CN117281899A

CN117281899A - Adjuvant system and preparation method and application thereof

Info

Publication number: CN117281899A
Application number: CN202310748409.0A
Authority: CN
Inventors: 袁楚晓; 姚文荣; 王维龙; 张琼月; 梁婧; 陈健平; 洪坤学; 刘勇
Original assignee: Abzymo Biosciences Co ltd; Jiangsu Ruike Biotechnology Co ltd
Current assignee: Abzymo Biosciences Co ltd; Jiangsu Ruike Biotechnology Co ltd
Priority date: 2022-06-24
Filing date: 2023-06-24
Publication date: 2023-12-26

Abstract

The invention relates to an adjuvant system containing aluminum nano particles and emulsion, a preparation method and application thereof. The adjuvant system and various antigens are combined to effectively enhance the immune response of the vaccine, the enhancement effect of the adjuvant system is obviously better than that of a single aluminum adjuvant, and the adjuvant system can still generate the effect equivalent to that of an AS04 adjuvant under the condition of reducing the cost. The adjuvant system has low cost and good safety, can be used as a candidate adjuvant for various vaccines, and provides a new idea for developing and applying novel vaccine adjuvants.

Description

Adjuvant system and preparation method and application thereof

Technical Field

The application belongs to the field of biomedical engineering, and in particular relates to an adjuvant system, a preparation method thereof and application thereof in the field of immunotherapy and prevention.

Background

Adjuvants (Adjuvants) are nonspecific immunomodulators, and can be added into vaccine to improve immunity, change immunity type, and prolong immunity duration. Particularly, for vaccines with weaker immunogenicity (such as inactivated vaccines, subunit vaccines, recombinant protein vaccines, polypeptide vaccines and the like), the reasonable application of the adjuvant can improve the positive transfer rate of the vaccine inoculated in different immune background populations. In addition, the adjuvant can reduce the dosage of antigen in the vaccine, reduce the number of vaccine needles, and help to reduce the vaccine cost and solve the productivity problem in epidemic outbreak.

Aluminum adjuvants have been used for a long time as immunoadjuvants, and their safety and immunopotentiation have been confirmed. The traditional aluminum adjuvant is mainly aluminum salt (such as aluminum sulfate and aluminum phosphate) or aluminum hydroxide coarse particles, and researches show that compared with the aluminum coarse particles, the nano aluminum adjuvant (nano aluminum for short) has smaller particles, increased specific surface area, stronger adsorption capacity and adjuvant activity, can improve the immune response of organisms, and greatly reduces the side effects of the adjuvant.

Although low cost, aluminum adjuvants lack adjuvant effect or only weak adjuvant effect on certain vaccine candidate antigens from an immunological perspective, can enhance humoral immune response, but have no enhancement effect on cellular immunity, and have no obvious enhancement effect on immune response in many human vaccines (especially recombinant protein vaccines and polypeptide vaccines). Thus, with the advent of more and more candidate vaccines, the need for new clinically useful adjuvants is rapidly growing.

Emulsion-type adjuvants (including oil-in-water emulsions, water-in-oil emulsions, etc.) are an important branch of new adjuvants, and emulsions can be used in combination with a variety of weak antigens (recombinant proteins, polypeptides, etc.) and elicit highly or lowly antigen-specific antibodies. Milk-type adjuvants generally comprise an oil phase component, an aqueous phase component and an emulsifier. The oil-in-water emulsion mainly comprises water phase components, has high tolerance to human body and has good compatibility with most vaccine antigens. After mixing different oil phase components, water phase components and emulsifying agents, the interaction modes of the components are complex and various, so that the adjuvant activity of the emulsion prepared by adopting different formulas is difficult to predict.

And researches show that the antigen alone or only one adjuvant is used, the strength of the immune response induced by the antigen is low and is biased to a certain type, the effect of improving the reaction strength, the immunity maintaining time, the immune tolerance and the like of the antigen is limited, and the vaccine using the composite adjuvant is expected to more comprehensively activate the immune response through the synergistic effect.

Three HPV vaccines currently on the market, only Cervarix of gladin smith uses a complex adjuvant (aluminium hydroxide and MPL) and shows higher immunogenicity, but MPL is currently a chemically treated "attenuated" salmonella lipopolysaccharide, which is complex in process and costly, and the productivity is easily limited by the source of MPL. In addition, the product is immature in domestic large-scale production condition, and can not meet the subsequent clinical use requirements. Whereas the mersartorial HPV vaccine Gardasil and the nine-valent HPV vaccine Gardasil 9 both use a single aluminum phosphate adjuvant, the antibody titer produced is far lower than Cervarix.

The composite immune adjuvant is developed by matching different types of adjuvants, but the mixed application of the adjuvants with different mechanisms has complementarity, the safety and the adjuvant activity of the adjuvant are changed, the immunostimulation activity can be maximized on the premise of ensuring the safety by preparing the components of the adjuvant according to the proportion and adopting the preparation process, the problems cannot be predicted, the experiment and verification are needed, and the technical problem which is urgently needed to be solved by the technicians in the field is also realized.

Disclosure of Invention

The invention aims to provide a novel adjuvant system with good safety and immunostimulation activity and human application prospect, and a preparation method and application thereof. The aluminum adjuvant is mixed with water phase to be used as water phase of the new emulsion, and the water phase and the oil phase are dispersed and homogenized to obtain the composite nano emulsion system.

The inventor surprisingly discovers that the composite nanoemulsion adjuvant system taking nano aluminum and tween 80 AS an oil-water interface stabilizer is obtained by combining the oil-in-water emulsion and the aluminum adjuvant in a proper compatibility mode and dispersing and homogenizing, has stronger immunostimulating activity, and can obviously improve the immunogenicity of the vaccine when being combined with HPV L1 VLP vaccine, thereby generating the beneficial effect equivalent to AS04 adjuvant.

The technical scheme of the invention is as follows:

in one aspect, the invention provides an adjuvant system comprising an oil-in-water emulsion and aluminum nanoparticles uniformly dispersed in the oil-in-water emulsion, wherein the oil-in-water emulsion encapsulates the aluminum nanoparticles to form emulsion droplets, and the emulsion droplets have a particle size of 100-250 nm.

In some embodiments, the aluminum nanoparticle is an aluminum nanoparticle formed from a material selected from aluminum phosphate, aluminum sulfate, aluminum hydroxide, or a mixture thereof. In some embodiments, the aluminum particles are aluminum hydroxide nanoparticles. The particle size of the aluminum particles is 100 to 1000nm, preferably 100 to 800nm, 100 to 500nm, 100 to 400nm or 100 to 250nm. Preferably, the aluminum hydroxide nano-particles have an aluminum content of 0.5mg/ml to 2mg/ml in the emulsion. More preferably, the aluminum hydroxide nanoparticles have an aluminum content of 2mg/ml in the emulsion.

In some embodiments, the emulsion comprises an oil phase and an aqueous phase.

In some embodiments, the ratio of aqueous phase to oil phase is 5-15ml aqueous phase to 1g oil phase.

In some embodiments, the oil phase comprises a metabolizable oil, preferably, the metabolizable oil is squalene.

In some embodiments, wherein the oil phase further comprises alpha-tocopherol.

In some embodiments, the weight ratio of squalene to alpha-tocopherol is 0.8-1, e.g., 0.85-0.95, preferably 0.9.

In some embodiments, wherein the oil phase further comprises span 85.

In some embodiments, wherein the weight ratio of squalene to span 85 is 8-10, preferably 8.3.

In some embodiments, wherein the oil phase further comprises an additional immunostimulant.

In some embodiments, wherein the immunostimulant is a TLR4 agonist selected from any one of LPS, MPL, 3D-MPL or GLA.

In some preferred embodiments, the immunostimulant is MPL.

In some embodiments, the aqueous phase contains tween 80; the aluminum particles are dissolved in the aqueous phase.

In some embodiments, the weight ratio of aluminum particles to tween 80 is 0.1 to 0.6, preferably 0.13.

In some embodiments, wherein the aqueous phase further comprises an additional immunostimulant.

In some embodiments, wherein the immunostimulant is selected from any one or more of CpG, polyIC, R837, R848.

In some preferred embodiments, the immunostimulant is CpG.

In another aspect, the invention provides a method of preparing the adjuvant system comprising the steps of:

a) Preparing aluminum nano particles;

b) Adding the aluminum nano particles in the step a) into a buffer solution containing an emulsifier to prepare a composite water phase;

c) Preparing an oil phase comprising a metabolisable oil;

d) Mixing the composite aqueous phase of the step b) with the oil phase, and dispersing and homogenizing to obtain the adjuvant system.

In some embodiments, the aluminum nanoparticle is an aluminum nanoparticle selected from aluminum phosphate, aluminum sulfate, aluminum hydroxide, or a mixture of at least two thereof.

In some embodiments, the metabolizable oil is squalene.

In some embodiments, the oil phase further comprises alpha-tocopherol or span 85.

In some embodiments, the weight ratio of squalene to alpha-tocopherol is from 0.8 to 1, such as from 0.85 to 0.95, preferably 0.9.

In some embodiments, the weight ratio of squalene to span 85 is 8-10, preferably 8.3.

In some embodiments, the buffer solution is a phosphate buffer solution or a citrate buffer solution.

In some embodiments, the emulsifier is tween 80 and the weight ratio of the aluminum adjuvant to tween 80 is from 0.1 to 0.6, preferably 0.13.

In some embodiments, the oil phase further comprises an additional immunostimulant; preferably, the immunostimulant is a TLR4 agonist selected from any one of LPS, MPL, 3D-MPL or GLA.

In some embodiments, the aqueous phase further comprises an additional immunostimulant; preferably, the immunostimulant is selected from any one or more of CpG, polyIC, R837, R848.

In some embodiments, the dispersing step comprises stirring at 8000-10000rpm for 10-25min.

In some embodiments, the homogenizing step comprises sequentially homogenizing at a pressure of from 0.5 to 1.2bar across a slit-type homogenizing valve and from 80 to 160MPa across a microfluidic valve.

In some embodiments, the homogenizing step is repeated for 5-20 cycles.

In some embodiments, the homogenizing step comprises sequentially performing a first homogenizing at a pressure of 0.5-1bar across the slit-type homogenizing valve and 80-140MPa across the microfluidic valve, and then performing a second homogenizing at a pressure of 1-1.2bar across the slit-type homogenizing valve and 120-160MPa across the microfluidic valve, the second homogenizing pressures being greater than the first homogenizing pressures, respectively.

In some embodiments, the first homogenizing is performed for 5-10 cycles and the second homogenizing is performed for 5-10 cycles.

In another aspect, the invention provides an immunogenic composition comprising the adjuvant system described above, further comprising at least one antigen adsorbed on the aluminium particles and homogeneously dispersed in the emulsion.

In some embodiments, the antigen is one or more antigens derived from bacteria, viruses, parasites, fungi, tumors, human autoantigens, and/or allergens.

In some embodiments, the antigen is derived from at least one of human immunodeficiency virus, human papilloma virus HPV, varicella zoster virus, human herpes simplex virus, respiratory syncytial virus, hepatitis b virus, hand-foot-mouth virus, coxsackie virus, human cytomegalovirus, influenza virus, coronavirus, and neocoronavirus SARS-CoV-2.

In some embodiments, the antigen is derived from varicella zoster virus.

Varicella Zoster Virus (VZV) is one of the human herpesviruses. The subunit vaccine of VZV glycoprotein E (gE) is the current mainstream research direction of varicella vaccine, gE is encoded by the ORF68 gene of virus, and the gene consisting of 1872 bases is located in the short fragment region of VZV genome. In the preparation of recombinant VZV gE proteins using modern biological molecular techniques, the gE protein will typically be truncated such that it lacks a carboxy-terminal hydrophobic anchor region. The shintrix developed by gelan smith is a subunit vaccine based on recombinant gE protein supplemented with a novel adjuvant AS01B, and three-phase clinical trial data show that the subunit vaccine has immunogenicity and efficacy superior to that of Zostavax in the elderly and was approved by the FDA in 2017. The production of VZV gE proteins is usually achieved by expression in cultured cells or by chemical synthesis. Host cells that are often used and suitable for producing proteins include E.coli, yeast, insects, and mammals. As used herein, gE proteins are well known to those skilled in the art (see, e.g., NCBI Genbank database accession number: Q9J3M 8).

Such antigens are readily available using conventional techniques of modern molecular biology, typical methods include:

(1) Cloning the gE protein gene (subjected to codon optimization) into an expression vector;

(2) Transfecting the expression vector obtained in the step (1) into CHO cells;

(3) Obtaining a cell strain stably expressing gE protein through cell population screening and monoclonal screening;

(4) And (3) using the cell strain obtained in the step (3) to express so as to obtain the VZV gE protein.

The protein obtained above can be processed by conventional methods such as hydrophobic chromatography, anion exchange chromatography, and hydroxyapatite chromatography to obtain purer antigen protein.

In some embodiments, the antigen is derived from a coronavirus, such as the middle east respiratory syndrome coronavirus (MERS CoV), the severe acute respiratory syndrome coronavirus (SARS CoV), and in particular the novel coronavirus SARS-CoV-2.

The Receptor Binding Domain (RBD) of SARS-CoV-2 spike protein (S protein) is considered to be the most predominant antigen target region for inducing the production of neutralizing antibodies by the body. The RBD can be used as a vaccine to focus the neutralizing antibodies generated by the stimulation of the organism on the receptor binding aiming at the virus, so that the immunogenicity and the immune efficiency of the vaccine can be improved. The N-terminal domain (NTD) of SARS-CoV-2 spike protein (S protein) is a sequence N-terminal to the viral S protein that binds to a protein or glycoprotein of a host cell, mediating viral invasion of the host cell, and thus may comprise an epitope that induces the production of neutralizing antibodies. For the purpose of developing the present invention, in the examples of the present invention, the inventors employed a fusion protein comprising the Receptor Binding Domain (RBD) of SARS-CoV-2 spike protein (S protein) or a functionally active fragment thereof and/or the N-terminal domain (NTD) of SARS-CoV-2 spike protein (S protein) or a functionally active fragment thereof as an antigen. The fusion protein further comprises a foldon domain or functionally active fragment thereof.

In some preferred embodiments, the antigen is selected from at least one of human papilloma virus HPV types 6, 11, 18, 31, 33, 45, 52, 58, 68.

The capsid of HPV is composed of a major capsid protein L1 and a minor capsid protein L2. The existing vaccines are all vaccines based on HPV L1 Virus-like particles (VLP) as antigens, and the L1 protein expressed by gene recombination can form Virus-like particles under certain conditions, so that the vaccine has better immunogenicity. In the NCBI database, there are many existing sequences of HPV-type L1 VLP proteins (HPV 16L1, 18L1, 6L1, 11L1, 31L1, 33L1, 45L1, 52L1, 58L 1) available for selection by those skilled in the art, which can be used as the basis for the ideal selection of antigenic proteins. For the purpose of developing the present invention, in the examples of the present invention, the inventors mostly employed sequences having high conservation from the prior art, in particular, as follows: the amino acid sequence of HPV 6L1 was recorded in NCBI database in 1995 under accession No. AAA74218; the amino acid sequence of HPV 11L1 was recorded in NCBI database in 1994 under accession No. AAA46935; the amino acid sequence of HPV 16L1 was recorded in NCBI database, accession No. AAC09292.1 in 1998; the amino acid sequence of HPV 18L1 protein was recorded in NCBI database in 2003 under accession number AAQ92369.1; the amino acid sequence of HPV 31L1 protein has been recorded in NCBI database in 1994 under accession No. AAA46956; the amino acid sequence of HPV 33L1 protein has been recorded in the NCBI database in 2009 under accession No. ACL12333.1; the amino acid sequence of HPV 45L1 protein was recorded in the NCBI database in 2009 under accession number ABP99831.1 (N-terminal 26 amino acids were truncated, the 26 amino acids being hydrophobic regions that could affect the formation of the L1 protein into VLPs, thus truncated); the amino acid sequence of HPV 52L1 protein was recorded in NCBI database 2005 under accession number CAA52590.1 (truncated by 27 amino acids at the N-terminus, the 27 amino acids being hydrophobic regions that may affect the formation of VLP from the L1 protein, thus truncated); the amino acid sequence of HPV 58L1 protein was recorded in the NCBI database in 2009 under accession number CAX48979.1.

Such antigens are readily available using conventional techniques of modern molecular biology, typical methods include: a method for expressing the HPV types of L1 VLP proteins in pichia pastoris, comprising the steps of:

(1) Cloning the HPV each type L1 protein gene (subjected to codon optimization) into an expression vector;

(2) Transforming the expression vector obtained in the step (1) into pichia pastoris host bacteria;

(3) Bacterial strain screening is carried out to obtain bacterial strains which stably express the L1 proteins of each HPV type;

(4) And (3) using the strain obtained in the step (3) to express so as to obtain the L1 protein of each HPV type.

The method for stably expressing each type of L1 protein of HPV using the Pichia pastoris expression system is well known in the art, and specific reference is made to the "molecular cloning test guidelines" and other documents. Other expression patterns, such as E.coli, saccharomyces cerevisiae, hansenula, CHO cells, insect cells, etc., may also be selected by those skilled in the art to obtain L1 proteins of each HPV type.

In some embodiments, the antigen content is between 20-40 μg, and the mass ratio of the aluminum particles to the emulsion is 1:23-1:53.

In some preferred embodiments, the antigen content is 20 or 40 μg, the mass ratio of the aluminum particles to the emulsion is 1:52.66,

in some preferred embodiments, the antigen content is 20 or 40 μg and the mass ratio of the aluminum particles to the emulsion is 1:29.48.

In some preferred embodiments, the antigen content is 20 or 40 μg and the mass ratio of the aluminum particles to the emulsion is 1:23.62.

In another aspect, the present invention provides a method of preparing the above immunogenic composition comprising the steps of:

a) Preparing aluminum nano particles;

c) Preparing an oil phase comprising a metabolisable oil;

d) Mixing the composite aqueous phase of the step b) with the oil phase, and dispersing and homogenizing to obtain the adjuvant system;

e) And adding the antigen into the adjuvant system, and uniformly mixing to obtain the immunogenic composition.

In some embodiments, the aluminum adjuvant used in step a) is a commercially available micron-sized aluminum Adjuvant (ALHYDROGEL). In some embodiments, the aluminum adjuvant used in step a) is a nanoscale aluminum adjuvant.

In some embodiments, step a) is specifically to add 1mol/L AlCl after stirring benzalkonium bromide, n-octanol and cyclohexane in a mass ratio of 1:1:1 at high speed ₃ The solution was stirred thoroughly. Slowly dropwise adding ammonia water to maintain the pH of the reaction system>10 reacting for 2 hours, adding acetone after finishing, demulsifying and centrifuging, discarding supernatant, then repeatedly washing for 6 times by using distilled water and ethanol respectively, and drying the centrifuged precipitate to obtain the bulked nano aluminum hydroxide particles.

In some embodiments, step b) is specifically to add the nano aluminum hydroxide particles obtained in step a) into a buffer solution containing an emulsifier and mix them thoroughly as a complex aqueous phase solution.

In some embodiments, step c) is specifically to thoroughly mix squalene and alpha-tocopherol as the oil phase.

In some embodiments, step c) is specifically to use squalene alone as the oil phase.

In some embodiments, step c) is specifically to thoroughly mix squalene and span 85 as the oil phase.

In some embodiments, step d) is specifically to thoroughly mix the aqueous composite solution prepared in step b) with the oil phase prepared in step c), stirring at high speed, and homogenizing to reduce the particle size. After multiple cycles, the emulsion was finally filtered using a 0.22 μm PES filter to obtain the adjuvant system.

The invention provides an adjuvant system comprising an aluminum adjuvant and an emulsion, in particular to an adjuvant system comprising an oil-in-water emulsion in which aluminum hydroxide nano particles are uniformly dispersed, and a preparation method and application thereof.

Compared with the prior art, the invention has the following beneficial effects:

(1) The application range is wide, and nanometer aluminum can adsorb antigens of different pathogens, so that vaccines aiming at different pathogens are formed.

(2) The nano aluminum has high stability, larger specific surface area and stronger adsorption capacity, is uniformly dispersed in the oil-in-water emulsion, protects antigen from being damaged by inoculation environment substances, and enhances the in-vivo and in-vitro stability.

(3) The adjuvant system of the invention greatly reduces the dosage of aluminum adjuvant, and nano aluminum is dispersed in an oil-in-water emulsion system, so that adverse reaction at an inoculation part can be obviously reduced.

(4) The adjuvant system provided by the invention can effectively enhance the cellular immune response of the vaccine, and the enhancement effect is obviously better than that of a single emulsion or aluminum adjuvant. Particularly when applied to HPV antigens, the anti-HPV vaccine can still obtain the effect which is equivalent to or even better than that of AS04 adjuvant specially designed for HPV vaccine under the condition of reducing cost.

Drawings

The following drawings are only for purposes of illustration and explanation of the present invention and are not intended to limit the scope of the invention.

Wherein:

FIG. 1 shows the results of detection of neutralizing antibody titers against L1 VLP proteins of HPV types in serum using a pseudovirus neutralization assay 28 days after immunization of mice in example 11 of the invention;

FIG. 2 is a graph showing layering of the adjuvant system prepared in comparative example 2 of the present invention;

FIG. 3 is a graph showing layering of the adjuvant system according to comparative example 3 of the present invention;

FIG. 4 shows the results of the detection of neutralizing antibody titer in serum of mice immunized with the adjuvant system and HPV 45 antigen prepared by different methods in example 13 of the present invention.

Detailed Description

The invention will be further illustrated by the following non-limiting examples, which are well known to those skilled in the art, that many modifications can be made to the invention without departing from the spirit thereof, and such modifications also fall within the scope of the invention. The following examples are merely illustrative of the present invention and should not be construed as limiting the scope of the invention as embodiments are necessarily varied. The terminology used in the description is for the purpose of describing particular embodiments only and is not intended to be limiting, the scope of the present invention being defined in the appended claims.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Preferred methods and materials of the invention are described below, but any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the invention. The following experimental methods are all methods described in conventional methods or product specifications unless otherwise specified, and the experimental materials used are readily available from commercial companies unless otherwise specified.

Definition of terms

Reference throughout this application to "one embodiment" means that a particular parameter, step, etc. described in that embodiment is at least included in one embodiment according to the present invention. Thus, references to "one embodiment according to the present invention," "in an embodiment," and the like, are not intended to be interpreted as referring to the same embodiment, nor are references to features intended to be included in a particular embodiment, unless references to "in another embodiment," "in a different embodiment according to the present invention," and the like are used in this application. It will be appreciated by those of skill in the art that the specific parameters, steps, etc. disclosed in one or more of the embodiments of the invention can be combined in any suitable manner.

In the present application, the term "adjuvant" refers to a substance having an immune response enhancing function that is clinically applicable to the human body or has a prospect of application to the human body, and includes various adjuvants that are currently approved and may be approved in the future, such as, but not limited to, aluminum adjuvants, MF59, and various forms of adjuvant compositions.

In the present application, the terms "nano-aluminum", "aluminum nano-particles", "aluminum particles" or "aluminum adjuvants" refer to commercially available nano-or micro-sized aluminum Adjuvants (ALHYDROGEL) directly employed or nano-or micro-particles prepared from aluminum salts, which may be selected from aluminum chloride, aluminum phosphate, aluminum sulfate or aluminum hydroxide.

In the present application, the term "emulsion" generally refers to a heterogeneous liquid dispersion system formed by mixing oil phase components, water phase components and emulsifying agents in proper proportion and emulsifying, and the heterogeneous liquid dispersion system can be an oil-in-water emulsion or a water-in-oil emulsion. The aqueous phase component includes, but is not limited to, phosphate buffer, citrate buffer, tris buffer, acetate buffer, or citric acid-phosphate buffer. The oil phase component is a metabolizable lipid including, but not limited to, vegetable oil, fish oil, animal oil, synthetic oil, and other lipid components (e.g., but not limited to squalene, peanut oil, linseed oil, phospholipids, etc.); the emulsifier is a surfactant having a suitable HLB, such as, but not limited to, sorbitan trioleate (Span 85), polysorbate 80 (Tween 80), sorbitan fatty acid esters (Span 80/Span 80), triethylene glycol monolauryl ether (Bridgy 30/Brij 30), polyethylene glycol hexadecyl ether (Bridgy 56/Brij 56), polyethylene glycol octylphenyl ether (Triton X-100), and the like. In some embodiments of the invention, "emulsion" refers to an oil-in-water emulsion.

In this application, the term "metabolizable oil" generally refers to an oil substance that can be converted by metabolism of the body, which may be any vegetable, animal or synthetic oil that is non-toxic to the recipient immunized and has the ability to be converted by metabolism. Nuts, seeds, and grains are common sources of vegetable oils; synthetic oils are also part of the present invention, including various oils commercially available. One suitable metabolisable oil is squalene, an unsaturated oil present in major amounts in shark liver oil, an intermediate product of cholesterol biosynthesis, and at a lower level in olive oil, wheat germ oil, rice bran oil and yeast.

In a preferred embodiment, the oil-in-water emulsion comprises squalene, alpha tocopherol and polyoxyethylene sorbitan monooleate (Tween 80 or Tween 80). In a preferred embodiment, the oil-in-water emulsion comprises squalene and tween 80. In another preferred embodiment, the oil-in-water emulsion comprises squalene, tween80 and sorbitan trioleate (Span 85 or Span 85).

In some embodiments of the invention, the adjuvant system comprises an oil-in-water emulsion consisting of squalene, alpha tocopherol and tween80 components. For example, an oil-in-water emulsion comprises 2-10% squalene, 0.3-3% tween80 and 2-10% alpha-tocopherol (percentages refer to percentages by total volume of the immunogenic composition) and can be prepared according to the preparation methods described in WO 95/17210. The amount of squalene may be equal to or less than the amount of alpha-tocopherol to provide a more stable emulsion. The oil-in-water emulsion may also comprise span85 and/or lecithin, for example at a level of 1% by total volume of the immunogenic composition. In some embodiments of the present invention, a mass of squalene and alpha-tocopherol is first weighed and mixed as an oil phase, and then mixed with phosphate buffer containing an emulsifier and emulsified by stirring at a high speed of 10000rpm to obtain a colostrum. Finally, the primary emulsion is homogenized for 5-8 cycles by a pre-stage valve (namely a slit type homogenizing valve, the same applies below) under the pressure of 0.8bar and a micro-jet valve of 100-150 MPa, and the particle size is further reduced to 180+/-10 nm. In some preferred embodiments of the present application, the microfluidic valve circulates a 100MPa pressure homogeneous colostrum 5. In some embodiments of the present application, only a certain mass of squalene is used as the oil phase in the emulsion, and then the emulsion is mixed with phosphate buffer solution containing an emulsifier and stirred at a high speed of 10000rpm for emulsification to obtain colostrum. Finally, the particle size is further reduced to within 180nm, preferably about 105nm, by circulating the homogenized colostrum 8 under a pressure of 0.8bar at the backing valve and 140MPa at the microfluidic valve. An oil-in-water emulsion comprising squalene, alpha-tocopherol and tween80 or an oil-in-water emulsion comprising squalene and tween80 is obtained.

In some embodiments of the invention, an oil-in-water emulsion comprising squalene, tween 80 and span 85 is prepared. In these embodiments, tween 80 is first weighed and dissolved in citrate buffer to prepare a water phase, squalene and span 85 are then weighed and mixed uniformly to obtain an oil phase, the oil phase and the water phase are mixed uniformly and stirred at high speed at 10000rpm to obtain colostrum, and finally the colostrum is homogenized through a pre-stage valve of 0.8bar and a micro-jet valve of 140MPa pressure, homogenized through a pre-stage valve of 1.2bar and a micro-jet valve of 160MPa pressure, and further reduced in particle size to within 240nm, preferably to within 235nm, after 6 cycles, to obtain the oil-in-water emulsion comprising squalene, tween 80 and span 85.

In the present application, the term "antigen" refers to a variety of substances recognized by the immune system under appropriate circumstances, which may originate from pathogens, the human body itself, tumors, etc.

In the present application, the term "immunogenic composition" refers to a composition comprising an immunogenic component capable of stimulating an immune response in an individual, such as a human. Accordingly, in one embodiment of the invention, the immunogenic composition of the invention is a vaccine, i.e., the immunogenic composition may be administered to an individual to enhance an immune response against the corresponding virus, which is capable of preventing or treating the corresponding viral infection in the individual. The virus is selected from the group consisting of human immunodeficiency virus HIV-1, human papilloma virus, varicella zoster virus, human herpes simplex virus, respiratory syncytial virus, hepatitis B virus, hand-foot-and-mouth virus, coxsackie virus, human cytomegalovirus, influenza virus, coronavirus and novel coronavirus SARS-CoV-2. In some embodiments of the invention, preferably, the virus is human papilloma virus. Accordingly, the term "vaccine" as used herein refers to both therapeutic vaccines (for the treatment of disease) and prophylactic vaccines (for the prevention of disease).

In this application, the terms "comprises," "comprising," and "includes" are used in their plain, inclusive, and open-ended meaning. In some cases, the meaning of "as", "consisting of … …" is also indicated.

EXAMPLE 1 preparation of aluminium adjuvant

Taking benzalkonium bromide, n-octanol and cyclohexane with the mass ratio of 1:1:1, stirring at high speed, pouring into a triangular flask, placing a stirrer, placing the triangular flask on a magnetic stirrer, and adding 1mol/L AlCl ₃ 10ml of the solution was stirred for about 20 minutes. Slowly adding 10ml of ammonia water dropwise into a separating funnel to adjust the dropping speed, keeping 30 drops per minute, and keeping the pH value of the reaction system>10 reacting for 2 hours, adding acetone after the reaction is finished, demulsifying and centrifuging (R=500 rpm), discarding supernatant, then repeatedly washing with distilled water and ethanol for 6 times respectively, collecting the centrifuged precipitate in a beaker, and drying in a drying oven at 70 ℃ for 12 hours to obtain the bulk nano aluminum hydroxide adjuvant. The aluminum hydroxide prepared in this example was examined to have a particle diameter of 139.9nm,PDI 0.106.

Example 2 preparation of adjuvant System BFA-EAL-1

The aluminum adjuvant of this example was a commercially available micron-sized aluminum adjuvant (ALHYDROGEL, CAS:21645-51-2, manufacturer: CRODA, product number: AJV3012, lot number: 0001678865), and the aluminum hydroxide particle diameter was 1051nm and PDI was 0.095.

Adjuvant ingredients: aluminum content 2mg/ml, squalene 42.76mg/ml, alpha-tocopherol 47.44mg/ml, tween 80 content 15.12mg/ml.

Preparing an aqueous phase: 12ml of aluminum adjuvant (10 mg/ml), 42ml of phosphate buffer solution with Tween 80 content of 21.6mg/ml were added and mixed uniformly to obtain a composite aqueous phase.

Preparing an oil phase: 2.58g squalene and 2.94g alpha-tocopherol are weighed and mixed evenly to form an oil phase.

Preparing colostrum: mixing the water phase and the oil phase, and dispersing at 10000rpm in water bath at 20deg.C for 15min to obtain colostrum.

Homogenizing to prepare emulsion: the backing valve 0.8bar and the micro-jet valve 100MPa pressure homogenized colostrum 5 is circulated to obtain an adjuvant system containing aluminum adjuvant and oil-in-water emulsion.

The particle size of aluminum particles in the adjuvant system obtained after a series of dispersion and homogenization is obviously reduced, and the composite emulsion has the particle size of 188.9nm and the PDI of 0.205 through detection. BFA-EAL-1 is identified.

Example 3 preparation of adjuvant System BFA-EAL-2

Adjuvant ingredients: aluminum content 2mg/ml, squalene 42.76mg/ml, tween 80 content 16.2mg/ml.

Preparing an aqueous phase: 12ml of aluminum adjuvant (10 mg/ml), 45ml of phosphate buffer solution with tween 80 content of 21.6mg/ml was added and mixed evenly to form a composite water phase.

Preparing an oil phase: 2.57g squalene was weighed out as oil phase.

Preparing colostrum: mixing the water phase and the oil phase, and dispersing at 10000rpm in water bath at 40deg.C for 15min to obtain colostrum.

Homogenizing to prepare emulsion: the backing valve 0.8bar and the micro-jet valve 140MPa pressure homogenized colostrum 8 are circulated to obtain an adjuvant system containing aluminum adjuvant and oil-in-water emulsion.

The particle size of aluminum particles in the adjuvant system obtained after a series of dispersion and homogenization is obviously reduced, and the composite emulsion has the particle size of 105.5nm and the PDI of 0.193 according to detection. BFA-EAL-2 is identified.

Example 4 preparation of adjuvant System BFA-EAL-3

Adjuvant ingredients: aluminum content 2mg/ml, squalene 39mg/ml, span 854.7mg/ml, tween 80 content 3.5mg/ml.

Preparing an aqueous phase: 12ml of aluminium adjuvant (10 mg/ml) and 45ml of citrate buffer solution with 4.7mg/ml Tween 80 content are added and mixed evenly to form a composite water phase.

Preparing an oil phase: 2.35g squalene and 0.28g span 85 are weighed and mixed evenly to form an oil phase.

Homogenizing to prepare emulsion: the backing valve 0.8bar and the micro-jet valve 140MPa pressure homogeneous colostrum 8 are circulated. Homogenizing and circulating at a pressure of 1.2bar in a backing valve and 160MPa in a microfluidic valve for 6 cycles to obtain an adjuvant system containing the aluminum adjuvant and the oil-in-water emulsion.

The particle size of aluminum particles in the adjuvant system obtained after a series of dispersion and homogenization is obviously reduced, and the particle size of the composite emulsion is 234.7nm and the PDI is 0.436 through detection. BFA-EAL-3 is identified.

Example 5 preparation of adjuvant System BFA-ENAL-1

This example used the nano aluminum adjuvant prepared in example 1.

Adjuvant ingredients: aluminum content 2mg/ml, squalene 42.76mg/ml, alpha-tocopherol 47.44mg/ml, tween 80 content 19.44mg/ml.

Preparing an aqueous phase: 36ml of aluminum adjuvant (4.48 mg/ml) and 36ml of phosphate buffer solution with Tween 80 content of 43.2mg/ml are added and uniformly mixed to obtain a composite water phase.

Preparing an oil phase: 3.43g squalene and 3.83g alpha-tocopherol are weighed and mixed evenly to form an oil phase.

Preparing colostrum: mixing the water phase and the oil phase, and dispersing at 10000rpm in water bath at 20deg.C for 20min to obtain colostrum.

Homogenizing to prepare emulsion: the backing valve 0.8bar and the micro-jet valve 120MPa pressure homogeneous colostrum 6 are circulated. Homogenizing and circulating at a pressure of 0.8bar in a backing valve and 140MPa in a microfluidic valve for 6 cycles to obtain an adjuvant system containing the aluminum adjuvant and the oil-in-water emulsion.

The particle size of aluminum particles in the adjuvant system obtained after a series of dispersion and homogenization is obviously reduced, and the particle size of the composite emulsion is 166.7nm and the PDI is 0.239. BFA-ENAL-1 is identified.

Example 6 preparation of adjuvant System BFA-ENAL-2

This example used the nano aluminum adjuvant prepared in example 1.

Adjuvant ingredients: aluminum content 0.5mg/ml, squalene 42.76mg/ml, alpha-tocopherol 47.44mg/ml, tween 80 content 19.44mg/ml.

Preparing an aqueous phase: 9ml of aluminum adjuvant (4.48 mg/ml), 36ml of phosphate buffer solution with Tween 80 content of 43.2mg/ml, and 27ml of water for injection are added and uniformly mixed to obtain a composite water phase.

Preparing an oil phase: 3.42g squalene and 3.81g alpha-tocopherol were weighed and mixed to be an oil phase.

Homogenizing to prepare emulsion: the backing valve 0.8bar and the micro-jet valve 140MPa pressure homogenized colostrum 6 are circulated to obtain an adjuvant system containing aluminum adjuvant and oil-in-water emulsion.

The particle size of aluminum particles in the adjuvant system obtained after a series of dispersion and homogenization is obviously reduced, and the particle size of the composite emulsion is 153.6nm and the PDI is 0.099. BFA-ENAL-2 is identified.

Example 7 preparation of the adjuvant System BFA-ENAL-1-GpG

This example used the nano aluminum adjuvant prepared in example 1.

Adjuvant ingredients: aluminum content 0.5mg/ml, squalene 42.76mg/ml, alpha-tocopherol 47.44mg/ml, tween 80 content 19.44mg/ml, gpG (0.2 mg/ml).

Preparing an aqueous phase: 9ml of aluminum adjuvant (4.48 mg/ml), 36ml of phosphate buffer solution with Tween 80 content of 43.2mg/ml and 27ml of water for injection are added into 16mg of GpG, and the mixture is uniformly mixed into a composite water phase.

The emulsion particle size was determined to be 159.1nm and the PDI was determined to be 0.356. The BFA-ENAL-1-CpG is identified.

Example 8 preparation of the adjuvant System BFA-ENAL-1-MPL

This example used the nano aluminum adjuvant prepared in example 1.

Adjuvant ingredients: aluminum content 2mg/ml, squalene 42.76mg/ml, alpha-tocopherol 47.44mg/ml, MPL 0.2mg/ml.

Preparing an oil phase: 3.43g squalene, 3.83g alpha-tocopherol and 16mg MPL were weighed and mixed to be an oil phase.

The emulsion particle size was measured to be 161.2nm and the PDI was measured to be 0.252. The identification BFA-ENAL-1-MPL.

EXAMPLE 9 preparation of immunogenic compositions containing HPV antigens

In order to study the technical effect of the adjuvant system provided by the invention. The inventors of the present invention made the following immunogenic compositions (0.5 ml/dose) containing HPV type 6/11/16/18L 1 VLP protein, aluminum adjuvant, BFA04 adjuvant or adjuvant system described in examples 2-4. The specific preparation method comprises the following steps: HPV type 6/11/16/18L 1 VLP antigen was thoroughly mixed with aluminium adjuvant, BFA04 adjuvant or the adjuvant system described in examples 2-4, respectively, in amounts of 20. Mu.g, 40. Mu.g.

The formulation of the BFA-EAL-1 adjuvant system in this example is: 500 μg of aluminum hydroxide, 10.69mg of squalene, 11.86mg of alpha-tocopherol, 3.78mg of Tween 80,3.53mg of sodium chloride, 0.09mg of potassium chloride, 0.51mg of disodium hydrogen phosphate and 0.09mg of potassium dihydrogen phosphate, and the preparation was stored in a 0.25ml adjuvant bottle.

The formulation of the BFA-EAL-2 adjuvant system in this example is: 500 μg of aluminum hydroxide, 10.69mg of squalene, 4.05mg of Tween 80,3.53mg of sodium chloride, 0.09mg of potassium chloride, 0.51mg of disodium hydrogen phosphate and 0.09mg of potassium dihydrogen phosphate, and the preparation is contained and stored in a 0.25ml adjuvant bottle.

The formulation of the BFA-EAL-3 adjuvant system in this example is: 500 mug of aluminum hydroxide, 9.75mg of squalene, 85.18 mg of span, 0.88mg of tween 80, 0.66mg of trisodium citrate dihydrate, 0.04mg of citric acid monohydrate, and the preparation is contained and stored in a 0.25ml adjuvant bottle.

In this example, the formulation of BFA04 adjuvant: 50. Mu.g MPL, 500. Mu.g aluminum hydroxide, 150mM sodium chloride, 8mM sodium dihydrogen phosphate dihydrate, and the preparation was stored in 0.25ml adjuvant bottles.

Al (OH) in this example ₃ A commercially available micron-sized aluminum Adjuvant (ALHYDROGEL) was used at a concentration of 2mg/ml Al (OH) ₃ 。

EXAMPLE 10 preparation of immunogenic compositions containing HPV antigens

In order to study the technical effect of the adjuvant system provided by the invention. The inventors of the present invention made the following immunogenic compositions (0.5 ml/dose) containing HPV type 6/11/16/18L 1 VLP protein, aluminium adjuvant, BFA04 adjuvant or adjuvant system as described in examples 5-7. The specific preparation method comprises the following steps: HPV type 6/11/16/18L 1 VLP antigen was thoroughly mixed with aluminium adjuvant, BFA04 adjuvant or adjuvant systems as described in examples 5-7, respectively, in amounts of 20. Mu.g, 40. Mu.g.

The formulation of the BFA-ENAL-1 adjuvant system in this example is: 500 μg of aluminum hydroxide, 10.69mg of squalene, 11.86mg of alpha-tocopherol, 4.86mg of tween 80, 3.53mg of sodium chloride, 0.09mg of potassium chloride, 0.51mg of disodium hydrogen phosphate and 0.09mg of potassium dihydrogen phosphate, and the preparation is contained and stored in a 0.25ml adjuvant bottle.

The formulation of the BFA-ENAL-2 adjuvant system in this example is: 125 μg of aluminum hydroxide, 10.69mg of squalene, 11.86mg of alpha-tocopherol, 4.86mg of tween 80, 3.53mg of sodium chloride, 0.09mg of potassium chloride, 0.51mg of disodium hydrogen phosphate and 0.09mg of potassium dihydrogen phosphate, and the preparation is contained and stored in a 0.25ml adjuvant bottle.

The formulation of the BFA-ENAL-1-CpG adjuvant system in this example is: 125 mug of aluminum hydroxide, 10.69mg of squalene, 11.86mg of alpha-tocopherol, 80 4.86mg,CpG 500 mug of tween, 3.53mg of sodium chloride, 0.09mg of potassium chloride, 0.51mg of disodium hydrogen phosphate and 0.09mg of monopotassium phosphate, and the preparation is contained and stored in a 0.25ml adjuvant bottle.

In this example, the formulation of BFA03 adjuvant: squalene 10.69mg, alpha-tocopherol 11.86mg, tween 80 4.86mg, sodium chloride 3.53mg, potassium chloride 0.09mg, disodium hydrogen phosphate 0.51mg and potassium dihydrogen phosphate 0.09mg, and the preparation was stored in a 0.25ml adjuvant bottle.

In this example, the formulation of BFA04 adjuvant: preparation was completed by holding 50. Mu.g of MPLA, 500. Mu.g of aluminum hydroxide, 150mM sodium chloride, 8mM sodium dihydrogen phosphate dihydrate in 0.25ml adjuvant bottles.

Al (OH) in this example ₃ Using the nanoscale aluminum adjuvant prepared in example 1, al (OH) was present at a concentration of 2mg/ml ₃ 。

EXAMPLE 11 evaluation of adjuvant Effect of adjuvant System

For the recombinant human papillomavirus vaccine composition obtained in example 9, the inventors conducted an immunogenicity study using BALB/c mice as animal models, and examined the immunogenicity of the adjuvant system of the present application in combination with HPV tetravalent antigen. The immunogenicity of the vaccine composition provided by the invention was studied with HPV6/11/16/18 type L1 VLP protein as antigen and the adjuvant system of examples 2-4 as adjuvant, thereby evaluating the effect of the adjuvant system of the invention. The BALB/c mice were randomly grouped at 6-8 weeks, 5 mice per group, and the vaccine prepared by intramuscular injection of HPV 6/11/16/18L 1 VLP protein in combination with the adjuvant system was injected at a volume of 0.05ml (i.e., 1/10 HD), and a self-made vaccine group, a commercial aluminum adjuvant control group, and a BFA04 adjuvant control group were set.

Blood was collected 14 days after the second immunization using an immunization program at intervals of 3 weeks to isolate serum, and the neutralizing antibody titer against each type of L1 VLP protein of HPV in the serum was detected using pseudovirus neutralization assay (pseudovirus-Based Neutralization Assay, PBNA) and the geometric mean antibody titer (GMT) was calculated and the grouping and immunization protocol of mice was as shown in Table 1.

Table 1 grouping table of mice

BFA-EAL-1 (also called BFA-EAL-1-uf in the figure), BFA-EAL-2 (also called BFA-EAL-2-uf in the figure), BFA-EAL-3 (also called BFA-EAL-3-uf in the figure), BFA04 and Al (OH) ₃ The adjuvant formulation is described in reference to example 9.

The immunogenicity evaluation method is a line-standard technical means in the field, and by way of example, the pseudovirus neutralization experimental method is more specifically operated as follows:

(1) Taking 293FT cells with good growth state, digesting the 293FT cells into single cells by 0.25% Trypsin-EDTA, and diluting the cells to a proper concentration by using a DMEM complete medium; counting by a cell counter;

(2) Based on the cell count, 293FT cells were diluted to 1.5X10 s in DMEM complete medium ⁵ Per ml, pre-plated in 96 well cell culture plates with 100. Mu.L of cell fluid per well, 5% CO at 37 ℃ ₂ Culturing in an incubator until the cells adhere to the wall;

(3) Diluting the inactivated serum sample to a proper initial dilution by using a DMEM complete culture medium, and then carrying out continuous double dilution for a plurality of gradients, wherein each dilution is provided with double wells;

(4) Pseudovirus liquid was diluted to log (TCID) with DMEM complete medium ₅₀ 0.1 ml) =3.2±0.5, and mixing;

(5) Taking serum diluent, adding the same volume of pseudovirus diluent, uniformly mixing, incubating for 60 minutes at room temperature, and simultaneously setting positive control holes, wherein each hole is the whole DMEM culture medium, and adding the same volume of pseudovirus diluent; setting blank control holes, wherein each hole is a DMEM complete culture medium;

(6) After the incubation is finished, accurately sucking 100 mu L of serum-pseudovirus mixed solution, and slowly and carefully adding the solution into a pre-paved 96-well cell plate;

(7) At 5% CO ₂ Culturing at 37deg.C in incubator for 72 hr, observing with fluorescence microscope or reading with spot analyzer,and analyzed and processed.

TABLE 2 detection results 35 days after immunization of mice

The results of 35 days after immunization of mice with HPV-associated antigens using the adjuvant systems prepared in examples 2-4 of the present application are shown in Table 2 and FIG. 1. The results show that, firstly, the three adjuvant systems prepared by using the commercial micron-sized aluminum adjuvants in examples 2-4 of the application can induce mice to generate neutralizing antibody titer which is obviously higher than that generated by using the micron-sized aluminum adjuvants alone in combination with HPV antigens, and all the three adjuvant systems have good immune activity.

Second, the BFA-EAL-3 adjuvant system is superior to the BFA-EAL-2 adjuvant system in comparison, demonstrating that span 85, which is used as an oil phase component in emulsions, makes a certain contribution.

More importantly, the BFA-EAL-1 adjuvant system showed optimal immunopotentiating effect, no matter whether HPV6, 11, 16 or 18 type, resulting in a neutralizing antibody titer against HPV6/11/16/18 of 7-9 times that of aluminum adjuvant alone, and at comparable levels to BFA04 adjuvant, indicating that the BFA-EAL-1 adjuvant system is sufficient to replace the use of BFA04 adjuvant in HPV vaccines.

EXAMPLE 12 evaluation of adjuvant Effect of adjuvant System

The inventors conducted an immunogenicity study with respect to the recombinant human papillomavirus vaccine composition obtained in example 10, using BALB/c mice as animal models, and examined the immunogenicity of the adjuvant system in combination with HPV tetravalent antigen.

The immunogenicity of the vaccine composition provided by the invention was studied with HPV6/11/16/18 type L1 VLP protein as antigen and the adjuvant system of examples 5-7 as adjuvant, thereby evaluating the effect of the adjuvant system of the invention. The BALB/c mice of 6-8 weeks were randomly grouped, 5 mice per group, and the vaccine prepared by the HPV 6/11/16/18L 1 VLP protein combined adjuvant system was intramuscular injected in a volume of 0.05ml (i.e., 1/10 HD), and a self-made vaccine group, a self-made aluminum adjuvant control group, a BFA03 adjuvant control group, and a BFA04 adjuvant control group were set.

Blood was collected 14 days after the second immunization using an immunization program at intervals of 3 weeks to isolate serum, and the neutralizing antibody titer against each type of L1 VLP protein of HPV in the serum was detected using pseudovirus neutralization assay (pseudovirus-Based Neutralization Assay, PBNA) and the geometric mean antibody titer (GMT) was calculated and the grouping and immunization protocol of mice was as shown in Table 3.

TABLE 3 grouping of mice table

BFA-ENAL-1, BFA-ENAL-2, BFA-ENAL-1-CpG, BFA03, BFA04 and Al (OH) ₃ The adjuvant formulation is described in reference to example 10.

The results of 35 days after immunization of mice with HPV-associated antigens using the adjuvant systems prepared in examples 5-7 of the present application are shown in Table 4. The results show that the titer of the neutralizing antibodies generated by the combined immunization of mice by the three adjuvant systems prepared by the self-made nanoscale aluminum adjuvant is obviously higher than that generated by the independent use of the nanoscale aluminum adjuvant, and the neutralizing antibodies have good immune activity.

The inventors have surprisingly found that the BFA-ENAL-2 adjuvant system, whether for HPV type 6, 11, 16 or 18 group vaccines, achieves a comparable or even slightly superior level to the BFA04 adjuvant, i.e. achieves an adjuvant effect comparable to the BFA-EAL-1 adjuvant system with the best results in the immunoassay of example 11, whereas the aluminum content in the BFA-ENAL-2 adjuvant system is only 1/4 of the BFA-EAL-1 adjuvant system, indicating that the adjuvant system formulated with the nano-sized aluminum adjuvant made by the invention works better than the micro-sized aluminum adjuvant. On the premise of ensuring good adjuvant effect, the dosage of the aluminum adjuvant can be greatly saved.

In particular, the BFA-ENAL-1-CpG adjuvant system of the present invention exhibits a more excellent immunopotentiating effect than the BFA-ENAL-2 adjuvant system. Of particular interest, the neutralizing antibody titres produced in combination with HPV6 antigen are 2.3 times that of BFA04 adjuvant. The low aluminum content adjuvant system added with a small amount of immunostimulant such as CpG can replace BFA04 adjuvant, which lays an important foundation for the development of novel vaccine adjuvant.

The inventors have surprisingly found that the aluminium content-enhanced BFA-ENAL-1 adjuvant system in combination with HPV antigens produces a generally higher neutralizing antibody titer than BFA03 adjuvant, and at the same time, shows a more excellent immune enhancing effect than BFA-ENAL-2 adjuvant system, and the neutralizing antibody titer produced by immunization is 1-2 times as high as that of BFA04 adjuvant. In addition, the preparation cost of the self-made nanoscale aluminum adjuvant is far lower than that of the CpG adjuvant, so that the BFA-ENAL-1 adjuvant system is a vaccine candidate adjuvant with higher potential, and the BFA-ENAL-1 adjuvant system can be particularly applied to research and development of new-generation HPV vaccines.

TABLE 4 detection results 35 days after immunization of mice

Three HPV vaccines currently on the market, only Cervarix of gladin smith uses a complex adjuvant (aluminium hydroxide and MPL), showing higher immunogenicity, but MPL is a chemically treated "attenuated" salmonella lipopolysaccharide, which is complex in process and costly, and the productivity is easily limited by the source of MPL. In addition, the product is immature in domestic large-scale production condition, and can not meet the subsequent clinical use requirements. Whereas the moesadong tetravalent HPV vaccine And nine-valent HPV vaccine Gardasil->All using a single aluminium phosphate adjuvant, the antibody titer produced is much lower than +.>

In contrast, the adjuvant system of the invention has strong immunity induction effect, the enhancement effect is obviously better than that of a single aluminum adjuvant, the adjuvant system of the invention is a composite adjuvant system, the particle size of aluminum particles in the system is further reduced to the nanometer level through the preparation of the inventor, the aluminum particles are uniformly dispersed at the water-oil interface of emulsion, the nanometer aluminum adjuvant particles are smaller, the specific surface area is increased, the adsorption capacity and the adjuvant activity are stronger, the immune response of organisms can be further improved, the side effect of the adjuvant is greatly reduced, and the adjuvant system can be used as a new-generation vaccine composition adjuvant. Especially when applied to HPV antigen, can obtain the peptide with the gram of the gramThe composite adjuvant AS04 (aluminum hydroxide and MPL) has quite even better effect, and the cost is far lower than that of the AS04 adjuvant because the raw materials are easy to obtain.

In further researches, the inventor finds that different preparation methods have great influence on the stability of an adjuvant system and the immunity enhancing effect. In the following studies, the inventors prepared adjuvant systems in different ways and performed immunization experiments in combination with HPV 45 antigen.

Comparative example 1

4.29g squalene, 4.74g alpha-tocopherol, 1.94g tween 80 were weighed out and mixed as oil phase. 10ml of aluminum hydroxide adjuvant was added to 80ml of phosphate buffer to give a white crushed flocculent, milky liquid. Dispersing at 20 ℃ at 8000rpm for 10min, homogenizing by a 1bar pre-stage valve and a 120MPa micro-jet valve, and circulating for 8 times to obtain the oily emulsion. Mu.l of HPV 45 antigen was mixed with 873. Mu.l of phosphate buffer as an aqueous phase, and the oily emulsion and the aqueous phase were mixed in equal volumes to obtain a vaccine composition. Since the adjuvant system forms an oily emulsion, it is mixed with an aqueous phase containing the antigen and allowed to stand for a long period of time to find delamination.

Comparative example 2

1.28g squalene, 1.43g alpha-tocopherol, 0.58g tween 80 were weighed out and mixed as oil phase. 360. Mu.l of aluminum hydroxide adjuvant, 108. Mu.l of HPV 45 antigen were added to 132. Mu.l of phosphate buffer and mixed as an aqueous phase. Mu.l of the aqueous phase was added to about 2.5ml of the oil phase and phacoemulsified (300W, 4min,5s/5 s) to give a water-in-oil emulsion, which was finally diluted with the same volume of phosphate buffer. Similarly to comparative example 1, the vaccine composition of comparative example 2 was a water-in-oil emulsion, and white flocculence, precipitation and delamination were found after dilution with a phosphate buffer solution and standing, as shown in fig. 2.

Comparative example 3

1.29g squalene and 1.43g alpha-tocopherol were weighed out and mixed as an oil phase. 400. Mu.l of aluminum hydroxide nanoparticles were added to 3080. Mu.l of phosphate buffer, 120. Mu.l of HPV 45 antigen stock solution was added and mixed to aqueous phase. Mixing 300 μl of oil phase with 2.7ml of water phase, performing ultrasonic emulsification (300W, 4min,5s/5 s) to obtain Pickering emulsion, mixing Pickering emulsion with phosphate buffer solution, obtaining vaccine composition, and centrifuging to obtain sample which is obviously layered. The Pickering emulsion prepared in this comparative example is an emulsion system with solid particles instead of surfactant. The emulsion stabilizing mechanism is mainly that solid particles are adsorbed on an oil-water interface to form a solid particle single-layer or multi-layer structure, so that the emulsion is stabilized. However, this technique requires high conditions for the preparation process and the emulsion stability is poor, and the inventors found that there is a significant delamination phenomenon after long-term standing or centrifugation, as shown in fig. 3.

In addition to the methods described in the above comparative examples, the inventors prepared an immune composition by sequentially mixing three components of an aluminum adjuvant, an oil-in-water emulsion, and an antigen in another study. It was found that droplets formed by the oil phase and the surfactant and the antigen were adsorbed onto the aluminium adjuvant sequentially. The adsorption between the liquid drops formed by the oil phase and the surfactant and the aluminum adjuvant can compete with the adsorption between the antigen and the aluminum adjuvant, the aluminum adjuvant is difficult to effectively adsorb and protect the antigen, on the other hand, the aluminum adjuvant can destroy the balance of the water-oil interface of the emulsion, thereby destroying the form of the emulsion, and the advantages of the emulsion per se can not be exerted after the emulsion is mixed with the antigen.

EXAMPLE 13 evaluation of the adjuvant Effect of adjuvant systems prepared by different methods

Reference to the procedure of example 12, the preparation of example 5 and comparative examples 1-3, respectively, was usedAdjuvant System tuberculosis HPV 45 antigen for immunization experiments, 10 mice per group were injected with vaccine prepared by HPV 45 type L1 VLP protein in combination with adjuvant System, HPV 45 protein dose 5 μg, injection volume 0.05ml (i.e. 1/10 HD), and Al (OH) was set up ₃ Control group.

The results of the tests for each mouse in each group are shown in Table 5 and FIG. 4. It can be found that the immune compositions prepared in example 5 and comparative example 1 each induced a significantly higher titer of neutralizing antibodies than Al (OH) ₃ Control group, but there was no significant difference between the two groups. The immune compositions prepared in comparative examples 2 and 3 each induced a significantly lower neutralizing antibody titer than Al (OH) ₃ In the control group, this may be due to the poor stability of the immune composition in the two comparative examples on the one hand, and the fact that the antigen and the aluminum adjuvant are emulsified as an aqueous phase together with an oil phase, the emulsion formation process may have a great destructive effect on the antigen, and may affect the immune effect of the antigen.

In a comprehensive view, the aluminum adjuvant is added into the water phase and then emulsified with the oil phase to prepare the adjuvant system, so that the stability of the adjuvant system can be improved on one hand, and the immune effect of the antigen can be obviously enhanced on the other hand.

TABLE 5

* And (3) injection: when the antibody titer is lower than the detection lower limit 80, it is recorded as half of the lower limit, namely 40.

The foregoing description of the embodiments has been provided for the purpose of illustrating the general principles of the invention, and is not meant to limit the invention thereto, but to limit the invention thereto, and any modifications, equivalents, improvements and equivalents thereof may be made without departing from the spirit and principles of the invention.

Claims

1. An adjuvant system comprising an oil-in-water emulsion and aluminum nanoparticles homogeneously dispersed in the oil-in-water emulsion, wherein the oil-in-water emulsion encapsulates the aluminum nanoparticles to form emulsion droplets, and the emulsion droplets have a particle size of 100-250 nm.

2. An adjuvant system according to claim 1, wherein the aluminium nanoparticles are aluminium nanoparticles selected from aluminium phosphate, aluminium sulphate, aluminium hydroxide or a mixture of at least two thereof; preferably, the aluminium content in the adjuvant system is between 0.5mg/ml and 2mg/ml.

3. An adjuvant system according to claim 1, wherein said oil-in-water emulsion comprises an oil phase and an aqueous phase; preferably, the ratio of the aqueous phase to the oil phase is 5-15ml of aqueous phase to 1g of oil phase.

4. An adjuvant system according to claim 3, wherein the oil phase comprises a metabolisable oil, preferably squalene.

5. An adjuvant system according to claim 4 wherein said oil phase further comprises alpha-tocopherol or span 85; preferably, the weight ratio of squalene to alpha-tocopherol is from 0.8 to 1, for example from 0.85 to 0.95, preferably 0.9; the weight ratio of squalene to span 85 is 8-10, preferably 8.3.

6. An adjuvant system according to claim 4 wherein said oil phase further comprises an additional immunostimulant; preferably, the immunostimulant is a TLR4 agonist selected from any one of LPS, MPL, 3D-MPL or GLA.

7. An adjuvant system according to claim 3, wherein said aqueous phase comprises tween 80; the aluminum nanoparticles are dissolved in the aqueous phase; preferably, the weight ratio of the aluminum nanoparticles to tween 80 is 0.1-0.6, preferably 0.13.

8. An adjuvant system according to claim 7, wherein the aqueous phase further comprises an additional immunostimulant; preferably, the immunostimulant is selected from any one or more of CpG, polyIC, R837 and R848.

9. A method of preparing an adjuvant system according to any one of claims 1 to 8, comprising the steps of:

a) Preparing aluminum nano particles;

c) Preparing an oil phase comprising a metabolisable oil;

10. The method of claim 9, wherein the aluminum nanoparticle is an aluminum nanoparticle selected from aluminum phosphate, aluminum sulfate, aluminum hydroxide, or a mixture of at least two thereof, and the emulsifier is tween 80; preferably, the weight ratio of the aluminum nanoparticles to tween 80 is 0.1-0.6, preferably 0.13.

11. The method of claim 9, wherein the ratio of aqueous phase to oil phase is from 5 to 15ml of aqueous phase to 1g of oil phase.

12. The method of claim 9, wherein the metabolizable oil is squalene; preferably, the oil phase further comprises alpha-tocopherol or span 85.

13. A method according to claim 12, characterized in that the weight ratio of squalene to α -tocopherol is 0.8-1, such as 0.85-0.95, preferably 0.9; the weight ratio of squalene to span 85 is 8-10, preferably 8.3.

14. The method of claim 9, wherein the aqueous solution is a phosphate buffer solution or a citrate buffer solution.

15. The method of claim 9, wherein the dispersing step comprises stirring at 8000-10000rpm for 10-25 minutes.

16. The method of claim 9, wherein the homogenizing step comprises sequentially homogenizing at a pressure of from 0.5 to 1.2bar slit-type homogenizing valve and from 80 to 160MPa microfluidic valve; preferably, the homogenizing step is repeated for 5-20 cycles.

17. The method of claim 9, wherein the homogenizing step comprises sequentially performing a first homogenization at a pressure of from 0.5 to 1bar across a slit-type homogenizing valve and from 80 to 140MPa across a microfluidic valve, followed by a second homogenization at a pressure of from 1 to 1.2bar across a slit-type homogenizing valve and from 120 to 160MPa across a microfluidic valve, the second homogenization being at a pressure greater than the pressure of the first homogenization; preferably, the first homogenisation is carried out for 5-10 cycles and the second homogenisation is carried out for 5-10 cycles.

18. An immunogenic composition comprising the adjuvant system of any one of claims 1-8 and at least one antigen adsorbed on the aluminium particles and homogeneously dispersed in the emulsion.

19. The immunogenic composition of claim 18, wherein the antigen is one or more antigens derived from bacteria, viruses, parasites, fungi, tumors, human autoantigens, and/or allergens; preferably, the antigen is derived from at least one of human immunodeficiency virus, human papilloma virus HPV, varicella zoster virus, human herpes simplex virus, respiratory syncytial virus, hepatitis b virus, hand-foot-and-mouth virus, coxsackie virus, human cytomegalovirus, influenza virus, coronavirus and novel coronavirus SARS-CoV-2; more preferably, the antigen is selected from at least one of human papilloma virus HPV types 6, 11, 16, 18, 31, 33, 45, 52, 58.

20. A method of preparing the immunogenic composition of any one of claims 18-19, comprising the steps of:

a) Preparing an adjuvant system according to the method of any one of claims 9-17;

b) And adding the antigen into the adjuvant system, and uniformly mixing to obtain the immunogenic composition.