CN115725482A - Attenuated gram-negative bacteria and OMV antigen delivery systems prepared therefrom - Google Patents

Abstract

The invention discloses an attenuated gram-negative bacterium and an OMV antigen delivery system prepared by the attenuated gram-negative bacterium, and the attenuated gram-negative bacterium is a strain formed after knocking out or silencing genes fliC, fljB and a phosphoethanolamine transferase gene EptA on the basis of a wild type or prototype strain; the delivery system formed by the strain has low toxicity and T cell adjuvant effect, can be normally amplified and cultured, and has important industrial value. Can be used for preparing preventive vaccines and tumor vaccines, and can realize administration such as injection or nasal spray.

Description

Attenuated gram-negative bacteria and OMV antigen delivery systems prepared therefrom

Technical Field

The invention relates to the fields of biological genetic engineering technology and vaccine manufacture, in particular to attenuated gram-negative bacteria and an OMV antigen delivery system prepared by the attenuated gram-negative bacteria.

Background

OMVs are outer membrane vesicles released by gram-negative bacteria, mainly containing the bacterial outer membrane and periplasmic components. The outer membrane consists of phospholipids and lipopolysaccharides, which are located on the outside of the membrane with membrane proteins interspersed between them. The inner lumen of the vesicle may contain a variety of substances derived from the periplasm or cytoplasm of the bacteria, such as proteins, RNA or DNA, peptidoglycans. Because OMVs were found to induce T cell immune responses, a number of studies have been conducted with the goal of investigating the potential immunity of OMV components in vivo. Vesicles enter cells using receptor-mediated endocytosis. OmpA, a common component of vesicles, also acts to enter cells and globally activate macrophage proliferation. OMV can extract surface protein into APC cell, and its self-characteristics determine the function of adjuvant, and it is an ideal vaccine carrier. To date, the first generation OMV vaccine, bexsero (Novartis), has been approved for marketing to prevent epidemic cerebrospinal meningitis group B bacterial infection. There are also several OMV-based products focusing on infectious diseases, in clinical stages, including pneumonia, meningitis, pertussis, etc. Furthermore, OMVs display a range of functions during helper bacterial infection of host cells, the most unique being that OMVs serve as delivery systems for pathogen-associated molecular patterns (PAMPs), which are delivered to distant cells by PAMP-loaded OMVs and other membrane components that influence the response of infection-associated hosts. The immune properties of OMVs are directed to the generation of protective mucosal immunity and bactericidal antibody responses, which can provide a platform for vaccine development. The use of OMVs from e.coli, salmonella, neisseria meningitidis as vaccine antigen delivery systems to elicit potent B cell responses has been well studied in many publications.

Compared with the traditional whole pathogen vaccine, the subunit vaccine based on single antigen provides more accurate targeting and excellent safety. However, this also determines their poor immunogenicity, requiring additional adjuvants to enhance the immunogenicity of the antigen and to activate the immune response. Unfortunately, few clinically applicable adjuvants with high efficacy and low toxicity are available, and new safe and potent adjuvants are urgently needed. However, microbial pathogens include a variety of bacterial cell wall components such as Lipopolysaccharides (LPS), nucleic acids, peptidoglycans (PGNs) and lipopeptides, as well as flagellins, bacterial DNA and viral double stranded RNA, etc., which cause a variety of different cellular responses, such as interferon production, proinflammatory cytokine release, sepsis resulting from an immune response, etc., limiting clinical use.

Salmonella is widely distributed in nature, often colonizing humans and animals, and belongs to the family enterobacteriaceae, gram-negative enterobacteriaceae. Nearly 1000 species (or strains) are found, and can be divided into basic groups such as A, B, C, D, E and the like according to antigen components, wherein the basic groups are mainly A paratyphoid bacillus of the A group, B paratyphoid bacillus and typhus bacillus of the B group, C paratyphoid bacillus and hog cholera bacillus of the C group, T typhoid bacillus and enteritis bacillus of the D group and the like related to human diseases. Salmonella has a complex antigen structure and can be generally divided into 3 types of somatic antigens, flagellar antigens and surface antigens. Salmonella typhimurium is an invasive intracellular bacterium that mainly causes intestinal infections. The attenuated salmonella typhimurium VNP20009 is an auxotrophic bacterium with pathogenic genes delta msbB and purine delta purI genes knocked out on the basis of wild salmonella 14028s, has good tumor targeting and tumor inhibiting effects, has a plurality of related tumor treatment drugs developing clinical research, is clinically safe and verified salmonella typhimurium, but flagella, exotoxin LPS and the like existing in the cell membrane of the salmonella VNP20009 have strong toxicity, are easy to cause septicemia, and are not suitable for serving as adjuvants. While there are many genes related to expression of flagella and exotoxin LPS, the mechanism is complex, fig. 1 is a mechanism, structure and modification diagram of LPS biosynthesis (derived from naturereviws miscrobiology RevIeWS volume 17JULY 2019) reviewed in NATuRe 2019, fig. 2 is a salmonella typhimurium flagellar structural component and control gene (derived from NATuRe RevIeWS MiCRobioLogy volume 6june 2008 457), it can be seen from fig. 1 and fig. 2 that the difficulty in selecting a gene editing strategy related to LPS biosynthesis and flagella structure is very great, the phenomenon of low survival rate of thalli and even death of thalli often occur in the gene knockout process, and the effect on delivery can also be influenced. Pathological section researches on liver, spleen and the like of mice inoculated with the VNP20009 strain in the abdominal cavity show that the VNP20009 strain is not sufficiently attenuated. Therefore, how to construct a safe and effective outer membrane vesicle delivery system has important significance in vaccine research.

Disclosure of Invention

In response to the above-described deficiencies of the prior art, the present application provides an attenuated gram-negative bacterium and an OMV antigen delivery system prepared therefrom. The attenuated gram-negative bacteria form an antigen delivery system with low toxicity and T cell adjuvant effect.

An object of the present invention is to provide an attenuated gram-negative bacterium, which is a strain formed after knocking out or silencing genes fliC, fljB and phosphoethanolamine transferase gene EptA on the basis of a wild-type or prototype strain; in one embodiment, the strain is formed after knocking out or silencing the genes fliC, fljB and the phosphoethanolamine transferase gene EptA in sequence on the basis of a wild-type strain.

The modified bacteria of the invention are gram-negative bacteria. Gram-negative bacteria generally refer to bacteria which are red in gram staining reaction, in a gram staining experiment, gentian violet is firstly added for primary staining, then iodine solution is added for secondary staining, after the first-step staining is completed, both gram-positive bacteria and gram-negative bacteria are stained purple, then color washing is carried out, only gram-negative bacteria can be washed out and become colorless, while positive bacteria are still purple, another secondary staining dye (generally safranin or fuchsin is used) is added, all gram-negative bacteria are stained red or pink, while positive bacteria are still purple, and through the test, two bacteria with different cell wall structures can be distinguished. The gram-negative bacterium of the present invention may be a gram-negative bacterium commonly used in the art, and may be, for example, salmonella, escherichia coli, or neisseria. The invention takes salmonella as an example to illustrate the preparation process of an OMV delivery system, and is also suitable for other gram-negative bacteria such as escherichia coli, neisseria and the like.

The invention also aims to provide an attenuated salmonella typhimurium GDB520, which is a strain formed after genes fliC, fljB and a phosphoethanolamine transferase gene EptA are sequentially knocked out or silenced in a salmonella VNP20009 strain.

The salmonella VNP20009 strain used in the invention is from ATCC strain preservation center (ATCC 202165).

In some embodiments, the gene fliC of the invention has a sequence shown in SEQ ID NO.1, and the encoded protein has a sequence shown in SEQ ID NO. 2.

In some embodiments, the gene fljB of the invention has a sequence as shown in SEQ ID NO.3, and the encoded protein has a sequence as shown in SEQ ID NO. 4.

In some embodiments, the phosphoethanolamine transferase gene, eptA, of the present invention has the sequence shown in SEQ ID No.5, and the encoded protein has the sequence shown in SEQ ID No. 6.

The knockdown or silencing described herein can be performed according to methods routine in the art, and in some embodiments:

the sequence of an upstream homology arm of the knocked-out gene fliC is shown in SEQ ID NO.7, and the sequence of a downstream homology arm is shown in SEQ ID NO. 8;

the sequence of an upstream homology arm of the knocked-out gene fljB is shown as SEQ ID NO.9, and the sequence of a downstream homology arm is shown as SEQ ID NO. 10;

the sequence of an upstream homology arm of the knocked-out phosphoethanolamine transferase gene EptA is shown as SEQ ID No.11, and the sequence of a downstream homology arm is shown as SEQ ID No. 12.

The invention also provides outer membrane vesicles formed from the attenuated gram-negative bacteria or the attenuated salmonella typhimurium GDB520 of the invention. The outer membrane vesicles of the invention have a bilayer membrane structure, and in some embodiments, the outer membrane vesicles have a diameter of 20-200 nm.

The outer membrane vesicles of the present invention may be prepared by extracting outer membrane OMVs according to conventional methods in the art, for example, using deoxycholate or lithium chloride or similar techniques, and then removing cell debris and purifying to form the desired outer membrane vesicles according to conventional methods.

The invention also provides a cell outer membrane vesicle OMV antigen delivery system, comprising the outer membrane vesicle and a target antigen or nucleic acid. The OMV antigen delivery system can be coupled or adsorbed with target protein antigen or nucleic acid on the outer side of the OMV, and target protein antigen or nucleic acid can be coated in the OMV vesicle and delivered to cells in vivo.

The invention also provides a vaccine formulation comprising the antigen delivery system of claim 8. In some embodiments, the vaccine formulation is formed by embedding or coupling an antigenic protein or nucleic acid with an outer membrane vesicle OMV antigen delivery system of the invention.

The embedding or coupling of the antigenic protein according to the invention with the outer membrane vesicle OMV antigen delivery system according to the invention can be carried out according to conventional methods in the art.

The antigen protein of the invention can be common antigen protein in the field, such as antigen protein of preventive vaccine or antigen protein of tumor vaccine, and can be antigen protein of virus or bacteria expressed and purified by adopting a genetic engineering method.

The attenuated salmonella typhimurium GDB520 provided by the invention has the advantages that genes fliC, fljB and phosphoethanolamine transferase gene EptA are knocked out or silenced according to a specific sequence, so that the improved strain does not contain flagellin, the molecular structure of LPS is changed, the attenuated salmonella typhimurium GDB520 has low toxicity and T cell adjuvant effect, can be normally amplified and cultured, and has important industrial value. Can be used for preparing preventive vaccines and tumor vaccines, and can realize administration by injection or nasal spray.

Drawings

FIG. 1 shows lipopolysaccharide LPS biosynthesis, structure and modification;

FIG. 2 is a Salmonella typhimurium flagella assembly;

FIG. 3 is a diagram showing the discrimination by PCR electrophoresis in example 1;

FIG. 4 is a wild-type electron micrograph of VNP 20009;

FIG. 5 is an electron micrograph of strain GDB520 (Δ FliC + Δ FljB + Δ eptA) obtained after gene knockout;

FIG. 6 is an electron micrograph of OMV vesicles from example 2;

FIG. 7 is the cytokine expression level of example 3.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified.

The salmonella VNP20009 adopted in the embodiments of the present invention is derived from ATCC strain preservation center (ATCC 202165), and it should be specifically noted that the method used in the embodiments of the present invention is also applicable to gram-negative bacteria such as escherichia coli and neisseria meningitidis.

Example 1 construction of attenuated Salmonella typhimurium GDB520

1. Construction of a.DELTA.fliC Strain

And (3) carrying out FliC gene knockout on VNP20009 by adopting a Cas9 lambda red homologous recombination plasmid. The Cas9 lambda red plasmid has Kan resistance and expresses recombinant protein; the CRISPR gRNA plasmid has Amp resistance and carries a gRNA sequence. The sequence of the gene fliC is shown in SEQ ID NO.1, and the sequence of the encoded protein is shown in SEQ ID NO. 2.

Designing 20bp gRNA sequence targeting as follows: 5 'AACGAAATCGACCTGTGTATC-3', the sequence of the upstream homology arm of the knockout gene fliC is shown as SEQ ID NO.7, and the sequence of the downstream homology arm is shown as SEQ ID NO. 8.

VNP preparation of Cas9 λ Red-containing homologous recombination plasmids: VNP monoclonal was inoculated into 2ml LB medium, cultured at 37 ℃ to OD600=0.6, the cells were collected by centrifugation, washed 3 times with pre-cooled 10% glycerol, and resuspended in 100 μ l with 10% glycerol. Cas9 plasmid 10 μ l was added to the cells for electroporation, conditions set: 2400V, 200. Omega., 1mm. Then coating the plate at 30 ℃, carrying out Kan resistance culture, and selecting the single clone overnight to obtain VNP-cas9.

VNP-cas9 transformation with gRNA plasmids to prepare competent cells: inoculating VNP-cas9 monoclonal into 2ml LB culture medium, culturing at 30 deg.C until OD600= 0.3-0.5, adding arabinose for induction, the concentration is 3mg/ml, incubating for 1hr, centrifuging to collect thallus, washing 3 times with 10% glycerol, and re-suspending 100. Mu.l. The gRNA plasmid and the homologous sequence were added to competent cells, and the cells were electroporated at 2400V, 200. Omega., 2mm. Then spread on LB plate, kan + and Amp +, incubate for more than 16hr, pick out the single clone, expand and culture.

Removal of single plasmids: and adding IPTG into a culture medium, culturing overnight, transferring for 2 generations, removing gRNA plasmids, removing Amp resistance of VNP at the moment, and selecting monoclonal culture to subculture and maintain seeds to obtain VNP delta FliC.

2. Construction of a.DELTA.fljB Strain on the basis of VNP.DELTA.FliC

The sequence of the gene fljB is shown as SEQ ID NO.3, and the sequence of the encoded protein is shown as SEQ ID NO. 4.

Designing 20bp gRNA sequence targeting as follows: 5 'GTTTACGGTATTGCCCAGGT-3', the sequence of the upstream homology arm of the knock-out gene fljB is shown as SEQ ID NO.9, and the sequence of the downstream homology arm is shown as SEQ ID NO. 10.

Transformation of VNP Δ FliC with gRNA plasmid preparation of competent cells: VNP. DELTA. FliC monoclonal (containing cas9 plasmid, kan-resistant) was inoculated into 2ml of LB medium, cultured at 30 ℃ until OD600= 0.3-0.5, induced with arabinose at a concentration of 3mg/ml, incubated for 1hr, centrifuged to collect cells, washed 3 times with 10% glycerol, and resuspended 100. Mu.l. The gRNA plasmid and the homologous sequence were added to competent cells, and the cells were electroporated at 2400V, 200. Omega., 2mm. Then spread on LB plate, kan + and Amp +, incubate for more than 16hr, pick out the single clone, expand and culture.

Removal of single plasmid: and adding IPTG into a culture medium, culturing overnight, transferring for 2 generations, removing gRNA plasmids, removing Amp resistance of VNP at the moment, and selecting monoclonal culture to subculture and maintain seeds to obtain VNP delta FliC + delta FljB.

3. Knock-out of eptA gene on the basis of VNP delta FliC delta FljB

The sequence of the phosphoethanolamine transferase gene EptA is shown as SEQ ID NO.5, and the sequence of the encoded protein is shown as SEQ ID NO. 6.

Designing 20bp gRNA sequence targeting as follows: 5 'GGCGAATCATTGGGTGAAAA-3', the sequence of the upstream homology arm of the knocked-out phosphoethanolamine transferase gene EptA is shown as SEQ ID NO.11, and the sequence of the downstream homology arm is shown as SEQ ID NO. 12.

Transformation of VNP Δ FliC Δ FljB with gRNA plasmid preparation of competent cells: VNP. DELTA. FliC. DELTA. FljB monoclonal (containing cas9 plasmid, kan-resistant) was inoculated into 2ml of LB medium, cultured at 30 ℃ to OD600= 0.3-0.5, induced with arabinose at a concentration of 3mg/ml, incubated for 1hr, centrifuged to collect the cells, washed 3 times with 10% glycerol, and resuspended in 100. Mu.l. The gRNA plasmid and the homologous sequence were added to competent cells, and the cells were electroporated at 2400V, 200. Omega., 2mm. Then spread on LB plate, kan + and Amp +, incubate for more than 16hr, pick out the single clone, expand and culture.

Removing the double plasmids: and adding IPTG into a culture medium, culturing overnight, transferring for 2 generations, removing gRNA plasmids, selecting a monoclonal for culturing and subculturing when the Amp resistance of VNP is lost, raising the culture temperature to 37 ℃ overnight, removing cas9 plasmids, and maintaining seeds to obtain VNP delta FliC + delta FljB + delta eptA.

The obtained strain formed after sequentially knocking out the genes fliC, fljB and the phosphoethanolamine transferase gene EptA is named as GDB520 strain.

4. Identification of Gene knockout events

The eptA knockout was identified by the following primers:

an upstream primer: 5' tccagtcagcagtatgtcgcc-3

A downstream primer: 5' actgcctgcctgagcatacac 3

The sequence interval of the PCR sequencing wild strain is as follows: 4440721-4443323, theoretically 2603nt in total, and 1059nt in actual sequencing result, wherein the sequence is shown in SEQ ID NO. 13.

The PCR electrophoretic identification pattern is shown in FIG. 3, indicating that eptA has been successfully knocked out. Through electron microscope observation, as shown in fig. 4, flagella can be seen by VNP20009 wild-type electron microscope (fig. 4), strain GDB520 (Δ FliC + Δ FljB + Δ eptA) obtained after gene knockout, and flagella can be seen to be knocked out by electron microscope (fig. 5).

Example 2 extraction, preparation and characterization of Outer Membrane Vesicles (OMVs)

The strain GDB520 obtained in example 1 was cultured in an expanded state, and the seed solution was inoculated into a fermentation basal medium (mixture ratio: 20g/L phytone, 20g/L yeast extract, glucose. H) at a ratio of 1 ₂ O5 g/L, dipotassium hydrogen phosphate 3g/L, potassium dihydrogen phosphate 2g/L, anhydrous magnesium sulfate 2g/L, and sodium chloride 14 g/L). The fermentation parameters are set to be pH 7.0, rotation speed 150rpm, temperature 37 ℃, tank pressure 0.02MPa and ventilation capacity 60L/min.

During the fermentation, the OD600 value was measured every 1 hour; microscopic examination is carried out every 2 hours; the pH value is respectively adjusted by ammonia water and 6M hydrochloric acid and is controlled to be 6.8-7.2; the dissolved oxygen is controlled to be more than 30 percent. And (5) fermenting for 6 hours, and harvesting the thalli when the bacteria enter a logarithmic phase. Adding 0.01M PBS into a centrifugal cylinder filled with the thallus precipitate, stirring, resuspending and washing the thallus, washing for 2 times, centrifuging, collecting the precipitate, and freezing and storing at-20 ℃. Freezing for at least 24hr, taking out thallus from-20 deg.C, and thawing at 2-8 deg.C for 18-24 hr. The bacterial body is re-suspended according to the proportion of 1. Then 8000g of the suspension was centrifuged to remove cell debris, and the supernatant was collected by concentration by repeatedly washing 10 volumes with a 300Kda tangential flow ultrafilter. And adding nuclease to remove nucleic acid impurities, performing column chromatography sepharose 4FF to collect pure OMV products, namely OMV vesicles (spheres) which can be used as a delivery system, and then performing embedding and coupling to deliver the antigen. The sample was taken by electron microscope and characterized as shown in FIG. 6, with a diameter of 20-200 nm.

Example 3 immune cell response assay

The method comprises the steps of adding 10% serum into MEM culture medium to culture Raw264.7 macrophages, stimulating the macrophages by using OMVs prepared in example 2 for 12 hours, using PBS sucrose solution as a control group, using TRIZOI to lyse cells to extract RNA, carrying out reverse transcription on the RNA to obtain cDNA, detecting expression quantity by Q-PCR, and calculating the expression level of the cytokine by a delta ct method. The results are shown in fig. 7, the expression level of the inflammatory factor IL-6 is significantly reduced, and IFN-gamma is retained and improved, and it can be seen that the OMV prepared in example 2 completely meets the requirements of vaccine adjuvant design and antigen delivery system.

Claims (10)

1. An attenuated gram-negative bacterium, wherein said gram-negative bacterium knocks out or silences a strain formed after genes fliC, fljB and phosphoethanolamine transferase gene EptA on the basis of a wild-type or prototype strain; preferably, the wild-type gram-negative bacterium is salmonella, escherichia coli or neisseria.

2. An attenuated salmonella typhimurium GDB520, characterized in that a strain formed after knocking out or silencing genes fliC, fljB and phosphoethanolamine transferase gene EptA in sequence in a salmonella VNP20009 strain.

3. The attenuated salmonella typhimurium GDB520 of claim 2, wherein the sequence of the gene fliC is shown in SEQ ID No.1, and the sequence of the encoded protein is shown in SEQ ID No. 2; the sequence of the gene fljB is shown as SEQ ID NO.3, and the sequence of the encoded protein is shown as SEQ ID NO. 4; the sequence of the phosphoethanolamine transferase gene EptA is shown as SEQ ID NO.5, and the sequence of the encoded protein is shown as SEQ ID NO. 6.

4. The attenuated salmonella typhimurium GDB520 of claim 2, wherein the upstream homology arm sequence of the knock-out gene fliC is shown in SEQ ID No.7, and the downstream homology arm sequence is shown in SEQ ID No. 8.

5. The attenuated salmonella typhimurium GDB520 of claim 2, wherein the upstream homology arm sequence of the knockout gene fljB is shown as SEQ ID No.9, and the downstream homology arm sequence is shown as SEQ ID No. 10.

6. The attenuated salmonella typhimurium GDB520 of claim 2, wherein the upstream homology arm sequence of the knocked-out phosphoethanolamine transferase gene EptA is shown as SEQ ID No.11, and the downstream homology arm sequence is shown as SEQ ID No. 12.

7. Outer membrane vesicles formed from the strain of any one of claims 1 to 6, preferably said outer membrane vesicles have a bilayer membrane structure, more preferably said outer membrane vesicles have a diameter of 20 to 200nm.

8. An outer membrane vesicle OMV antigen delivery system comprising the outer membrane vesicle of claim 7 and an antigen or nucleic acid of interest.

9. A vaccine formulation comprising the antigen delivery system of claim 8.

10. The vaccine formulation of claim 9, wherein an antigenic protein or nucleic acid is embedded or conjugated to the outer membrane vesicle OMV antigen delivery system.

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