Novel Kdo-MPLA adjuvant

Abstract

The invention discloses a novel Kdo-MPLA adjuvant, and relates to the technical field of synthetic biology, the adjuvant is formed by connecting monophosphate lipid A (MPLA) and Kdo sugar through an alpha-glucosidic bond, MPLA is of a structure formed by modifying salmonella lipid A through 4 '-site monophosphorylation, and the Kdo sugar is endowed with amphipathy, so that the Kdo sugar is self-assembled into liposome nanoparticles in an aqueous solution; the rfaC and eptA genes are double knocked out through a gene editing salmonella attenuated strain, wherein the rfaC knockout blocks the synthesis of LPS core polysaccharide, and lipid A-Kdo connection is reserved; knocking out eptA to obtain a monophosphorylated lipid A; after the engineering bacteria are fermented, high-purity Kdo-MPLA is obtained by adopting a phenol-chloroform-petroleum ether extraction method, the process is simplified, and the product is uniform; the adjuvant can activate a TLR4 signal channel and induce Th1 type immune response, the levels of inflammatory factors IL-6 and IL-1beta are remarkably lower than those of natural LPS, and the adjuvant is suitable for a mixed preparation of vaccine antigens such as HPV and malaria; the method overcomes the defects of hydrolysis uncontrollability and high chemical synthesis cost of the traditional MPLA chemical extraction process.

Description

Technical Field

[0001] The present invention relates to the technical field of synthetic biology, and in particular to a novel Kdo-MPLA adjuvant. Background Art

[0002] Monophosphoryl Lipid A (MPLA), a derivative of lipopolysaccharide from Salmonella, has strong immunostimulatory activity and weak toxicity, and is widely used as a vaccine adjuvant. MPLA is a structural component of the bacterial endotoxin lipopolysaccharide (LPS) molecule and is the main component of the cell wall of Gram-negative bacteria. It consists of three parts: O-antigen, core sugar, and lipid A. Early studies found that LPS could cause strong immune responses such as fever and shock, so LPS was called endotoxin. When studying Salmonella, it was found that the LPS of Salmonella had immunogenicity and could induce antibody production. Further purification of LPS proved that its toxicity mainly came from the lipid A part. Analyzing the precise structure of lipid A, it was found that the lipid A of Salmonella consisted of a disaccharide backbone of glucosamine and multiple fatty acid chains, and its toxicity depended on the degree of phosphorylation and the structure and position of fatty acids.

[0003] In 1984, the Edgar Ribi team found that by weak acid hydrolysis or enzymatic treatment of LPS, one phosphate group could be removed to obtain monophosphoryl lipid A, i.e., MPLA, with significantly reduced toxicity but still retaining the immunostimulatory ability. In 1985, David et al. proved that MPLA could activate macrophages and induce the release of cytokines (TNF-α, IL-6, IL-1β), but would not cause a strong inflammatory response like LPS. These findings indicated that MLPA was a safer candidate molecule for immunoadjuvants. In 1998, the mechanism of action of MPLA was explored. It was found that LPS and MPLA activated the innate immune system by binding to Toll-like receptor (TLR4), which provided a molecular basis for the mechanism of MPLA as an adjuvant. LPS activated the TLR4 / MD-2 complex, leading to a strong inflammatory response such as sepsis. The signal of MPLA activating TLR4 was weak, mainly inducing Th1-type immune responses without causing excessive inflammation. Based on these studies, MPLA is an ideal vaccine adjuvant suitable for enhancing the immunogenicity of recombinant protein vaccines.

[0004] The first MPLA-adjuvanted vaccine is the HPV vaccine (Cervarix®). The HPV vaccine launched by GSK in 2009 uses the AS04 adjuvant system, namely MPLA + aluminum adjuvant. This is the world's first approved human vaccine containing MPLA adjuvant, thus confirming the safety and effectiveness of MPLA. The shingles vaccine (Shingrix®) approved in 2017 uses MPLA + QS21 adjuvant, with a protection rate exceeding 90%. The malaria vaccine (Mosquirix®) recommended by the WHO in 2021 contains an MPLA derivative adjuvant.

[0005] Salmonella is widely distributed in nature and often inhabits the human and animal bodies. It belongs to the Enterobacteriaceae family and is a Gram-negative intestinal bacillus. Nearly 1000 species (or strains) have been discovered. According to antigenic components, it can be divided into basic groups such as A, B, C, D, and E. Among them, the main ones related to human diseases are Salmonella paratyphi A of group A, Salmonella paratyphi B and Salmonella typhimurium of group B, Salmonella paratyphi C and Salmonella choleraesuis of group C, Salmonella typhi and Salmonella enteritidis of group D, etc. Salmonella has a complex antigenic structure and can generally be divided into three types: somatic antigen, flagellar antigen, and surface antigen. Salmonella typhimurium is an invasive intracellular bacterium that mainly causes intestinal infections.

[0006] MPLA can be extracted from the cell membrane of Salmonella and chemically modified to obtain a product for adjuvants. GSK acquired the Ribi company and established the AS adjuvant system using the obtained production technology. The process of producing MPLA is to culture Salmonella strains, extract LPS, purify lipid A, and then hydrolyze and recover MPLA. Specifically, the attenuated and non-pathogenic Salmonella strain R595 is used, and a high-nutrient medium such as LB or TSB is used. After culturing to the logarithmic phase, the bacterial cells are collected, washed 2 - 3 times with physiological saline or PBS to remove the medium components, and then inactivated by heating at 60°C for 1 hour. The bacterial cells are lysed by the hot phenol method. The bacterial cell suspension is suspended in deionized water, and an equal volume of phenol (90%) is added. Stir at 65°C for 30 - 60 minutes and centrifuge at 10000g for 20 minutes. LPS is mainly distributed in the aqueous phase, and proteins and nucleic acids are mainly distributed in the phenol phase. The aqueous phase is dialyzed against deionized water for 48 hours and centrifuged at 100000g to precipitate LPS, obtaining a crude white LPS product. Next, acid hydrolysis is carried out to remove the O-antigen and core polysaccharide. Heat 1% acetic acid to 100°C for 1 hour, and lipid A precipitates. The hydrolysis solution is cooled and centrifuged at 10000g for 20 minutes, and the precipitate is crude lipid A. Repeated washing with an organic solvent combination (chloroform - methanol 2:1) can further purify lipid A. Two phosphate groups are attached to the 1 and 4' positions of the two sugar rings of lipid A, and hydrolysis is required to remove them. Specifically, the weak acid method is used, and 0.1M HCl is used to treat at 30°C for 30 minutes, which can selectively remove the phosphate group at the 1 position. After the reaction, it is neutralized with NaOH, and chloroform - methanol extraction is carried out to obtain the crude MPLA product.

[0007] The above describes the MPLA chemical extraction process. Although this process has been successfully applied to commercial vaccines, the defects in the MPLA production process are obvious. First, in the process of hydrolyzing and removing the core sugar, the hydrolysis of the core polysaccharide with the structural formula: [lipid A]-(GlcN)-6'-α-Kdo-(core polysaccharide)-O-antigen is non-specific. The core polysaccharide is usually composed of heptose (Hep), glucose (Glc), galactose (Gal), etc., connected by α- or β-glycosidic bonds. The hydrolysis products of the broken glycosidic bonds are mixtures. In addition to the hydrolysis of Kdo sugar, other glycosidic bonds in the core polysaccharide will also be gradually hydrolyzed to produce monosaccharides and short-chain oligosaccharides. Insufficient hydrolysis of Kdo will inevitably lead to the connection of core polysaccharides of various lengths with lipid A. Therefore, a very big defect of this hydrolysis process is that the degree of hydrolysis is uncontrollable, and the molecular weight of the target product is distributed within a range, and the quality cannot be quantitatively controlled. Second, in the process of further hydrolyzing diphospholipid A into monophospholipid A, over-hydrolysis may cause the phosphate group at the 1 position of lipid A to fall off. Even if the reaction conditions are strictly controlled, it is inevitable that some products do not carry phosphates. Lipid A without phosphate has no pharmacological activity. Therefore, the quality of MPLA products is unstable. In short, this production process is affected by multiple factors. More specifically: the acidic condition pH 4.5 breaks the Kdo bond, and if excessive, it will degrade lipid A; the reaction temperature is 100 °C, and if it exceeds 105 °C, the structure of lipid A will be damaged; the reaction time is 30-60 min. If the time is short, the hydrolysis is insufficient, and if the time is long, lipid A decomposes; low ionic strength is beneficial to the reaction, and if the salt concentration is high, it will inhibit the attack reaction of protons on the glycosidic bond. It is very difficult to achieve batch-to-batch consistency in industrial production by precisely controlling pH, temperature, and time and balancing the yield and quality. In summary, the method of bacterial culture, chemical extraction, and then chemical modification is not a good process for preparing MPLA.

[0008] In response to the above-mentioned process defects, the American Institute of Infectious Diseases invented a chemical total synthesis process. The adjuvant prepared by this process is an MPLA analogue and has been used in the development of various infectious diseases and tumor vaccines. Specifically, D-glucosamine is used as the basic unit of the lipid A skeleton; long-chain fatty acids (such as lauric acid and palmitic acid) are used for acylation modification; phosphorylation reagents (such as POCl3 and H3PO4) introduce phosphate groups. The core steps of chemical synthesis are: acylation reaction of glucosamine, introduction of fatty acid chains at positions 2 and 3 of glucosamine; phosphorylation reaction, introduction of phosphate groups at position 4' (key step, determining TLR4 binding ability); further modification of fatty acid chains, condensation of hydroxyl at position 6' with activated fatty acids (such as palmitic acid-NHS ester). Compared with the extraction method, the fully chemically synthesized structure has the characteristics of high uniformity, no risk of LPS residues, and good batch-to-batch consistency. However, there are disadvantages such as high cost and high process complexity; use of DMF / pyridine and chloroform / methanol organic solvents in the preparation process; and low phosphorylation efficiency.

[0009] The above two technical routes for preparing MPLA have been described. In addition to their respective process defects, they also have a common disadvantage, that is, they are both non-polar molecules, insoluble in water, and need to be dispersed in organic solvents before preparing liposomes with cholesterol and DOPE. Poor solubility often leads to uneven dispersion. In addition, when the preparation is used, the adjuvant is packaged separately from the antigen and mixed before use, which makes it inconvenient to use.

[0010] As a highly efficient and low-toxic TLR4 agonist, MPLA has become an important part of the modern vaccine adjuvant system. With the success of GSK's shingles vaccine in the market, many vaccine companies in China have begun to follow up on the preparation of MPLA. In terms of application, in addition to the malaria vaccines, HPV and VZV mentioned above, there are also COVID-19 vaccines, tuberculosis vaccines and HIV vaccines. The main suppliers are GSK, the US Institute of Infectious Diseases IDRI, and Avanti Polar Lipids. The annual production capacity is about 500kg, which can meet the demand for 100 million doses of vaccines. As clinical value continues to expand. In the next five years, MPLA is expected to achieve greater breakthroughs in the fields of mRNA vaccines and universal influenza vaccines, and the global market size is expected to reach US$2.5 billion (2028).

[0011] The latest direction of MPLA internationally is to develop an E. coli synthetic biology production platform. Due to the involvement of multiple gene modifications and expression regulation issues, the E. coli synthetic biology route is still under exploration. The design of the present invention belongs to the field of synthetic biology. We use the attenuated strain of Salmonella as the starting strain, perform gene editing, and cultivate the edited strain on a large scale. TLR4 agonists can be directly extracted from the cell membrane to prepare a new MPLA derivative, namely Kdo-MPLA. Summary of the Invention

[0012] In view of the above existing problems, the present invention is proposed.

[0013] The present invention provides a novel Kdo-MPLA adjuvant to solve the problems of uncontrollable hydrolysis, non-uniform products and high chemical synthesis cost in the traditional MPLA production process.

[0014] To solve the above technical problems, the present invention provides the following technical solutions: In a first aspect, an embodiment of the present invention provides a novel Kdo-MPLA adjuvant. The adjuvant is a complex of MPLA and Kdo sugar. Its hydrophobic part is monophosphoryl lipid A, MPLA, and its hydrophilic part is Kdo sugar. The structure of MPLA is the form of lipid A derived from Salmonella phosphorylated at the 1-hydroxy group and 4'-monophosphate, and the Kdo sugar is linked to the 6'-hydroxy group of MPLA through an α-glycosidic bond. The Kdo-MPLA has amphiphilicity and forms liposome nanoparticles after dissolving in water.

[0015] As a preferred embodiment of the novel Kdo-MPLA adjuvant of the present invention, wherein: the Kdo sugar is 3-deoxy-D-manno-octulosonic acid, Kdo, the connection between MPLA and Kdo sugar is achieved by gene editing the LPS synthesis pathway of Salmonella, and the rfaC gene and eptA gene of the Salmonella are knocked out.

[0016] As a preferred embodiment of the novel Kdo-MPLA adjuvant of the present invention, wherein: the Salmonella is a attenuated strain, preferably the R595 strain (ATCC9700) or the VNP2000 strain.

[0017] In a second aspect, the present invention provides a preparation method of a novel Kdo-MPLA adjuvant, the following steps: (a) Genetically edit Salmonella, knock out the rfaC gene to block the synthesis of LPS core polysaccharide, so that lipid A is only linked to Kdo sugar; (b) Knock out the eptA gene to remove the phosphate group at the 1-position of lipid A and retain the 4'-monophosphorylated structure; (c) Culture the genetically edited Salmonella and extract Kdo-MPLA from the cell membrane of the bacterial cells.

[0018] As a preferred embodiment of the method for preparing a novel Kdo-MPLA adjuvant according to the present invention, wherein: the knockout of the rfaC gene is achieved by the CRISPR-Cas9 system, and the homologous recombination fragment contains the sequences shown in SEQ ID NO.4 and SEQ ID NO.5; the knockout of the eptA gene is achieved by the CRISPR-Cas9 system, and the homologous recombination fragment contains the sequences shown in SEQ ID NO.9 and SEQ ID NO.10.

[0019] As a preferred embodiment of the method for preparing a novel Kdo-MPLA adjuvant according to the present invention, wherein: the extraction process of the Kdo-MPLA includes: (i) The bacterial cells are pretreated with ethanol, acetone, and ether to remove impurities; (ii) The lipid components are extracted with a phenol-chloroform-petroleum ether mixture PCP; (iii) The Kdo-MPLA is purified by the acetone / ether precipitation method.

[0020] As a preferred embodiment of a novel Kdo-MPLA adjuvant according to the present invention, it self-assembles into nanoscale liposome nanoparticles in an aqueous solution and presents an "onion ring" - like structure under a transmission electron microscope.

[0021] As a preferred embodiment of a novel Kdo-MPLA adjuvant according to the present invention, the adjuvant is a TLR4 agonist, which is used to activate Th1 - type immune responses, and the induced levels of inflammatory factors IL-6 and IL-1β are significantly lower than those of natural LPS.

[0022] In a third aspect, the present invention provides a vaccine composition, which includes a novel Kdo-MPLA adjuvant and a vaccine antigen, and the adjuvant and the antigen are directly mixed to form a homogeneous preparation.

[0023] As a preferred embodiment of a novel Kdo-MPLA adjuvant according to the present invention, wherein: the antigen is an HPV antigen, a varicella-zoster virus antigen, a malaria antigen, a tuberculosis antigen, or an HIV antigen.

[0024] The beneficial effects of the present invention are as follows: The present invention uses gene editing methods to transform Salmonella. By knocking out the rfaC gene, the connection of the core polysaccharide of LPS in the Salmonella cell membrane is blocked, and only a structure with lipid A connected to Kdo sugar is formed; by knocking out the eptA gene, lipid A is monophosphorylated at the 4' position while retaining a hydroxyl group at the 1 position. Culturing the gene-edited Salmonella can directly extract Kdo-MPLA with TLR4 agonist activity, wherein MPLA is connected to Kdo sugar at the 6' - position hydroxyl group, and Kdo sugar has hydrophilicity, endowing Kdo-MPLA with water solubility. The prepared adjuvant presents a nano-liposome structure and can be directly mixed with the vaccine antigen preparation.

[0025] The Salmonella strains used in the present invention are preferably attenuated strains, such as strain R595 and strain VNP2000, and more preferably R595 is derived from the ATCC strain preservation center (ATCC 9700).

[0026] In the present invention, the O antigen and core polysaccharide structure in LPS are removed by knocking out the rfaC gene. The rfaC gene, also known as the waaC gene, is a key gene involved in the synthesis of the core oligosaccharide of lipopolysaccharide in bacteria, encoding heptosyltransferase, and its main function is to add heptose to the core oligosaccharide of LPS.

[0027] The eptA gene described in the present invention is a phosphoethanolamine transferase gene, which is mainly involved in the modification of lipopolysaccharide (LPS) in bacteria and regulates the addition of phosphoethanolamine to LPS. Knocking out this gene can selectively remove the phosphate group at position 1 and retain the hydroxyl group.

[0028] The engineered bacteria with double knockout have good growth performance and can fully meet the production requirements.

[0029] The present invention has established a process for freeze-drying Kdo-MPLA bacteria, extracting Kdo-MPLA, and its quality standard. The obtained product is a vaccine adjuvant. After extracting Kdo-MPLA according to the described method and then performing comparative research after weak acid hydrolysis treatment with MPLA, the Rf values (retention factors) on the thin layer plate are the same for both, indicating the same structure. Inoculating Kdo-MPLA into immune cells shows the same response as MPLA reported in the literature, with significantly reduced inflammatory factors, meeting the usage requirements of vaccine adjuvants. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0031] Figure 1 It is an electron micrograph of the Kdo-MPLA engineered bacteria of the present invention.

[0032] Figure 2 It is a growth curve of the Kdo-MPLA engineered bacteria of the present invention (squares represent WT, and diamonds represent Kdo-MPLA engineered bacteria).

[0033] Figure 3 It is an electron micrograph of Kdo-MPLA in an aqueous solution (a photo of a single nanoparticle) of the present invention.

[0034] Figure 4Electron micrograph of Kdo-MPLA of the present invention in aqueous solution (multiple nanoparticle photos).

[0035] Figure 5 Thin layer comparative analysis diagram of MPLA standard of the present invention and MPLA derived from Kdo-MPLA (the MPLA in the first and third lanes is derived from the engineered bacteria, and the MPLA in the second lane is the MPLA standard).

[0036] Figure 6 Comparison diagram of cytokine expression levels before and after gene editing of the present invention. Detailed implementation manners

[0037] To make the above objects, features and advantages of the present invention more obvious and understandable, the following detailed description of the specific implementation manners of the present invention will be given in conjunction with the accompanying drawings of the specification.

[0038] In the following description, many specific details are set forth to fully understand the present invention. However, the present invention can also be implemented in other ways different from those described herein. Those skilled in the art can make similar generalizations without departing from the connotation of the present invention. Therefore, the present invention is not limited by the specific embodiments disclosed below.

[0039] Secondly, the so-called "one embodiment" or "embodiment" herein refers to a specific feature, structure or characteristic that can be included in at least one implementation manner of the present invention. The "in one embodiment" that appears in different places in this specification does not all refer to the same embodiment, nor is it an embodiment that is separate or selectively exclusive of other embodiments.

[0040] The experimental methods in the examples and experimental examples are all conventional methods unless otherwise specified. The test materials used in the following examples are all obtained from regular biochemical reagent stores unless otherwise specified. The Salmonella R595 used in the embodiments of the present invention is derived from the ATCC strain preservation center (ATCC 9700). It should be particularly noted that the methods used in the embodiments of the present invention are equally applicable to other strains of Salmonella and Gram-negative bacteria such as Escherichia coli.

[0041] Example 1, the first example of the present invention, provides the knockout of the rfaC gene: The rfaC gene of R595 was knocked out using the Cas9 λ red homologous recombination plasmid. The Cas9 λ red plasmid has Kan resistance and expresses recombinant proteins; the CRISPR gRNA plasmid has Amp resistance and carries the gRNA sequence. The sequence of the rfaC gene is shown in SEQ ID NO.1, and the sequence of the encoded protein is shown in SEQ ID NO.2.

[0042] Design a 20bp gRNA sequence SEQ ID NO.3 for targeting, with the sequence: 5’-tgcgcttaccgacgcgcaac-3’. The upstream homologous arm sequence for knocking out the gene rfaC is as shown in SEQ ID NO.4, and the downstream homologous arm sequence is as shown in SEQ ID NO.5.

[0043] Preparation of R595 containing the Cas9 λRed homologous recombination plasmid: Inoculate the monoclonal into 2 ml of LB medium and culture at 37°C until OD600 = 0.6. Centrifuge to collect the bacteria, wash the bacteria 3 times with pre-cooled 10% glycerol, and then resuspend in 100 μl of 10% glycerol. Add 10 μl of the Cas9 plasmid to the cells for electroporation, with the conditions set as: 2400V, 200Ω, 1mm. Then plate and culture at 30°C with Kan resistance, and pick monoclonal colonies overnight, which is R595-cas9.

[0044] Prepare competent cells by transforming R595-cas9 with the gRNA plasmid: Inoculate the R595-cas9 monoclonal into 2 ml of LB medium and culture at 30°C until OD600 = 0.3 - 0.5. Add arabinose for induction at a concentration of 3 mg / ml, incubate for 1 hr, centrifuge to collect the bacteria, wash 3 times with 10% glycerol, and resuspend in 100 μl. Add the gRNA plasmid and the homologous sequence to the competent cells, and perform electroporation at 2400V, 200Ω, 2mm. Then spread on an LB plate with + Kan + and Amp

[0045] Remove the single plasmid: Add IPTG to the medium, culture overnight, passage 2 generations to remove the gRNA plasmid. At this time, R595 loses Amp resistance. Pick monoclonal colonies, culture and passage to preserve the seeds, which is R595 ΔrfaC.

[0046] Example 2, referring to Figure 1 and Figure 2 is the second example of the present invention. This example provides knocking out the eptA gene based on R595ΔrfaC: The sequence of the phosphoethanolamine transferase gene eptA is as shown in SEQ ID NO.6, and the sequence of the encoded protein is as shown in SEQID NO.7.

[0047] Design a 20bp gRNA sequence for targeting as SEQ ID NO.8: 5’-GGCGAATCATTGGGTGAAAA-3’. The upstream homologous arm sequence for knocking out the phosphoethanolamine transferase gene eptA is as shown in SEQ ID NO.9, and the downstream homologous arm sequence is as shown in SEQID NO.10.

[0048] Transform R595ΔrfaC with gRNA plasmid to prepare competent cells: Inoculate R595ΔrfaC monoclonal (containing cas9 plasmid, Kan resistance) in 2ml LB medium, culture at 30℃ until OD600=0.3~0.5, add arabinose induction, concentration 3mg / ml, incubate for 1hr, collect bacteria by centrifugation, wash 3 times with 10% glycerol, and resuspend in 100μl. Add gRNA plasmid and homologous sequence to competent cells, electrotransform at 2400V, 200Ω, 2mm. Then spread on LB plate, Kan + and Amp + , incubate for more than 16 hours, pick out single clones, and expand and culture.

[0049] Remove the double plasmids: Add IPTG to the culture medium, culture overnight, pass 2 generations, remove the gRNA plasmid, at this time VNP loses Amp resistance, pick a single clone for culture and pass, increase the culture temperature to 37℃ overnight, remove the cas9 plasmid, keep the seed, that is, R595ΔrfaC+ΔeptA. Take the engineered bacteria for transmission electron microscopy, see Figure 1 The results showed that the bacteria were intact and morphologically normal. Continuous culture monitoring was performed in LB medium and growth curves were drawn. Figure 2 ,The results showed that compared with the wild type, the ,engineered bacteria grew well, with obvious logarithmic growth phase and ,plateau phase, meeting the predetermined standards.

[0050] Example 3 is the third example of the present invention, which provides a method for identifying gene knockout: The rfaC knockout was identified by the following primers: Upstream primer: 5'-ctctgcaaggcagcgaagt-3' Downstream primer: 5'-cgttggccgcactatcacta-3' The sequence range of the wild strain by PCR sequencing is: 3813820-3815699, theoretically a total of 1880nt, and the actual sequencing result is 926nt.

[0051] eptA knockout was identified using the following primers: Upstream primer: 5'-tccagttcagcagtatgtcgcc-3' Downstream primer: 5'-actgcctgccttgagcatcaac-3' The sequence range of the wild strain by PCR sequencing is: 4440721-4443323, theoretically a total of 2603nt, and the actual sequencing result is 1059nt.

[0052] Example 4, the fourth example of the present invention, provides the fermentation and extraction of the target substance from the engineered bacteria of Kdo-MPLA: The strain obtained in Example 2 was expanded in culture, and the seed liquid was inoculated into the fermentation basal medium (formulation: peptone 20 g / L, yeast extract 20 g / L, glucose•H2O 5 g / L, dipotassium hydrogen phosphate 3 g / L, potassium dihydrogen phosphate 2 g / L, anhydrous magnesium sulfate 2 g / L, sodium chloride 14 g / L) at a ratio of 1:100 (volume ratio). The fermentation parameters were set at pH 7.0, rotation speed 150 rpm, temperature 37°C, tank pressure 0.02 MPa, and ventilation volume 60 L / min.

[0053] During the fermentation process, the OD600 value was measured every 1 hour; microscopy was performed every 2 hours; the pH value was adjusted with ammonia water and 6M hydrochloric acid, and should be controlled between 6.8 and 7.2; the dissolved oxygen was controlled > 30%. At the 6th hour of fermentation, the bacteria entered the logarithmic growth phase, and the bacterial cells were harvested. 0.01M PBS was added to the centrifuge tube containing the bacterial cell precipitate, and the cells were stirred and resuspended for washing, washed 2 times, and the precipitate was collected by centrifugation and stored frozen at -20°C. After freezing for at least 24 hr, the bacterial cells were taken out from -20°C for the extraction of Kdo-MPLA; 1. The dried bacteria were dissolved in 90% ethanol at a concentration of 20 mg / ml, stirred for 1 h, and then the ethanol was removed using a vacuum filter, and the bacterial cells were recovered. The above operation was repeated once. (Purpose: reduce the phospholipid content) 2. The recovered bacterial cells were dissolved in acetone at a concentration of 40 mg / mL (initial weight of the bacterial cells), stirred for 15 minutes, and then the acetone was removed using a vacuum filter, and the bacterial cells were recovered. The above operation was repeated once.

[0054] 3. The recovered bacterial cells were dissolved in diethyl ether at a concentration of 40 mg / mL (initial weight of the bacterial cells), stirred for 15 minutes, and then the ethanol was removed using a vacuum filter, and the bacterial cells were recovered. The above operation was repeated once.

[0055] 4. The diethyl ether-treated bacterial cells were dried overnight in air.

[0056] 5. Prepare a mixed solution of 89% phenol:chloroform:petroleum ether = 19:45:72 (abbreviated as PCP), and let it stand overnight.

[0057] 6. The bacterial cell powder was suspended in PCP at a concentration of 70 mg / mL, stirred for 30 minutes, and then centrifuged at 4°C, 3000 g for 15 minutes.

[0058] 7. Take the upper layer supernatant. Repeat step 6, and then combine the upper layer liquids from the two times. Most of the liquid was removed using a rotary evaporator, leaving a small amount of liquid. Measure the volume, and then add water drop by drop until a persistent turbidity appears.

[0059] 8. Then add 5 volumes of acetone cooled in an ice bath and 1 volume of cooled ether to the turbid liquid, mix rapidly, and let the mixture stand in the ice bath for 30 minutes.

[0060] 9. Centrifuge at 5000 g for 15 minutes at 4 °C, take the supernatant, and recover Kdo-MPLA.

[0061] 10. Remove the ether by rotary evaporation, and wash the precipitate once more with cold acetone (Steps 7 and 8). A yield of 4 - 5% of the initial weight is obtained.

[0062] Example 5, referring to Figure 3 , Figure 4 and Figure 5 , is the fifth example of the present invention, which provides the characterization of Kdo-MPLA: Take the Kdo-MPLA powder, resuspend it in water at 10 mg / ml, and perform ultrasonic bath at 45 - 55 °C. Take one portion for transmission electron microscopy examination, and see Figure 3 and Figure 4 , the results show that Kdo-MPLA presents nano-scale particles in solution with an "onion ring" - like structure, which has not been reported in relevant literature before; add 1 volume of 0.2 N HCl to another portion, boil in water for 15 min, and terminate the reaction in an ice bath. Add 5 volumes (calculated based on the starting volume) of chloroform: methanol 2:1 (v / v), vortex, centrifuge at 500 - 1000 g, harvest the bottom layer, evaporate the solvent under N2 gas protection, harvest the crude MPLA, and perform thin-layer chromatography (TLC) analysis. The specific method is as follows: Sample treatment: Dissolve the sample with chloroform: methanol = 4:1, draw a straight line with a pencil 1 cm from the bottom of the thin plate, and spot the sample on the line with a capillary tube; Developing solvent: chloroform: methanol: water: ammonium hydroxide solution = 40:25:4:2; When the solvent reaches 1 cm from the upper part of the thin layer plate, take out the thin layer plate from the developing solvent, volatilize the solvent, spray 10% sulfuric acid ethanol solution on the surface with a spray bottle, and heat for color development at 105 °C. The results are shown in Figure 5 , and the Rf value is ~0.6. The MPLA derived from the engineered bacteria is consistent with the standard product. The standard product is from Avanti Polar Lipids.

[0063] Experimental Example 1, referring to Figure 6 , is the experimental example of the present invention, which provides an immune cell response test: Raw264.7 macrophages were cultured in DMEM medium supplemented with 10% serum and stimulated with Kdo-MPLA prepared in Example 4 for 12 hours. The control group was PBS sucrose solution. Then, cells were lysed with TRIzol to extract RNA, which was reverse transcribed into cDNA, and the expression levels were detected by Q-PCR. The cytokine expression levels were calculated by the ΔΔct method. The results are as Figure 6 shown. The expression levels of inflammatory factors IL-6 and IL-1β decreased significantly, and the ability to stimulate IFN-γ was retained, meeting the requirements for vaccine adjuvant design.

[0064] It should be noted that the above examples are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the spirit and scope of the technical solutions of the present invention, and they should all be covered by the scope of the claims of the present invention.

Claims

1. A novel Kdo-MPLA adjuvant, characterized in that, The adjuvant is a complex of MPLA and Kdo sugar, with its hydrophobic part being monophosphoryl lipid A, MPLA, and its hydrophilic part being 3-deoxy-D-manno-octulosonic acid Kdo sugar; the structure of the MPLA is the form of lipid A from Salmonella phosphorylated at the 1-hydroxy group and 4'-monophosphate, and the Kdo sugar is linked to the 6'-hydroxy group of MPLA through an α-glycosidic bond; the Kdo-MPLA is amphiphilic and forms liposome nanoparticles after dissolving in water.

2. The novel Kdo-MPLA adjuvant according to claim 1, wherein The Kdo sugar is 3-deoxy-D-manno-octulosonic acid, Kdo, and the linkage between the MPLA and Kdo sugar is achieved through the LPS synthesis pathway of genetically engineered Salmonella, and the rfaC gene and eptA gene of the Salmonella are knocked out.

3. The novel Kdo-MPLA adjuvant according to claim 2, characterized in that, The Salmonella is a attenuated strain.

4. A preparation method of a novel Kdo-MPLA adjuvant. Based on the novel Kdo-MPLA adjuvant described in any one of claims 1 to 3, it is characterized in that, It includes the following steps: (a) Genetically engineer Salmonella to knock out the rfaC gene to block the synthesis of the LPS core polysaccharide, so that lipid A is only linked to Kdo sugar; (b) Knock out the eptA gene to remove the phosphate group at the 1-position of lipid A and retain the 4'-monophosphorylated structure; (c) Culture the genetically engineered Salmonella and extract Kdo-MPLA from the cell membrane of the bacteria.

5. The preparation method of a novel Kdo-MPLA adjuvant according to claim 4, wherein The knockout of the rfaC gene is achieved through the CRISPR-Cas9 system, and the homologous recombination fragment contains the sequences shown in SEQ ID NO.4 and SEQ ID NO.5; the knockout of the eptA gene is achieved through the CRISPR-Cas9 system, and the homologous recombination fragment contains the sequences shown in SEQ ID NO.9 and SEQ ID NO.

10.

6. The preparation method of a novel Kdo-MPLA adjuvant as described in claim 5, characterized in that, The extraction process of the Kdo-MPLA includes: (i) Pretreat the bacteria with ethanol, acetone, and ether to remove impurities; (ii) Extract the lipid components with a phenol-chloroform-petroleum ether mixture PCP; (iii) Purify the Kdo-MPLA by the acetone / ether precipitation method.

7. A novel Kdo-MPLA adjuvant according to claim 1, characterized in that, It self-assembles into nanoscale liposome nanoparticles in an aqueous solution.

8. A novel Kdo-MPLA adjuvant according to claim 1, characterized in that, The adjuvant is a TLR4 agonist and is used to activate Th1-type immune responses.

9. A vaccine composition, based on a novel Kdo-MPLA adjuvant and a vaccine antigen according to any one of claims 1 to 3, characterized in that, The adjuvant is directly mixed with the antigen to form a homogeneous preparation.

10. A vaccine composition according to claim 9, characterized in that, The antigen is an HPV antigen, a varicella-zoster virus antigen, a malaria antigen, a tuberculosis antigen, or an HIV antigen.

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