VNP outer membrane vesicle nano-delivery system, comprising the following steps:

[0022] (1) Preparing an attenuated Salmonella typhimurium EN-VNP strain;

[0023] (2) collecting the culture supernatant of the strain and extracting the outer membrane vesicles (EMV) through ultracentrifugation and purification process;

[0024] (3) Protein antigens were coupled to EMV vesicles using biotin-streptavidin coupling technology.

[0025] Furthermore, the diameter of the outer membrane vesicle EMV is 20-200nm; the outer membrane vesicle EMV does not contain flagella and LPS toxic components; the coupling step includes biotinylating the EMV vesicle and then coupling it with the target protein fusion expression peptide chain containing streptavidin.

[0026] The third aspect of the present application provides an application of an attenuated Salmonella EN-VNP outer membrane vesicle nanodelivery system in the preparation of a vaccine, which utilizes outer membrane vesicles to load and deliver target antigens or nucleic acids.

[0027] The fourth aspect of the present application provides a vaccine preparation comprising an attenuated Salmonella EN-VNP outer membrane vesicle nano-delivery system.

[0028] Furthermore, the antigen protein or nucleic acid is embedded or coupled with the EMV outer membrane vesicle antigen delivery system.

[0029] The EMV outer membrane vesicles of the present invention can be prepared according to conventional methods in the art, such as removing bacteria and bacterial fragments, concentrating the supernatant of bacterial culture fluid, ultracentrifuging and purifying similar processes to extract outer membrane vesicles.

[0030] The outer side of the vesicles of the EMV antigen delivery system of the present invention can be coupled or adsorbed with target protein antigens or nucleic acids, and the outer membrane vesicles can also be coated with target protein antigens or nucleic acids for delivery to cells in vivo.

[0031] The antigen protein of the present invention and the outer membrane vesicle EMV antigen delivery system of the present invention can be embedded or coupled according to conventional methods in the art.

[0032] The antigen protein of the present invention may be an antigen protein commonly used in the art, such as an antigen protein of a preventive vaccine or an antigen protein of a tumor vaccine.

[0033] Beneficial effects: The present invention provides a safe, efficient and stable antigen delivery system with broad application prospects, especially in the fields of vaccine development and tumor treatment. Compared with the unmodified strain, the EMV-induced inflammatory factor production ability is significantly reduced, and it has low toxicity characteristics.

[0034] Compared with the prior art, the present invention has the following advantages: (1) Improved safety: The outer membrane vesicles (EMV) of the present invention are derived from modified EN-VNP strains and do not contain flagella and LPS toxic components, thereby improving the safety of the delivery system.

[0035] (2) Versatility: The EMV delivery system is suitable for all types of protein antigens, including bacterial antigens, viral antigens or tumor antigens, and is a universal platform. Efficient antigen delivery: Through biotin-streptavidin coupling technology, EMV can efficiently achieve antigen loading and delivery.

[0036] (3) Long-term stability: EMV has long-term stability when stored at -20°C, including the stability of its composition and double-layer membrane structure.

[0037] (4) Immune activation: EMV can effectively activate the immune system, including long-term retention in lymph nodes and promoting the proliferation of germinal center B cells.

[0038] (5) Enhanced immune response: EMV as a protein antigen delivery system can increase the uptake of protein antigens by antigen presenting cells (APCs), increase the production of antigen-specific IgG antibodies, and induce cellular immune responses, including an increase in the proportion of CD4+ T cells and CD8+ T cells.

[0039] (6) Application of preventive vaccines and tumor vaccines: EMV can be used to manufacture preventive vaccines and tumor vaccines for injection. Prevention of tuberculosis infection: When EMV is used as a tuberculosis antigen delivery system, it can prevent tuberculosis infection and reduce the infection of tuberculosis to the lungs. Effective antigen delivery: The outer surface of EMV can be coupled or adsorbed with the target protein antigen, and the target protein antigen can also be encapsulated in the EMV vesicles and delivered to cells in the body. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0041] Figure 1 This is an electron microscope image of the EMV vesicle of Example 1 of the present invention.

[0042] Figure 2 This is a graph showing the expression levels of inflammatory factors in Example 2 of the present invention.

[0043] Figure 3 This is a diagram of germinal center B cell proliferation in Example 3 of the present invention.

[0044] Figure 4 This is a graph showing the long-term stability of EMV of Example 4 of the present invention.

[0045] Figure 5 This is a diagram of the EMV surface-coupled red fluorescent protein antigen according to Example 5 of the present invention.

[0046] Figure 6 This is a diagram showing the uptake of antigens by dendritic cells in Example 6 of the present invention.

[0047] Figure 7 This is a diagram of the antigen humoral immune response of Example 7 of the present invention.

[0048] Figure 8 CD4 of Example 8 of the present invention + Diagram of T cell immune response.

[0049] Fig. 9 CD8 of Example 8 of the present invention + Diagram of T cell immune response.

[0050] Fig.10 This is a diagram for preventing tuberculosis infection according to Example 9 of the present invention. DETAILED DESCRIPTION

[0051] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present application more clearly understood, the present application is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application.

[0052] In this application, the term "and / or" describes the association relationship of associated objects, indicating that there may be three relationships. For example, A and / or B can mean: A exists alone, A and B exist at the same time, and B exists alone. A and B can be singular or plural. The character " / " generally indicates that the associated objects are in an "or" relationship.

[0053] In this application, "at least one" means one or more, and "plurality" means two or more. "At least one of the following" or similar expressions refers to any combination of these items, including any combination of single items or plural items. For example, "at least one of a, b, or c", or "at least one of a, b, and c" can all mean: a, b, c, ab (i.e., a and b), ac, bc, or abc, where a, b, c can be single or multiple, respectively.

[0054] It should be understood that in the various embodiments of the present application, the size of the serial numbers of the above-mentioned processes does not mean the order of execution, some or all of the steps can be executed in parallel or sequentially, and the execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0055] The terms used in the embodiments of the present application are only for the purpose of describing specific embodiments, and are not intended to limit the present application. The singular forms "a", "said" and "the" used in the embodiments of the present application and the appended claims are also intended to include plural forms, unless the context clearly indicates other meanings.

[0056] The following examples are provided for better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from conventional biochemical reagent stores unless otherwise specified.

[0057] The following examples are provided for better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from conventional biochemical reagent stores unless otherwise specified.

[0058] The first aspect of the embodiment of the present application provides a nano-delivery system based on an outer membrane vesicle of attenuated Salmonella. The nano-delivery system based on the outer membrane vesicle of attenuated Salmonella EN-VNP includes an outer membrane vesicle carrier EMV, a protein antigen and a linker; the protein antigen is any one of a bacterial antigen, a viral antigen or a tumor antigen; the linker is a biotin-streptavidin coupling technology to achieve antigen coupling.

[0059] In some embodiments, the outer membrane vesicle EMV is derived from the outer membrane vesicle formed by the modified attenuated Salmonella typhimurium EN-VNP strain, does not contain flagella and LPS toxic components; the outer membrane vesicle has a double membrane structure, and the diameter of the outer membrane vesicle is 20-200 nm.

[0060] In some embodiments, a modified attenuated Salmonella typhimurium EN-VNP strain is prepared: based on the Salmonella VNP20009 strain, the flagellin genes fliC, fljB, the phosphoethanolamine transferase gene eptA, the 4-amino-4-deoxy-L-arabinose transferase gene arnT, the phosphotransferase yeiU are knocked out or silenced in sequence, and the phosphotransferase gene lpxE is knocked in or overexpressed to form a strain to obtain an attenuated Salmonella typhimurium EN-VNP strain.

[0061] In some embodiments, the nucleotide sequence of the gene fliC is shown in SEQ ID NO.1, and the amino acid sequence of the protein encoded by the gene fliC is shown in SEQ ID NO.2; the nucleotide sequence of the gene fljB is shown in SEQ ID NO.3, and the amino acid sequence of the protein encoded by the gene fljB is shown in SEQ ID NO.4; the nucleotide sequence of the phosphoethanolamine transferase gene eptA is shown in SEQ ID NO.5, and the amino acid sequence of the protein encoded by the phosphoethanolamine transferase gene eptA is shown in SEQ ID NO.6; the nucleotide sequence of the 4-amino-4-deoxy-L-arabinose transferase gene arnT is shown in SEQ ID NO.7, and the amino acid sequence of the protein encoded by the 4-amino-4-deoxy-L-arabinose transferase gene arnT is shown in SEQ ID NO.8; the nucleotide sequence of the phosphotransferase gene yeiU is shown in SEQ ID NO.9, and the amino acid sequence of the protein encoded by the phosphotransferase gene yeiU is shown in SEQ ID NO.10; the nucleotide sequence of the phosphotransferase gene lpxE is shown in SEQ ID NO. NO.11, the amino acid sequence of the protein encoded by the phosphotransferase gene lpxE is shown in SEQ ID NO.12; the nucleotide sequence of the upstream homologous arm of the knockout flagellin gene fliC is shown in SEQ ID NO.13, and the nucleotide sequence of the downstream homologous arm of the knockout flagellin gene fliC is shown in SEQ ID NO.14; the nucleotide sequence of the upstream homologous arm of the knockout flagellin gene fljB is shown in SEQ ID NO.15, and the nucleotide sequence of the downstream homologous arm of the knockout flagellin gene fljB is shown in SEQ ID NO.16; the nucleotide sequence of the upstream homologous arm of the knockout phosphoethanolamine transferase gene eptA is shown in SEQ ID NO.17, and the nucleotide sequence of the downstream homologous arm of the knockout phosphoethanolamine transferase gene eptA is shown in SEQ ID NO.18.

[0062] In some embodiments, the nucleotide sequence of the upstream homologous arm of the knock-out 4-amino-4-deoxy-L-arabinose transferase gene arnT is shown as SEQ ID NO.19, and the nucleotide sequence of the downstream homologous arm of the knock-out 4-amino-4-deoxy-L-arabinose transferase gene arnT is shown as SEQ ID NO.20; the nucleotide sequence of the upstream homologous arm of the knock-out phosphotransferase yeiU is shown as SEQ ID NO.21, and the nucleotide sequence of the downstream homologous arm of the knock-out phosphotransferase yeiU is shown as SEQ ID NO.22; the nucleotide sequence of the upstream homologous arm of the knock-in phosphotransferase gene lpxE is shown as SEQ ID NO.23, and the nucleotide sequence of the downstream homologous arm of the knock-in phosphotransferase gene lpxE is shown as SEQ ID NO.24.

[0063] In some embodiments, the LPS component is a non-toxic LPS modified by gene editing; the coupling of biotin to EMV vesicles is a chemical coupling of biotinylated EMV, and the connection of streptavidin to the target protein is fusion expressed on a peptide chain, with streptavidin at the N-terminus of the peptide chain.

[0064] The second aspect of the embodiment of the present application provides a method for preparing an attenuated Salmonella EN-VNP outer membrane vesicle EMV nano delivery system, comprising the following steps:

[0065] (1) Preparing an attenuated Salmonella typhimurium EN-VNP strain;

[0066] (2) collecting the culture supernatant of the strain and extracting the outer membrane vesicles (EMV) through ultracentrifugation and purification process;

[0067] (3) Protein antigens were coupled to EMV vesicles using biotin-streptavidin coupling technology.

[0068] In some embodiments, the diameter of the outer membrane vesicle EMV is 20-200 nm; the outer membrane vesicle EMV does not contain flagella and LPS toxic components; the coupling step includes biotinylating the EMV vesicle and then coupling it with the target protein fusion expression peptide chain containing streptavidin.

[0069] A third aspect of an embodiment of the present application provides an application of an attenuated Salmonella outer membrane vesicle nanodelivery system in the preparation of a vaccine, which utilizes outer membrane vesicles to load and deliver target antigens or nucleic acids.

[0070] The fourth aspect of the embodiments of the present application provides a vaccine preparation comprising an attenuated Salmonella outer membrane vesicle nano-delivery system.

[0071] In some embodiments, antigenic proteins or nucleic acids are embedded or coupled to the outer membrane vesicle EMV antigen delivery system.

[0072] The outer membrane vesicles EMV of the present invention can be prepared according to conventional methods in the art, such as removing bacteria and bacterial fragments, concentrating the supernatant of bacterial culture fluid, ultracentrifuging and purifying similar processes to extract the outer membrane vesicles.

[0073] The outer side of the outer membrane vesicle antigen delivery system EMV of the present invention can be coupled or adsorbed with the target protein antigen or nucleic acid, and the inner side of the outer membrane vesicle EMV can also be coated with the target protein antigen or nucleic acid for delivery to cells in the body.

[0074] The antigen protein of the present invention and the outer membrane vesicle EMV antigen delivery system of the present invention can be embedded or coupled according to conventional methods in the art.

[0075] The antigen protein of the present invention may be an antigen protein commonly used in the art, such as an antigen protein of a preventive vaccine or an antigen protein of a tumor vaccine.

[0076] The Salmonella EN-VNP strain described in the present invention is derived from the results previously successfully constructed by this laboratory and has been properly preserved in the strain preservation facility of this laboratory.

[0077] Example 1

[0078] The invention discloses an attenuated Salmonella EN-VNP outer membrane vesicle nano-delivery system, which comprises an outer membrane vesicle carrier EMV, a protein antigen and a linker; the protein antigen is any one of a bacterial antigen, a viral antigen or a tumor antigen; and the linker is a biotin-streptavidin coupling technology to achieve antigen coupling.

[0079] The EMV vesicles are prepared from a modified attenuated Salmonella typhimurium EN-VNP strain and do not contain flagella and LPS toxic components; the outer membrane vesicles formed by the attenuated Salmonella typhimurium EN-VNP strain have a double-layer membrane structure and a diameter of 20-200nm.

[0080] The LPS component is a non-toxic LPS modified by gene editing; the coupling of biotin to EMV vesicles is a chemical coupling of EMV biotinylation, and the connection of streptavidin to the target protein is fusion expressed on a peptide chain, with streptavidin at the N-terminus of the peptide chain.

[0081] Example 2

[0082] The preparation method of the attenuated Salmonella outer membrane vesicle nano-delivery system of the present invention comprises the following steps:

[0083] (1) Preparing an attenuated Salmonella typhimurium EN-VNP strain;

[0084] The attenuated Salmonella typhimurium EN-VNP strain was prepared by knocking out or silencing the flagellin genes fliC and fljB, the phosphoethanolamine transferase gene eptA, the 4-amino-4-deoxy-L-arabinose transferase gene arnT, the phosphotransferase gene yeiU in sequence and knocking in or overexpressing the phosphotransferase gene lpxE on the basis of the Salmonella VNP20009 strain.

[0085] The nucleotide sequence of the gene fliC is shown in SEQ ID NO.1, and the amino acid sequence of the protein encoded by the gene fliC is shown in SEQ ID NO.2; the nucleotide sequence of the gene fljB is shown in SEQ ID NO.3 As shown, the gene fljB The amino acid sequence of the encoded protein is shown in SEQ ID NO.4; the phosphate The nucleotide sequence of the ethanolamine transferase gene eptA is shown in SEQ ID NO.5, and the amino acid sequence of the protein encoded by the phosphoethanolamine transferase gene eptA is shown in SEQ ID NO.6; the nucleotide sequence of the 4-amino-4-deoxy-L-arabinose transferase gene arnT is shown in SEQ ID NO.7, and the amino acid sequence of the protein encoded by the 4-amino-4-deoxy-L-arabinose transferase gene arnT is shown in SEQ ID NO.8; the nucleotide sequence of the phosphotransferase gene yeiU is shown in SEQ ID NO.9, and the amino acid sequence of the protein encoded by the phosphotransferase gene yeiU is shown in SEQ ID NO.10; the nucleotide sequence of the phosphotransferase gene lpxE is shown in SEQ ID NO.11, and the amino acid sequence of the protein encoded by the phosphotransferase gene lpxE is shown in SEQ ID NO.12; the nucleotide sequence of the upstream homologous arm of the knockout flagellin gene fliC is shown in SEQ ID NO.13, and the nucleotide sequence of the downstream homologous arm of the knockout flagellin gene fliC is shown in SEQ ID NO. NO.14; the nucleotide sequence of the upstream homologous arm of the knockout flagellin gene fljB is shown in SEQ ID NO.15, and the nucleotide sequence of the downstream homologous arm of the knockout flagellin gene fljB is shown in SEQ ID NO.16; the nucleotide sequence of the upstream homologous arm of the knockout phosphoethanolamine transferase gene eptA is shown in SEQ ID NO.17, and the nucleotide sequence of the downstream homologous arm of the knockout phosphoethanolamine transferase gene eptA is shown in SEQ ID NO.18.

[0086] The nucleotide sequence of the upstream homologous arm of the knocked-out 4-amino-4-deoxy-L-arabinose transferase gene arnT is shown as SEQ ID NO.19, and the nucleotide sequence of the downstream homologous arm of the knocked-out 4-amino-4-deoxy-L-arabinose transferase gene arnT is shown as SEQ ID NO.20; the nucleotide sequence of the upstream homologous arm of the knocked-out phosphotransferase yeiU is shown as SEQ ID NO.21, and the nucleotide sequence of the downstream homologous arm of the knocked-out phosphotransferase yeiU is shown as SEQ ID NO.22; the nucleotide sequence of the upstream homologous arm of the knocked-in phosphotransferase gene lpxE is shown as SEQ ID NO.23, and the nucleotide sequence of the downstream homologous arm of the knocked-in phosphotransferase gene lpxE is shown as SEQ ID NO.24.

[0087] (2) collecting the culture supernatant of the strain and extracting the outer membrane vesicles (EMV) through ultracentrifugation and purification process;

[0088] Extraction, preparation and characterization of outer membrane vesicles

[0089] The strain EN-VNP was expanded and the seed solution was inoculated into the fermentation basal medium (ratio: 10 g / L plant peptone, 5 g / L yeast extract, 10 g / L sodium chloride) at a ratio of 1:100 (volume ratio). The fermentation parameters were set at pH 7.0, rotation speed 220 rpm, and temperature 37°C.

[0090] During the fermentation process, the OD600 value was measured every hour. At the 8th hour of fermentation, the OD 600 Centrifuge at about 15000rpm for 30min to remove bacteria and cell debris, and collect the culture supernatant. The culture supernatant is filtered through a 0.22μm filter membrane and concentrated with a 100Kda ultrafilter to collect the supernatant. Centrifuge at 100000rpm for 2h to collect pure EMV, which is the EMV vesicle (sphere) used in the delivery system, which can be embedded and coupled to deliver antigens later. Take samples and take electron microscopy for characterization. Figure 1 As shown, the diameter is 20-200nm.

[0091] The diameter of the outer membrane vesicle EMV is 20-200nm; the outer membrane vesicle EMV does not contain flagella and LPS toxic components; the coupling step includes biotinylating the EMV vesicle and then coupling it with the target protein fusion expression peptide chain containing streptavidin.

[0092] (3) Protein antigens were coupled to EMV vesicles using biotin-streptavidin coupling technology.

[0093] The invention discloses an application of an attenuated Salmonella outer membrane vesicle nano-delivery system in vaccine preparation, which utilizes outer membrane vesicles to load and deliver target antigens or nucleic acids.

[0094] The vaccine formulation of the present invention comprises an attenuated Salmonella EN-VNP outer membrane vesicle nano-delivery system.

[0095] The antigen protein or nucleic acid is embedded or coupled with the outer membrane vesicle EMV antigen delivery system.

[0096] The EMV outer membrane vesicles of the present invention can be prepared according to conventional methods in the art, such as removing bacteria and bacterial fragments, concentrating the supernatant of bacterial culture fluid, ultracentrifuging and purifying similar processes to extract outer membrane vesicles.

[0097] The outer side of the outer membrane vesicle antigen delivery system EMV of the present invention can be coupled or adsorbed with the target protein antigen or nucleic acid, and the inner side of the outer membrane vesicle EMV can also be coated with the target protein antigen or nucleic acid for delivery to cells in the body.

[0098] The antigen protein of the present invention and the outer membrane vesicle EMV antigen delivery system of the present invention can be embedded or coupled according to conventional methods in the art.

[0099] The antigen protein of the present invention may be an antigen protein commonly used in the art, such as an antigen protein of a preventive vaccine or an antigen protein of a tumor vaccine.

[0100] Test Example 1

[0101] Immune cell inflammatory factor release test

[0102] DMEM medium was supplemented with 10% complement inactivated serum, RAW246.7 macrophages were cultured, and the macrophages were stimulated with EMV prepared in Example 1 for 4 hours. The control group was the outer membrane vesicles (WT-OMV) prepared from the EN-VNP parent strain VNP 20009. The cells were then lysed with TRIZOI to extract RNA, and the RNA was reverse transcribed into cDNA. The expression of inflammatory factors was detected by Q-PCR, and the expression level of inflammatory factors was calculated by the ΔΔct method. The results are shown in Figure 2 As shown in the results, the expression levels of inflammatory factors IL-1β (WT-OMV: 274.88±4.89; EMV: 25.67±8.75), IL-6 (WT-OMV: 45.97±1.89; EMV: 14.97±1.69) and TNF-α (WT-OMV: 32.52±1.96; EMV: 13.45±0.17) were significantly decreased by 10.72, 3.07 and 2.42 times, respectively. The significant decrease in inflammatory factors such as IL-1β, IL-6 and TNF-α showed that EMV has better safety than WT-OMV.

[0103] Test Example 2

[0104] Germinal center B cell proliferation assay

[0105] Lymph node collection: BALB / C mice were immunized with EMV by intramuscular injection, and 9 days later, the inguinal lymph nodes of the mice were collected and placed in pre-cooled PBS.

[0106] Preparation of single cell suspension: Place lymph nodes on a 70 μm mesh and gently crush with a grinding rod to allow lymph node cells to filter through the mesh into PBS.

[0107] After sufficient grinding, the solution at the bottom of the sieve was collected into a 50 mL centrifuge tube, and centrifuged at 1000 rpm for 5 minutes at 4°C to remove the supernatant.

[0108] Red blood cell lysis: resuspend the cells in 1 mL of red blood cell lysis buffer and place on ice for 5 minutes, then add 10 mL of pre-cooled PBS to stop lysis and wash once with PBS.

[0109] Single cell counting and cell blocking: Use a cell counter to count the prepared single cell suspension, and block the cells with 1% BSA solution for half an hour on ice.

[0110] Cell labeling and flow cytometric analysis: B cell antibodies B220 and GL-7 were used for cell surface labeling, and the labeled cell suspension was subjected to flow cytometric analysis. Figure 3 As shown (PBS: 1.02±0.29; EMV: 4.65±1.73), EMV can significantly promote the proliferation of germinal center B cells, increasing it by 4.56 times, demonstrating that EMV is a good vaccine vector.

[0111] Test Example 3

[0112] EMV long-term stability test

[0113] The EMV prepared in Example 1 was placed in a -20°C storage device, and the EMV protein components were analyzed by SDS-PAGE on day 0, day 14, and day 28, and the EMV morphology was analyzed by transmission electron microscopy on day 0 and day 28. The results are shown in FIG. Figure 4 As shown, a, b, c are the protein components of EMV analyzed by SDS-PAGE after storage at -20°C for 0, 14 and 28 days, respectively. d and e are transmission electron microscopy images of EMV stored at -20°C for 0 and 28 days, respectively. The protein components of EMV remained stable during the 28-day storage period. The morphological analysis results showed that the morphology of EMV did not change significantly during the 28-day storage period.

[0114] Test Example 4

[0115] EMV surface coupled protein antigen test

[0116] Preparation of streptavidin-antigen protein fusion protein: composed of streptavidin protein and target antigen protein connected by a flexible linker.

[0117] Antigen protein coupling on the EMV surface: In phosphate-buffered saline, the biotin derivative Amine-PEG3-biotin, N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDAC) and EMV were incubated at 4°C for 2 hours, and Amine-PEG3-biotin was coated on the EMV surface. The 100kDa cutoff membrane ultrafiltration tube was washed to eliminate unbound Amine-PEG3-biotin and EDAC. The biotinylated EMV was mixed with the streptavidin-antigen protein fusion protein at a ratio of 2:1 (mass / mass) and incubated at 4°C for 2 hours. Ultracentrifugation was performed at 100,000×g and 4°C for 2 hours. The resulting particles were washed twice with PBS and finally resuspended in PBS buffer. The results are shown in Figure 5 As shown, the fusion protein of streptavidin protein and RFP (aRFP) was successfully loaded on the surface of EMV.

[0118] Test Example 5

[0119] Dendritic cell antigen uptake test

[0120] Dendritic cells DC2.4 were incubated with EMV (EMV-aRFP) or aRFP protein loaded with RFP protein prepared in Experimental Example 4 for 4 hours. The cells were fixed with 4% paraformaldehyde and then stained with DAPI for nuclei. The uptake was analyzed by fluorescence microscopy. The results are shown in Table 1. Figure 6 As shown, EMV, as an antigen delivery system, significantly increased the uptake of antigens by DC2.4 cells.

[0121] Test Example 6

[0122] Preparation of aluminum adjuvant loaded antigen

[0123] The aluminum adjuvant used was the most commonly used aluminum hydroxide adjuvant. The aluminum adjuvant and antigen were incubated at room temperature for 30 minutes at a mass ratio of 10:1.

[0124] Test Example 7

[0125] Humoral immune response test

[0126] The mice were immunized with EMV-RFP prepared in Experimental Example 4 and the antigen-loaded aluminum adjuvant AI-aRFP prepared in Experimental Example 6 on the 1st and 14th days, and the control groups were PBS solution, RFP solution, and EMV solution, respectively. The mouse serum was collected on the 28th day, and the RFP-specific antibody concentration in the serum was detected by ELISA. The results are shown in Figure 7As shown, the antibody concentration in the EMV-aRFP immunization group increased significantly, indicating that the EMV prepared in Experimental Example 4 fully meets the requirements of vaccine adjuvant design and antigen delivery system.

[0127] RFP is a commonly used tool protein in cell biology, and its immunogenicity is low, which is one of the reasons why it is widely used as a tool for cell biology research. Compared with aRFP (streptavidin-RFP fusion protein) as an antigen and EMV vector, the antibody concentration produced by the EMV-aRFP vaccine group (EMV: 0.0675±0.0192; aRFP: 0.0678±0.0105; EMV-aRFP: 0.3808±0.2552) was 5.61 times that of the EMV group and 5.62 times that of the aRFP group on the 14th day after immunization; on the 28th day after immunization, the antibody concentration produced by the EMV-aRFP vaccine group (EMV: 0.066±0.0077; aRFP: 0.197±0.0703; EMV-aRFP: 1.6045±0.2693) was 24.31 times that of the EMV group and 8.14 times that of the aRFP group. The above results show that the activity of EMV in stimulating immunity is lower than that of the low immunogenic antigen protein aRFP fusion protein, so EMV has better safety than aRFP; at the same time, although EMV itself has low immunogenicity, it has a good adjuvant effect and significantly enhances the immune effect of aRFP protein. On the 28th day after immunization, the antibody concentration produced by the EMV-aRFP vaccine group was about 94.4% of the traditional aluminum adjuvant (1.6998±0.2003), and the two effects were comparable.

[0128] Test Example 8

[0129] Cellular immune response assay

[0130] The mice were immunized with EMV-RFP prepared in Experimental Example 4 and the antigen-loaded aluminum adjuvant AI-aRFP prepared in Experimental Example 6 on the 1st and 14th days, and the control groups were PBS solution, RFP solution, and EMV solution, respectively. The spleens of the mice were collected on the 28th day. The spleen single cell suspension was prepared according to the method described in Example 3. CD3 / CD4 / CD8 antibodies were used for cell surface labeling, and the labeled cell suspension was subjected to flow cytometric analysis. The results are shown in FIG. Figure 8 and Fig. 9 As shown, EMV can significantly promote CD4 + T and CD8 + T cell proliferation. EMV-aRFP vaccine group promoted CD4 + The effect of T cell proliferation was significantly better than that of aluminum adjuvant (EMV-aRFP: 24.58±1.26; AI-aRFP: 18.91±0.68); promoting CD8 +The effect of T cell proliferation is also significantly better than that of aluminum adjuvant (EMV-aRFP: 10.45±0.24; AI-aRFP: 8.38±0.53). In terms of the overall immune effect of T cells and B cells, the vaccine effect of EMV as an immune adjuvant or carrier is better than that of traditional aluminum adjuvant.

[0131] Test Example 9

[0132] Mycobacterium tuberculosis challenge test

[0133] Preparation of streptavidin-Mercobacterium tuberculosis antigen fusion protein: According to the method in Experimental Example 4, a fusion protein (EMV-aEEC) was prepared using Mycobacterium tuberculosis antigens ESAT-6, CFP-10 and streptavidin.

[0134] Tuberculosis challenge: On days 0 and 21, BALB / C mice were inoculated with EMV-aEEC by intramuscular injection. The control groups were PBS, aEEC, EMV, and BCG. On day 42, the mice were injected with Mtb H37R tuberculosis via the tail vein. On day 84, the mouse lungs were collected, single cell suspensions were prepared, plated, and the number of colonies formed was counted on day 105. The experimental results are shown in Fig.10 As shown, EMV, as a delivery system for Mycobacterium tuberculosis antigens, can significantly reduce the infection of Mycobacterium tuberculosis in the lungs; its efficacy is comparable to that of the classic Mycobacterium tuberculosis vaccine BCG.

[0135] The present invention uses OVA as an antigen, couples it to EMV, immunizes B16-OVA melanoma mouse model, and compares it with aluminum adjuvant, and the two produce the same anti-tumor efficacy.

[0136] Therefore, EMV is a good vaccine adjuvant and immune carrier suitable for a variety of antigens. Its effect is comparable to that of existing vaccines and adjuvants, and its safety is better.

[0137] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, and the scope of protection of the present invention is defined by the attached claims, description and their equivalents.

Claims

1. A nano-delivery system based on attenuated Salmonella EN-VNP outer membrane vesicles, characterized in that: The attenuated Salmonella EN-VNP outer membrane vesicle nano-delivery system comprises an outer membrane vesicle EMV of EN-VNP used as a carrier, a protein antigen and a linker; the protein antigen is any one of a bacterial antigen, a viral antigen or a tumor antigen; and the linker is a biotin-streptavidin coupling technology to achieve antigen coupling.

2. The outer membrane vesicle nano-delivery system based on attenuated Salmonella carrier according to claim 1, characterized in that: The outer membrane vesicle EMV is derived from the outer membrane vesicle formed by the modified attenuated Salmonella typhimurium EN-VNP strain and does not contain flagella and LPS toxic components; the outer membrane vesicle has a double-layer membrane structure, and the diameter of the outer membrane vesicle is 20-200nm.

3. The method for preparing the attenuated Salmonella EN-VNP outer membrane vesicle nano-delivery system according to claim 2, characterized in that: Preparation of modified attenuated Salmonella typhimurium EN-VNP strain: Based on the Salmonella VNP20009 strain, the flagellin genes fliC, fljB, the phosphoethanolamine transferase gene eptA, the 4-amino-4-deoxy-L-arabinose transferase gene arnT, the phosphotransferase yeiU were knocked out or silenced in sequence, and the phosphotransferase gene lpxE was knocked in or overexpressed to form a strain to obtain the attenuated Salmonella typhimurium EN-VNP strain.

4. The method for preparing the attenuated Salmonella EN-VNP outer membrane vesicle nano-delivery system according to claim 2, characterized in that: The nucleotide sequence of the gene fliC is shown in SEQ ID NO.1, and the amino acid sequence of the protein encoded by the gene fliC is shown in SEQ ID NO.2; the nucleotide sequence of the gene fljB is shown in SEQ ID NO.3, and the amino acid sequence of the protein encoded by the gene fljB is shown in SEQ ID NO.4; the nucleotide sequence of the phosphoethanolamine transferase gene eptA is shown in SEQ ID NO.5, and the amino acid sequence of the protein encoded by the phosphoethanolamine transferase gene eptA is shown in SEQ ID NO.6; the nucleotide sequence of the 4-amino-4-deoxy-L-arabinose transferase gene arnT is shown in SEQ ID NO.7, and the amino acid sequence of the protein encoded by the 4-amino-4-deoxy-L-arabinose transferase gene arnT is shown in SEQ ID NO.8; the nucleotide sequence of the phosphotransferase gene yeiU is shown in SEQ ID NO.9, and the amino acid sequence of the protein encoded by the phosphotransferase gene yeiU is shown in SEQ ID NO.10; the nucleotide sequence of the phosphotransferase gene lpxE is shown in SEQ ID NO. NO.11, the amino acid sequence of the protein encoded by the phosphotransferase gene lpxE is shown in SEQ ID NO.12; the nucleotide sequence of the upstream homologous arm of the knockout flagellin gene fliC is shown in SEQ ID NO.13, and the nucleotide sequence of the downstream homologous arm of the knockout flagellin gene fliC is shown in SEQ ID NO.14; the nucleotide sequence of the upstream homologous arm of the knockout flagellin gene fljB is shown in SEQ ID NO.15, and the nucleotide sequence of the downstream homologous arm of the knockout flagellin gene fljB is shown in SEQ ID NO.16; the nucleotide sequence of the upstream homologous arm of the knockout phosphoethanolamine transferase gene eptA is shown in SEQ ID NO.17, and the nucleotide sequence of the downstream homologous arm of the knockout phosphoethanolamine transferase gene eptA is shown in SEQ ID NO.

18.

5. The method for preparing the attenuated Salmonella EN-VNP outer membrane vesicle nano-delivery system according to claim 2, characterized in that: The nucleotide sequence of the upstream homology arm of the knocked-out 4-amino-4-deoxy-L-arabinose transferase gene arnT is shown in SEQ ID NO.19, and the nucleotide sequence of the downstream homology arm of the knocked-out 4-amino-4-deoxy-L-arabinose transferase gene arnT is shown in SEQ ID NO.20; the nucleotide sequence of the upstream homology arm of the knocked-out phosphotransferase yeiU is shown in SEQ ID NO.21, and the nucleotide sequence of the downstream homology arm of the knocked-out phosphotransferase yeiU is shown in SEQ ID NO.22; the nucleotide sequence of the upstream homology arm of the knocked-in phosphotransferase gene lpxE is shown in SEQ ID NO.23, and the nucleotide sequence of the downstream homology arm of the knocked-in phosphotransferase gene lpxE is shown in SEQ ID NO. As shown in NO.24; the LPS component is a non-toxic LPS modified by gene editing; the coupling of biotin and EMV vesicles is the chemical coupling of EMV biotinylation, and the connection of streptavidin and target protein is fusion expressed on a peptide chain, and streptavidin is at the N-terminus of the peptide chain.

6. The method for preparing the attenuated Salmonella EN-VNP outer membrane vesicle nano-delivery system according to any one of claims 1 to 3, characterized in that The steps include: (1) Preparing an attenuated Salmonella typhimurium EN-VNP strain; (2) collecting the culture supernatant of the strain and extracting the outer membrane vesicles (EMV) through ultracentrifugation and purification process; (3) Protein antigens were coupled to EMV vesicles using biotin-streptavidin coupling technology.

7. The method for preparing the attenuated Salmonella EN-VNP outer membrane vesicle nano-delivery system according to claim 6, characterized in that: The diameter of the outer membrane vesicle EMV is 20-200nm; the outer membrane vesicle EMV does not contain flagella and LPS toxic components; the coupling step includes biotinylating the EMV vesicle and then coupling it with the target protein fusion expression peptide chain containing streptavidin.

8. Use of the attenuated Salmonella EN-VNP outer membrane vesicle nano-delivery system according to claim 1 in the preparation of vaccines, characterized in that: The outer membrane vesicles according to claim 6 are used to load and deliver target antigens or nucleic acids.

9. A vaccine formulation comprising the attenuated Salmonella EN-VNP outer membrane vesicle nano-delivery system according to claim 8.

10. The vaccine preparation according to claim 9, characterized in that: The antigen protein or nucleic acid is embedded or coupled with the outer membrane vesicle EMV antigen delivery system.

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