A kdo-mpla adjuvant

Abstract

The application discloses a novel Kdo-MPLA adjuvant, relates to the technical field of synthetic biology, and discloses that the adjuvant is composed of monophosphoryl lipid A (MPLA) and Kdo sugar through an alpha-glycosidic bond, the MPLA is a structure of Salmonella lipid A modified by monophosphorylation at a 4' position, and the Kdo sugar is endowed with amphiphilicity, so that the Kdo sugar is self-assembled into a liposome nanoparticle in an aqueous solution; the attenuated strain of Salmonella is edited by double knockout of rfaC and eptA genes: the rfaC knockout blocks the synthesis of LPS core polysaccharide and retains the lipid A-Kdo connection; the eptA knockout obtains monophosphorylated lipid A; after fermentation of the engineering bacteria, high-purity Kdo-MPLA is obtained by using a phenol-chloroform-petroleum ether extraction method, the process is simplified, and the product is uniform; the adjuvant can activate a TLR4 signal pathway and induce a Th1 type immune response, and the levels of inflammatory factors IL-6 and IL-1beta are significantly lower than those of natural LPS, so that the adjuvant is suitable for a mixed preparation of HPV, malaria and other vaccine antigens; and the application overcomes the uncontrollability of hydrolysis and the high cost defect of chemical synthesis of a traditional MPLA chemical extraction process.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of synthetic biology, and particularly relates to a novel Kdo-MPLA adjuvant. BACKGROUND

[0002] Monophosphoryl Lipid A (MPLA) is a derivative of Salmonella lipopolysaccharide, which has strong immune stimulating activity and weak toxicity, and is widely used as a vaccine adjuvant. MPLA is a structural component of the bacterial endotoxin LPS molecule and is the main component of the cell wall of Gram-negative bacteria, which is composed of three parts: O-antigen, core sugar and lipid A. Early studies found that LPS can cause strong immune responses such as fever and shock, so LPS is called endotoxin. People found that the LPS of Salmonella has immunogenicity and can induce antibody production when studying Salmonella. Further purification of LPS proved that the toxicity mainly comes from the lipid A part. The analysis of the precise structure of lipid A found that the lipid A of Salmonella is composed of a disaccharide backbone glucosamine and multiple fatty acid chains, and the toxicity depends on the degree of phosphorylation and the structure and position of the fatty acid.

[0003] In 1984, Edgar Ribi's team found that by weak acid hydrolysis or enzyme treatment of LPS, one phosphate group can be removed to obtain MPLA, and the toxicity is significantly reduced, but the immune stimulating ability is still retained. In 1985, David et al. proved that MPLA can activate macrophages and induce the release of cytokines (TNF-α, IL-6, IL-1β), but will not cause the strong inflammatory response of LPS. These findings indicate that MLPA is a safer immune adjuvant candidate molecule. In 1998, people explored the mechanism of MPLA and found that LPS and MPLA activate the innate immune system by binding to Toll-like receptor (TLR4), which provides a molecular basis for the mechanism of MPLA as an adjuvant. LPS activates the TLR4 / MD-2 complex, leading to strong inflammatory response such as sepsis. MPLA activates TLR4 with a weak signal, mainly inducing Th1 type immune response, and will not cause 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 was HPV vaccine (Cervarix®), GSK launched HPV vaccine in 2009 with AS04 adjuvant system, MPLA + aluminum adjuvant, which is the first MPLA adjuvanted vaccine approved for human use in the world, thus confirming the safety and effectiveness of MPLA. The zoster vaccine (Shingrix®) approved in 2017 uses MPLA + QS21 adjuvant, with a protection rate of more than 90%. The malaria vaccine (Mosquirix®) recommended by WHO in 2021 contains MPLA derivative adjuvant.

[0005] Salmonella is widely distributed in nature, often inhabiting the human and animal body, belonging to the Enterobacteriaceae family, gram-negative enteric bacilli. Nearly 1000 species (or strains) have been found, which can be divided into A, B, C, D, E basic groups according to antigen components. Among them, the main ones related to human diseases are Salmonella paratyphi A in group A, Salmonella paratyphi B and Salmonella typhimurium in group B, Salmonella paratyphi C and Salmonella choleraesuis in group C, Salmonella typhi and Salmonella enteritidis in group D, etc. Salmonella has a complex antigen structure, which can be generally divided into three types: bacterial antigen, flagellar antigen and surface antigen. Salmonella typhimurium is an invasive intracellular bacterium that mainly causes intestinal infection.

[0006] MPLA used as an adjuvant can be obtained by extracting MPLA from the cell membrane of Salmonella and chemically modifying it. GSK acquired Ribi's company and established the AS adjuvant system using the production technology obtained. The process of producing MPLA is to culture Salmonella strains, extract LPS, purify lipid A, and then hydrolyze to recover MPLA. Specifically, attenuated, non-pathogenic Salmonella R595 strain is used, high-nutrient medium LB or TSB is used, and the bacteria are collected after the logarithmic phase, washed with physiological saline or PBS for 2-3 times to remove the culture medium components, and then heated at 60°C for 1 hr to inactivate. The bacterial cells are lysed by hot phenol method, the bacterial cells are suspended in deionized water, an equal volume of phenol (90%) is added, stirred at 65°C for 30-60 min, centrifuged at 10000g for 20 min, and the LPS is mainly distributed in the water phase, and the proteins and nucleic acids are mainly distributed in the phenol phase. The water phase is dialyzed against deionized water for 48 hr, centrifuged at 100000g, and the LPS is precipitated to obtain white LPS crude product. Next, acid hydrolysis is performed to remove O-antigen and core polysaccharide, 1% acetic acid is heated to 100°C for 1 hr, and lipid A is precipitated. The hydrolysis solution is cooled and centrifuged at 10000g for 20 min to precipitate crude lipid A. Further purification of lipid A can be achieved by multiple washes with an organic solvent combination (chloroform-methanol 2:1). The 1 and 4' positions of the two sugar rings of lipid A have two phosphate groups, which need to be removed by hydrolysis. Specifically, the weak acid method is used, 0.1M HCl is used at 30°C for 30 min to selectively remove the 1 phosphate group. After the reaction, NaOH is used for neutralization, chloroform-methanol extraction, and MPLA crude product is obtained.

[0007] The above describes the MPLA chemical extraction process, which has been successfully applied to commercial vaccines, but the defects in the MPLA production process are obvious. First, in the process of removing the core sugar by hydrolysis, the hydrolysis of the core polysaccharide with the structural formula: [Lipid A]-(GlcN)-6'-α-Kdo-(core polysaccharide)-O-antigen is non-specific, and the core polysaccharide is usually connected by α- or β-glycosidic bonds by heptose (Hep), glucose (Glc), galactose (Gal), etc. The hydrolysis product of breaking the glycosidic bond is a mixture, in addition to Kdo sugar hydrolysis, other glycosidic bonds in the core polysaccharide will also be gradually hydrolyzed to generate monosaccharides and short-chain oligosaccharides, and insufficient Kdo hydrolysis will inevitably lead to various lengths of core polysaccharides connected with lipid A, so one of the biggest defects of this hydrolysis process is that the degree of hydrolysis is uncontrollable, the target product molecular weight distribution is in a range, and the quality cannot be quantitatively controlled. Second, in the process of further hydrolyzing the bisphosphate lipid A into monophosphate lipid A, excessive hydrolysis may lead to the loss of the 1-phosphate group of lipid A, and even if the reaction conditions are strictly controlled, part of the product without phosphate is inevitable, and the lipid A without phosphate has no pharmacological activity, so the quality of MPLA product is unstable. In summary, this production process is affected by many factors, more specifically: the Kdo bond is broken at pH 4.5 in acidic conditions, if it is excessive, it will degrade lipid A; the reaction temperature is 100°C, if it exceeds 105°C, it will destroy the structure of lipid A; the reaction time is 30-60 min, if the time is short, the hydrolysis is insufficient, if the time is long, the lipid A is decomposed; low ionic strength is beneficial to the reaction, if the salt concentration is high, it will inhibit the attack of protons on the glycosidic bond. Precise control of pH, temperature and time, balance yield and quality, it is difficult to achieve batch consistency in industrial production. In summary, the method of bacterial culture, chemical extraction, and chemical modification is not a good process for preparing MPLA.

[0008] To address the above process defects, the US Institute of Infectious Diseases has invented a chemical total synthesis process. The adjuvant prepared by this process belongs to the MPLA analogues, which has been used in the development of various infectious disease 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, palmitic acid) are used for acylation modification; and phosphating reagents (such as POCl3, H3PO4) are used to introduce phosphate groups. The core steps of chemical synthesis include: acylation reaction of glucosamine, introduction of fatty acid chains at positions 2 and 3 of glucosamine; phosphating reaction, introduction of phosphate groups at position 4' (key step, determines TLR4 binding ability); further modification of fatty acid chains, condensation of 6' hydroxyl group with activated fatty acid (such as palmitic acid-NHS ester). Compared with the extraction method, the chemical synthesis structure has the characteristics of high uniformity, no LPS residue risk, and good batch consistency. However, it has the disadvantages of high cost, high process complexity, use of DMF / pyridine and chloroform / methanol organic solvents in the preparation process, and low phosphating efficiency.

[0009] The above describes two technical routes to prepare MPLA. In addition to their respective process defects, they also have a common shortcoming, which is that they are all non-polar molecules and are not soluble in water. They need to be dispersed in organic solvents and then prepared into liposomes together with cholesterol and DOPE, etc. Poor solubility often leads to uneven dispersion. In addition, in the preparation application, the adjuvant is packaged separately from the antigen, and mixing before use leads to inconvenience in use.

[0010] MPLA, as a highly efficient and low-toxic TLR4 agonist, has become an important part of modern vaccine adjuvant systems. With the success of GSK's herpes zoster vaccine in the market, many domestic vaccine companies have begun to follow the preparation of MPLA. In terms of application, in addition to the above-mentioned malaria vaccine, HPV and VZV, there are COVID-19 vaccine and tuberculosis vaccine as well as HIV vaccine. The main suppliers are GSK, the US Institute of Infectious Diseases IDRI, and Avanti Polar Lipids. The annual production capacity is about 500 kg, which meets the demand of 100 million doses of vaccine. With the continuous expansion of clinical value, MPLA is expected to achieve greater breakthroughs in mRNA vaccines, universal influenza vaccines, etc. in the next five years, and the global market size is expected to reach 2.5 billion US dollars (2028).

[0011] The latest direction of MPLA internationally is to develop an Escherichia coli synthetic biology production platform. Due to the problems of multiple gene modification and expression regulation, the Escherichia coli synthetic biology route is still under exploration. The present invention belongs to the field of synthetic biology, and we use a Salmonella attenuated strain as the starting strain for gene editing. The edited strain is cultured on a large scale, and the TLR4 agonist can be directly extracted from the cell membrane to prepare a new MPLA derivative, Kdo-MPLA. SUMMARY

[0012] In view of the above-mentioned existing problems, the present application is proposed.

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

[0014] To solve the above technical problems, the present application provides the following technical solutions.

[0015] In a first aspect, the present application provides a new Kdo-MPLA adjuvant, which is a complex of MPLA and Kdo sugar, the hydrophobic part of which is monophosphoryl lipid A, MPLA, and the hydrophilic part of which is Kdo sugar; the structure of the MPLA is a monophosphorylated form of the lipid A derived from Salmonella at the 1-hydroxyl and 4' position, and the Kdo sugar is connected to the 6'-hydroxyl of the MPLA through an alpha-glycosidic bond; the Kdo-MPLA has amphiphilic properties and forms a liposome nanoparticle after dissolving in water.

[0016] As a preferred scheme of the new Kdo-MPLA adjuvant, the Kdo sugar is 3-deoxy-D-manno-octulosonic acid, Kdo, the connection between the MPLA and the Kdo sugar is achieved by gene editing the LPS synthesis pathway of Salmonella, and the rfaC gene and the eptA gene of the Salmonella are knocked out.

[0017] As a preferred scheme of the new Kdo-MPLA adjuvant, the Salmonella is an attenuated strain, preferably R595 strain (ATCC9700) or VNP2000 strain.

[0018] In a second aspect, the present application provides a preparation method of the new Kdo-MPLA adjuvant, comprising the following steps:

[0019] (a) gene editing of Salmonella to knock out the rfaC gene to block the synthesis of the LPS core polysaccharide, so that the lipid A is only connected to the Kdo sugar;

[0020] (b) knocking out the eptA gene to remove the phosphate group at the 1 position of the lipid A, while retaining the monophosphorylated structure at the 4' position;

[0021] (c) culturing the gene edited Salmonella, and extracting the Kdo-MPLA in the cell membrane of the bacterial body.

[0022] As a preferred scheme of the preparation method of the novel Kdo-MPLA adjuvant, wherein: the knockout of the rfaC gene is realized by the CRISPR-Cas9 system, and the homologous recombination fragment comprises the sequences shown in SEQ ID NO. 4 and SEQ ID NO. 5; the knockout of the eptA gene is realized by the CRISPR-Cas9 system, and the homologous recombination fragment comprises the sequences shown in SEQ ID NO. 9 and SEQ ID NO. 10.

[0023] As a preferred scheme of the preparation method of the novel Kdo-MPLA adjuvant, wherein: the extraction process of the Kdo-MPLA comprises:

[0024] (i) the bacterial cells are pretreated by ethanol, acetone and diethyl ether to remove impurities;

[0025] (ii) the lipid components are extracted by a phenol-chloroform-petroleum ether mixed solution PCP;

[0026] (iii) the Kdo-MPLA is purified by an acetone / diethyl ether precipitation method.

[0027] As a preferred scheme of the novel Kdo-MPLA adjuvant, the adjuvant self-assembles into nanoscale liposome nanoparticles in an aqueous solution, and presents an "onion ring" structure under a transmission electron microscope.

[0028] As a preferred scheme of the novel Kdo-MPLA adjuvant, the adjuvant is a TLR4 agonist, is used for activating a Th1 type immune response, and the induced levels of inflammatory factors IL-6 and IL-1β are significantly lower than those of natural LPS.

[0029] In a third aspect, the present application provides a vaccine composition comprising the novel Kdo-MPLA adjuvant and a vaccine antigen, wherein the adjuvant is directly mixed with the antigen to form a homogeneous preparation.

[0030] As a preferred scheme of the novel Kdo-MPLA adjuvant, wherein: the antigen is an HPV antigen, a herpes zoster virus antigen, a malaria antigen, a tuberculosis antigen or an HIV antigen.

[0031] The application has the advantages that: the Salmonella is reformed by gene editing method, the rfaC gene is knocked out to block the connection of the LPS core polysaccharide of the Salmonella cell membrane, only the structure of the lipid A and the Kdo sugar connection is formed; the eptA gene is knocked out to realize the monophosphorylation of the lipid A4', and the hydroxyl group is reserved at the 1 position. The Kdo-MPLA with TLR4 agonist activity can be directly extracted from the cultured gene edited Salmonella, wherein the hydroxyl group of the MPLA at the 6' position is connected with the Kdo sugar, the Kdo sugar has hydrophilicity, and the Kdo-MPLA has water solubility. The prepared adjuvant has a nano-liposome structure, and can be directly mixed with a vaccine antigen preparation.

[0032] The Salmonella strain used in the application preferably adopts an attenuated strain, such as the R595 strain and the VNP2000 strain, and more preferably the R595 is derived from the ATCC strain preservation center (ATCC 9700).

[0033] The rfaC gene, also known as the waaC gene, is a key gene involved in the synthesis of the lipopolysaccharide core oligosaccharide in bacteria, encodes heptose transferase, and the main function is to add heptose to the LPS core oligosaccharide.

[0034] The eptA gene described in the application is a phosphoethanolamine transferase gene, which mainly participates in the modification of the lipopolysaccharide LPS in bacteria, and regulates the addition of phosphoethanolamine on the LPS. Knocking out the gene can selectively remove the phosphate group at the 1 position and reserve the hydroxyl group.

[0035] The double-knockout engineering bacteria have good growth performance and can fully meet the production requirements.

[0036] The application establishes a Kdo-MPLA freeze-dried bacterial body, a Kdo-MPLA extraction process and quality standards, and the obtained product is a vaccine adjuvant. After the Kdo-MPLA is extracted according to the method, and then treated by weak acid hydrolysis, the Kdo-MPLA is compared with the MPLA, and the Rf value retention factors are consistent on the thin layer plate, indicating that the structures are consistent. The Kdo-MPLA is inoculated into immune cells, and the response is consistent with the MPLA reported in the literature, the inflammatory factors are significantly reduced, and the use requirements of the vaccine adjuvant are met. BRIEF DESCRIPTION OF DRAWINGS

[0037] In order to more clearly illustrate the technical solutions of the embodiments of the application, the drawings needed in the embodiment description will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the application, and those skilled in the art can also obtain other drawings according to these drawings without creative labor.

[0038] Figure 1 The Kdo-MPLA engineering bacteria of the present application.

[0039] Figure 2 The Kdo-MPLA engineering bacteria of the present application.

[0040] Figure 3 The Kdo-MPLA of the present application in aqueous solution.

[0041] Figure 4 The Kdo-MPLA of the present application in aqueous solution.

[0042] Figure 5 The Kdo-MPLA of the present application in aqueous solution.

[0043] Figure 6 The Kdo-MPLA of the present application in aqueous solution. DETAILED DESCRIPTION

[0044] In order to make the above objectives, features and advantages of the present application more apparent, specific embodiments of the present application will be described in detail below with reference to the accompanying drawings.

[0045] In the following description, a lot of specific details are set forth in order to facilitate a thorough understanding of the present application, but the present application can also be implemented in other ways different from those described herein, and those skilled in the art can make similar generalizations without departing from the spirit of the present application, so the present application is not limited to the specific embodiments disclosed below.

[0046] Secondly, the "one embodiment" or "embodiment" referred to herein can include specific features, structures or characteristics contained in at least one implementation of the present application. "In one embodiment" appearing in different places in the specification does not mean the same embodiment, nor is it an independent or alternative embodiment that excludes other embodiments.

[0047] In the experimental methods of the embodiments and experimental examples, if not otherwise specified, they are all conventional methods. The test materials used in the following embodiments, if not otherwise specified, are all purchased from conventional biochemical reagent stores. The Salmonella R595 used in the embodiments of the present application is from the ATCC strain preservation center (ATCC 9700), and it should be particularly pointed out that the methods used in the embodiments of the present application are also applicable to other strains of Salmonella and Gram-negative bacteria such as Escherichia coli.

[0048] Example 1, which is a first embodiment of the present application, provides rfaC gene knockout:

[0049] The Cas9 lambda red homologous recombination plasmid is used to knockout the rfaC gene of R595. The Cas9 lambda red plasmid has Kan resistance and expresses a recombination protein; the CRISPR gRNA plasmid has Amp resistance and carries a gRNA sequence. The sequence of the gene rfaC is shown in SEQ ID NO. 1, and the sequence of the encoded protein is shown in SEQ ID NO. 2.

[0050] A 20bp gRNA sequence SEQ ID NO. 3 is designed for targeting, and the sequence is: 5'-tgcgcttaccgacgcgcaac-3'. The upstream homologous arm sequence of the knockout gene rfaC is shown in SEQ ID NO. 4, and the downstream homologous arm sequence is shown in SEQ ID NO. 5.

[0051] Preparation of R595 containing Cas9 lambda red homologous recombination plasmid: inoculate a single colony into 2ml LB medium and cultivate at 37°C until OD600=0.6. Centrifuge to collect the bacterial cells, wash the bacterial cells with pre-cooled 10% glycerol 3 times, and then resuspend the bacterial cells in 100ul of 10% glycerol. Add 10ul of Cas9 plasmid to the cells for electroporation, and set the conditions as follows: 2400V, 200Ω, 1mm. Then, plate at 30°C, cultivate with Kan resistance, and pick single colonies overnight to obtain R595-cas9.

[0052] Preparation of competent cells by transforming R595-cas9 with gRNA plasmid: inoculate a single colony of R595-cas9 into 2ml LB medium and cultivate at 30°C until OD600=0.3-0.5. Add arabinose for induction at a concentration of 3mg / ml, incubate for 1hr, centrifuge to collect the bacterial cells, wash the bacterial cells with 10% glycerol 3 times, and then resuspend the bacterial cells in 100ul. Add the gRNA plasmid and the homologous sequence to the competent cells for electroporation at 2400V, 200Ω, and 2mm. Then, plate on LB plates with Kan + and Amp + , incubate for 16hr or more, pick single colonies, and amplify the culture.

[0053] Remove single plasmid: add IPTG to the culture medium and cultivate overnight. Pass the culture for 2 generations to remove the gRNA plasmid. At this time, R595 loses Amp resistance, so pick single colonies for culture, pass the culture for generations, and save the seeds, which are R595 ΔrfaC.

[0054] Example 2, which is a second embodiment of the present application, provides knockout of the eptA gene based on R595 ΔrfaC: Figure 1 and Figure 2 ​

[0055] The sequence of the phosphoethanolamine transferase gene eptA is shown as SEQ ID NO. 6, and the sequence of the encoded protein is shown as SEQ ID NO. 7.

[0056] The gRNA sequence targeting is designed as 20bp of SEQ ID NO. 8: 5'-GGCGAATCATTGGGTGAAAA-3', the upstream homologous arm sequence of the phosphoethanolamine transferase gene eptA is shown as SEQ ID NO. 9, and the downstream homologous arm sequence is shown as SEQ ID NO. 10.

[0057] Preparation of competent cells of R595ΔrfaC by gRNA plasmid transformation: inoculate R595ΔrfaC monoclonal (containing cas9 plasmid, Kan resistance) in 2ml LB medium, cultivate at 30℃ to OD600=0.3~0.5, add arabinose for induction, the concentration is 3mg / ml, incubate for 1hr, centrifugal collect the bacterial body, wash 3 times with 10% glycerol, resuspend 100ul. Add gRNA plasmid and homologous sequence to the competent cells, electrotransform 2400V, 200Ω, 2mm. Then coat on LB plate, Kan + and Amp + , incubate for 16hr or more, pick single colonies for amplification culture.

[0058] Remove double plasmid: add IPTG to the culture medium, cultivate overnight, pass 2 generations, remove the gRNA plasmid, at this time the VNP loses Amp resistance, pick single colonies for culture and passage, increase the culture temperature to 37℃ overnight, remove the cas9 plasmid, keep the seed, which is R595ΔrfaC+ ΔeptA. Take the engineering bacteria for transmission electron microscope examination, see Figure 1 , the results show that the bacterial body is complete and the morphology is normal. Perform continuous culture monitoring in LB medium, draw the growth curve, see Figure 2 , the results show that, compared with the wild type, the growth of the engineering bacteria is good, there is obvious logarithmic growth phase and plateau phase, which meets the predetermined standard.

[0059] Example 3 is a third embodiment of the present application, which provides a method for identifying the gene knockout condition:

[0060] The rfaC knockout condition is identified by the following primers:

[0061] Upstream primer: 5'-ctctgcaaggcagcgaagt-3'

[0062] Downstream primer: 5'-cgttggccgcactatcacta-3'

[0063] PCR sequencing wild strain sequence interval: 3813820-3815699, theoretically 1880 nt, actual sequencing result 926 nt.

[0064] eptA knockout was identified by the following primers:

[0065] Upstream primer: 5'-tccagttcagcagtatgtcgcc-3'

[0066] Downstream primer: 5'-actgcctgccttgagcatcaac-3'

[0067] PCR sequencing wild strain sequence interval: 4440721-4443323, theoretically 2603 nt, actual sequencing result 1059 nt.

[0068] Example 4 is the fourth embodiment of the present application, which provides Kdo-MPLA engineering bacteria fermentation and extraction of target objects:

[0069] The strain obtained in Example 2 was expanded and cultured, and the seed liquid was inoculated into the fermentation base medium (ratio: plant 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 as follows: pH 7.0, rotation speed 150 rpm, temperature 37°C, tank pressure 0.02 MPa, and aeration amount 60 L / min.

[0070] During the fermentation process, the OD600 value was measured every 1 hour, and the mirror was examined every 2 hours. The pH value was adjusted with ammonia and 6M hydrochloric acid, and should be controlled at 6.8-7.2. The dissolved oxygen was controlled to be greater than 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 bacterial cells were resuspended and washed by stirring. The bacterial cells were washed twice, and the precipitate was collected by centrifugation and stored at -20°C. The bacterial cells were taken out from -20°C after being frozen for at least 24 hours, and Kdo-MPLA was extracted;

[0071] 1. The dried bacteria were dissolved in 90% ethanol at 20 mg / ml, stirred for 1 h, and then the ethanol was removed by a vacuum filter, and the bacterial cells were recovered. The above operation was repeated once. (Purpose: to reduce the content of phospholipids)

[0072] 2. The recovered bacterial cells were dissolved in acetone at a concentration of 40 mg / mL (initial weight of bacterial cells), stirred for 15 minutes, and then the acetone was removed by a vacuum filter, and the bacterial cells were recovered. The above operation was repeated once.

[0073] 3. The recovered bacteria were dissolved in ether at a concentration of 40 mg / mL (original weight of bacteria) and stirred for 15 minutes. The ether was then removed by vacuum filtration and the bacteria were recovered. The above procedure was repeated once.

[0074] 4. The ether-treated bacteria were dried overnight in air.

[0075] 5. A mixture of 89% phenol: chloroform: petroleum ether = 19:45:72 (PCP) was prepared and left overnight.

[0076] 6. The bacteria powder was suspended in PCP at a concentration of 70 mg / mL and stirred for 30 minutes. The suspension was then centrifuged at 3000 g for 15 minutes at 4°C.

[0077] 7. The supernatant was removed. Step 6 was repeated and the supernatants from both runs were combined. Most of the liquid was removed by rotary evaporation and a small volume of liquid was left. The volume was measured and water was added dropwise until a persistent turbidity appeared.

[0078] 8. Five volumes of acetone cooled in an ice bath and one volume of cooled ether were then added to the turbid liquid and mixed rapidly. The mixture was left in an ice bath for 30 minutes.

[0079] 9. The mixture was centrifuged at 5000 g for 15 minutes at 4°C. The supernatant was removed and the Kdo-MPLA was recovered.

[0080] 10. The ether was removed by rotary evaporation and the pellet was washed once more with cold acetone (steps 7 and 8). A yield of 4-5% of the original weight was obtained.

[0081] Example 5, with reference to Figure 3 、 Figure 4 and Figure 5 is a fifth embodiment of the present application which provides a characterisation of Kdo-MPLA:

[0082] The Kdo-MPLA powder was resuspended in water at 10 mg / mL and sonicated in a water bath at 45-55°C. One portion was examined by transmission electron microscopy and is shown in Figure 3 and Figure 4 which shows that the Kdo-MPLA appears as nano-sized particles with an "onion ring" like structure. This has not been reported previously in the literature. The other portion was treated by adding one volume of 0.2N HCI and boiling in water for 15 minutes. The reaction was stopped in an ice bath. Five volumes (calculated on the starting volume) of chloroform:methanol 2:1 (v / v) were added and the mixture was vortexed and centrifuged at 500-1000 g. The bottom layer was harvested and the solvent was evaporated under a stream of N2. The crude MPLA was harvested and analysed by thin layer chromatography (TLC). The method was as follows:

[0083] Sample treatment: dissolve sample with chloroform:methanol=4:1, draw a straight line at 1cm from the bottom of the thin plate with a pencil, and point the sample on the straight line with a capillary;

[0084] Developing agent: chloroform:methanol:water:ammonium hydroxide solution=40:25:4:2;

[0085] When the solvent is at 1cm from the top of the thin plate, take the thin plate out of the developing agent, dry the solvent, spray 10% sulfuric acid ethanol solution on the surface with a sprinkler, and heat to develop color at 105℃. The result is shown in Table 1. Figure 5 , and the Rf value is ~0.6. The MPLA from the engineering bacteria is consistent with the standard product. The standard product is from Avanti Polar Lipids Company.

[0086] Experimental Example 1, referring to Figure 6 , is an experimental example of the present application, which provides an immune cell response test:

[0087] DMEM medium is added with 10% serum to culture Raw264.7 macrophages, the macrophages are stimulated with Kdo-MPLA prepared in Example 4 for 12 hours, the control group is PBS sucrose solution, and then the cells are lysed with TRIZOI to extract RNA, which is reversely transcribed into cDNA, and the expression amount is detected by Q-PCR, and the cytokine expression level is calculated by ΔΔct method. The result is shown in Table 2. Figure 6 , the expression levels of inflammatory factors IL-6 and IL-1β are significantly decreased, and the stimulation ability of IFN-γ is reserved, which meets the design requirements of vaccine adjuvant.

[0088] It should be noted that the above examples are only used to illustrate the technical solutions of the present application and not to limit, although the present application is described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can be modified or replaced equivalently without departing from the spirit and scope of the technical solutions of the present application, which should be covered in the scope of the claims of the present application.

Claims

1. A Kdo-MPLA adjuvant, characterized in that, The adjuvant is a complex of MPLA and Kdo sugar, the hydrophobic part of which is monophosphoryl lipid A, MPLA, and the hydrophilic part of which is 3-deoxy-D-manno-octulosonic acid Kdo sugar; the structure of the MPLA is a form of monophosphorylation of lipid A derived from a Salmonella R595 attenuated strain with a preservation number of ATCC 9700 at 1-hydroxyl and 4'-position, and the Kdo is connected to the 6'-hydroxyl of the MPLA through an alpha-glycosidic bond; the Kdo-MPLA has amphiphilicity and forms a liposome nanoparticle after being dissolved in water; The novel Kdo-MPLA adjuvant is prepared by the following steps: (a) gene editing of the Salmonella attenuated strain R595, ATCC 9700, knocking out the rfaC gene to block the synthesis of the LPS core polysaccharide, so that the lipid A is only connected to the Kdo, wherein the two ends of the homologous recombination fragment for knocking out the rfaC gene are a 5' homologous arm and a 3' homologous arm, respectively, the 5' homologous arm consists of the nucleotide sequence shown in SEQ ID NO. 4, and the 3' homologous arm consists of the nucleotide sequence shown in SEQ ID NO. 5; (b) knocking out the eptA gene to remove the phosphate group at the 1-position of the lipid A, retaining the monophosphorylation structure at the 4'-position, wherein the two ends of the homologous recombination fragment for knocking out the eptA gene are a 5' homologous arm and a 3' homologous arm, respectively, the 5' homologous arm consists of the nucleotide sequence shown in SEQ ID NO. 9, and the 3' homologous arm consists of the nucleotide sequence shown in SEQ ID NO. 10; (c) culturing the gene-edited Salmonella and obtaining Kdo-MPLA through the following extraction process: (i) removing impurities by pretreating the bacterial cells with ethanol, acetone, and diethyl ether; (ii) extracting the lipid component with a phenol-chloroform-petroleum ether mixed solution PCP; (iii) purifying the Kdo-MPLA through an acetone / ethyl ether precipitation method.

2. A Kdo-MPLA adjuvant as claimed in claim 1, wherein, The Salmonella attenuated strain R595 is derived from the ATCC strain preservation center with a preservation number of ATCC 9700.

3. A Kdo-MPLA adjuvant as claimed in claim 1, wherein, It self-assembles into a nanoscale liposome nanoparticle in an aqueous solution.

4. A Kdo-MPLA adjuvant as claimed in claim 1, wherein, The adjuvant is a TLR4 agonist for activating a Th1 type immune response.

5. A vaccine composition, characterized in that, The vaccine composition comprises the Kdo-MPLA adjuvant of any one of claims 1-4 and a vaccine antigen, and the adjuvant is directly mixed with the antigen to form a homogeneous preparation.

6. A vaccine composition as claimed in claim 5, wherein, 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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