KR102168661B1 - Virus-like particles comprising the inner membrane complex of plasmodium berghei, and vaccine compositions using thereof - Google Patents

Abstract

The present invention relates to a virus-like particle of malaria, a vaccine composition including the same, an expression vector, a host cell, and a method for producing the same. The present invention provides an immune and preventive effect against malaria.

Description

Virus-like particles containing the inner membrane complex of protozoal malaria and a vaccine composition using the same {VIRUS-LIKE PARTICLES COMPRISING THE INNER MEMBRANE COMPLEX OF PLASMODIUM BERGHEI, AND VACCINE COMPOSITIONS USING THEREOF}

The present invention relates to a virus-like particle of malaria, a vaccine composition comprising the same, an expression vector, a host cell, and a method for producing the same.

Malaria is a parasitic in human blood, causing malnutrition, and is a single disease that causes the most deaths (2.4 million per year) worldwide.

About 300 to 500 million people each year are infected with malaria with a relatively high morbidity and mortality. In particular, not only children but also adults who have not been exposed to malaria before, the prevalence and mortality of malaria infection is at a serious level.

The World Health Organization (WHO) estimates that two to three million children die of malaria each year in Africa alone.

In addition, the widespread incidence and increase in prevalence of malaria caused by drug-resistant parasites ( Plasmodium falciparum, Plasmodium vivax ) and pesticide-resistant mediators (Anopheles mosquitoes) imposes the need to develop new control methods for malaria in many countries. Emphasis.

In Korea, malaria has reappeared in the northern part of Gyeonggi Province and in the northwest of Gangwon, near the armistice line, and the area at risk of infection is gradually expanding. Although more than 1,000 patients occur every year in Korea, the current malaria treatments such as chloroquine and artemether induce resistance, so vaccination is evaluated as the most effective method in preventing malaria protozoa.

On the other hand, virus-like particles mean a kind of antigen that is morphologically similar to the actual virus structure, and has been proposed as a vaccine antigen for several viruses (Roldao A, Mellado MC, Castilho LR, Carrondo MJ, Alves PM: Expert review. of vaccines 2010, 9:1149-1176;Kang SM, Kim MC, Compans RW: Expert review of vaccines 2012, 11:995-1007;Kushnir N, Streatfield SJ, Yusibov V: Vaccine 2012, 31:58-83).

Virus-like particles are assembled in a form similar to a real virus through the combination of viral structural proteins, but they are very safe because there is no risk of infection because the virus gene is not included in the assembly process.

Conventionally, studies have been conducted focusing on two antigens (Pfs25 and Pvs25) provided as recombinant proteins or attenuated vaccinia virus in relation to malaria vaccines, but studies of malaria vaccines with improved effects as the effects are known to be insignificant. Development is in demand.

Korean Patent Publication No. 10-2019-0009691

An object of the present invention is influenza virus matrix protein 1 (Influenza virus matrix protein, M1); It is to provide a virus-like particle of malaria comprising; and the inner membrane complex (IMC) of the malaria protozoan.

Another object of the present invention is to provide a vaccine composition comprising the virus-like particles.

Another object of the present invention is to provide an expression vector for producing the virus-like particles of malaria and a host cell transformed by the expression vector.

Another object of the present invention is to transform a host cell with an expression vector; And culturing the host cell to express the virus-like particle. It is to provide a method for producing a virus-like particle of malaria comprising.

One aspect of the present invention, influenza virus matrix protein 1 (Influenza virus matrix protein, M1); And it provides a virus-like particles of malaria comprising a; and inner membrane complex (IMC) of the malaria protozoa.

In the present invention, the term "Virus-Like Particles (VLPs)" refers to a non-infectious viral subunit with or without a viral protein. For example, the virus-like particle completely lacks DNA or RNA genome, or a virus-like particle including a viral capsid protein may undergo spontaneous self-assembly.

The “malaria” refers to an acute febrile infectious disease caused by infection with protozoal malaria, and the symptoms and characteristics differ according to the type of the causative pathogen. Specifically, in the present invention, the malaria protozoa may be a rodent malaria protozoan ( Plasmodium berghei ANKA), but is not limited thereto.

In the present invention, the term “malaria virus-like particle” refers to a protein construct (particle) that induces a specific immune response against malaria and has a virus-like form. Specifically, the particle may have a form in which an antigen-determining site derived from malaria protozoa is bound outside a structural protein derived from a virus (FIG. 1A). Meanwhile, in the present invention, the term “malaria virus-like particles” does not mean that malaria is a disease caused by a virus, but is used to mean that it is similar to a virus in its form and use, and is appropriately changed as necessary. Can be applied.

The virus-like particles of malaria are assembled with structural proteins derived from the virus to produce particles in a form similar to a virus, and at the same time include an antigen-determining site derived from a malaria protozoan, thereby making the virus-like particles of malaria into a specific individual. It is characterized in that it can induce a specific immune response against malaria when inoculated with.

The virus-like particles may be produced by a genetic engineering method, and a separate causative infectious agent, that is, in the present invention, can be produced by a genetic engineering method without malaria protozoa, so high productivity and economy can be realized.

In the present invention, the virus-like particles may include influenza virus matrix protein (M1) as a core protein. The "influenza virus" may be composed of subtypes named A, B and C, and in the present invention, the "influenza virus subtype" is hemagglutinin (H) and neuraminidase (neuraminidase, N) refers to an influenza A virus variant characterized by a combination of viral surface proteins. Specifically, influenza virus subtypes are by their H number, for example "influenza virus comprising HA of H1 subtype", "influenza virus of H1 subtype" or "H1 influenza", or by H number And a combination of N numbers, for example “influenza virus subtype H1N1” or “H1N1”.

In the present invention, the term “influenza virus matrix protein (M1)” is a structural protein of an influenza virus, and is a matrix protein that forms a coat inside the fat layer, the envelope of the influenza virus. Means).

In the present invention, the term "subtype" specifically includes all individual "strains" within each subtype usually resulting from mutations, and exhibits different pathological profiles, including natural isolates and artificial mutants or rearrangements. I can. Specifically, in the present invention, the influenza virus may be an influenza A virus A/Puerto Rico/8/34 (H1N1) strain, but is not limited thereto.

In the present invention, the virus-like particles may include influenza virus matrix protein 1 as a structural protein, and at least one antigenic determining site derived from malaria may be included on the surface of the influenza virus matrix protein 1.

The virus-like particles can be prepared by methods well known in the art. For example, the virus-like particles can be prepared by transforming and culturing a predetermined host cell using a recombinant DNA molecule encoding the structural protein and antigen recognition site, and the protein expressed in the cell is assembled on the cell surface. It can then be discharged as a culture supernatant. At this time, since the surface protein contained in the virus-like particles maintains its natural form without undergoing an immobilization process, it is possible to induce a desired immune response to a specific pathogen in an individual.

In addition, the virus-like particles act as antigens in an individual and may label T or B immune cells by reacting with antigen-labeling cells such as dendritic cells.

The virus-like particles of malaria of the present invention contain antigen-determining sites derived from malaria, but do not contain genetic material other than that, so they cannot proliferate and are safe because they are not toxic, so they can be used as a vaccine against malaria. The antigen-determining site introduced on the surface of the virus-like particle has higher antigenicity compared to the purely isolated recombinant protein and can form an effective neutralizing antibody.

In the present invention, the term “antigen determining site (epitope)” is a basic element or minimum unit of recognition by each antibody or T cell receptor, and refers to a specific domain, region or molecular structure to which the antibody or T cell receptor binds. . The antigenic determining site may be derived from malaria and is not particularly limited as long as it can induce an immune activity against malaria.

Specifically, the antigen-determining site includes EBA, GLURP, RAP1, RAP2, Sequestrin, PO32, STARP, SALSA, PfEXPl, Pfs25, Pfs28, PFS27/25, Pfs48/45, Pfs230, or analogs thereof derived from malaria. can do. Specifically, in the present invention, the antigen-determining site may include an inner membrane complex (IMC) derived from a parasitic protozoan mouse malaria ( Plasmodium berghei , ANKA strain).

In the present invention, the term “intimal complex protein (IMC)” is a superficial membrane system constituting planarized cells forming the base of the plasma membrane, and is connected to a cytoskeletal network. The inner membrane complex protein plays an important role in parasitic replication, cell movement, and host cell penetration. The inner membrane complex protein is involved in the formation of new cells in the malaria protozoa, and the cloned chromatin and organelles are closely related to the assembly of the skeleton as a cell division process.

In particular, the present inventors believe that a virus-like particle vaccine containing an inner membrane complex (IMC) derived from the rodent malaria parasite ( Plasmodium berghei ANKA strain) is a fused form with influenza virus matrix protein 1 when introduced into an individual. It was confirmed that it can provide a high level of immunity against (FIGS. 2 and 3 ).

Specifically, the influenza virus matrix protein 1 (M1) may be composed of the amino acid sequence of SEQ ID NO: 1, and the inner membrane complex (IMC) of the malaria protozoan may be composed of the amino acid sequence of SEQ ID NO: 2.

In addition, the influenza virus matrix protein 1 (M1) or the malaria-derived inner membrane complex (IMC) antigen-like protein may include a functional equivalent of a protein composed of the amino acid sequence of SEQ ID NO: 1 or 2.

In the present invention, the term "functional equivalent" refers to at least 70% or more, specifically 80% or more, more specifically 90% or more, with the amino acid sequence of SEQ ID NO: 1 or 2 as a result of the addition, substitution or deletion of amino acids, Most specifically, it refers to a protein having a physiological activity substantially identical to the amino acid sequence of SEQ ID NO: 1 or 2, having 95% or more sequence homology.

In the present invention, the term "substantially equivalent physiological activity" refers to a specific immune response against malaria due to structural and functional homology with the influenza virus matrix protein 1 or malaria-derived inner membrane complex (IMC) antigen-like protein. It refers to the activity as a possible virus-like particle.

In addition, specifically, the virus-like particles of malaria are influenza virus matrix protein 1 (M1) encoded by a gene amplified by a primer pair consisting of the nucleotide sequences of SEQ ID NOs: 3 and 4 and the nucleotide sequences of SEQ ID NOs: 5 and 6 It may include an inner membrane complex (IMC) of a malaria protozoan encoded with a gene amplified by the formed primer pair.

In the present invention, "primer pair" is used as a term that refers to both forward and reverse primers together.

The design of primer pairs is subject to various restrictions, such as A, G, C, T content ratio, prevention of primer conjugate formation, and repetition of the same base sequence more than three times. In addition, conditions such as the amount of template DNA, the concentration of the primer, the concentration of dNTP, the concentration of Mg2+, the reaction temperature, and the reaction time should be appropriate.

The primer is a short nucleic acid sequence containing a free 3'hydroxyl group, and can form a base pair with a template of a complementary nucleic acid, and allows strand copying of the nucleic acid template. Can serve as a starting point for

The primer can initiate DNA synthesis in the presence of a reagent for polymerization reaction and four different nucleoside triphosphates at an appropriate buffer solution and temperature, and hybridizes with a nucleic acid present in the target substance to detect a specific gene of the target substance. Can be used to amplify.

In consideration of the efficiency of amplification, the primer may be specifically single-chain, deoxyribonucleotide, and naturally occurring dNMP (i.e., dAMP, dGMP, dCMP and dTMP), modified nucleotides or non-natural It may contain nucleotides. In addition, the primer may also include a ribonucleotide.

The hybridization means that two single-stranded nucleic acids form a duplex structure by pairing of complementary nucleotide sequences.

The hybridization may occur when the complementarity between single-stranded nucleic acid sequences is perfect or when some mismatch bases are present. The degree of complementarity required for the hybridization may vary depending on the conditions of the hybridization reaction, and in particular, may be controlled by temperature. In general, when the hybridization temperature is high, hybridization is likely to occur in the case of a perfect match, and when the hybridization temperature is low, hybridization may occur even if some mismatches exist.

The primer pair may be hybridized to a specific gene present in the target material, and if the target material is present in the sample, the base sequence of the characteristic gene is severely degraded by the polymerase chain reaction, so the amplified product (amplicon) is used. Through this, it is possible to determine the presence or absence of the raw material.

The primers can incorporate additional features that do not change the basic properties. That is, the nucleic acid sequence can be modified using a number of means known in the art. Examples of such modifications include methylation, encapsulation, substitution of one or more homologs of nucleotides, and uncharged linkers such as phosphonates, phosphotriesters, phosphoroamidates or carbamates, or phosphorothioates or phosphorodithioates. Modification of nucleotides into charged linkers of the back is possible. In addition, nucleic acids are one or more such as nucleases, toxins, antibodies, signal peptides, proteins such as poly L lysine, insertion agents such as acridine or psoralen, chelating agents such as metals, radioactive metals, iron oxidizing metals, and alkylating agents It may have additional covalently bonded moieties.

In addition, the primer sequence may be modified using a label that can directly or indirectly provide a detectable signal. The primer may include a label that can be detected using spectroscopy, photochemical, biochemical, immunochemical or chemical means. Useful labels include 32P, fluorescent dyes, electron congestion reagents, enzymes (commonly used in ELISA), biotin or hapten, and proteins for which antisera or monoclonal antibodies are available.

The primers are cloning of an appropriate sequence and restriction enzyme digestion and a phosphotriester method such as Narang (1979, Meth, Enzymol. 68:90-99), a diethylphosphoramidite method such as Beaucage. (1981, Tetrahedron Lett. 22: 1859-1862) and any other well known method, including direct chemical synthesis, such as the solids support method of U.S. Patent No. 4458066.

Another aspect of the present invention is to provide a vaccine composition comprising the virus-like particles of malaria.

The vaccine composition may be used in a form comprising the virus-like particles or a concentrate thereof, or in the form of a transformed host cell itself or a dry powder of transformed cells. In addition, the vaccine composition may be used together with other foods or food ingredients, and may be appropriately used according to a conventional method.

The vaccine composition is a stabilizer, emulsifier, aluminum hydroxide, aluminum phosphate, pH adjuster, surfactant, liposome, iscom (iscom) adjuvant, synthetic glycopeptide, extender, carboxypolymethylene, bacterial cell wall, derivative of bacterial cell wall, bacterial vaccine, Animal poxvirus protein, subviral particle adjuvant, cholera toxin, N, N-dioctadecyl-N',N'-bis(2-hydroxyethyl)-propanediamine, monophosphoryl lipid A, dimethyldi It may further include at least one second adjuvant selected from the group consisting of octadecyl-ammonium bromide and mixtures thereof.

In addition, the vaccine composition may include a medically acceptable carrier. The “medically acceptable carrier” may include any and all solvents, dispersion media, coating agents, adjuvants, stabilizers, diluents, preservatives, antibacterial and antifungal agents, isotonic agents, adsorption delaying agents, etc., but are limited thereto. It is not.

Carriers, excipients, and diluents that may be included in the vaccine composition are lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, maltitol, starch, glycerin, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose , Methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, and the like, but are not limited thereto.

In addition, the vaccine composition may be formulated in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, and sterile injectable solutions according to conventional methods.

When formulated, diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants may be used together.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and these solid preparations include at least one excipient such as starch, calcium carbonate (calcium carbonate), sucrose or lactose, gelatin in the lecithin-like emulsifier. The lights can be used together. In addition, a lubricant such as magnesium stearate talc may be used in addition to the above excipients.

As liquid preparations for oral administration, suspensions, solvents, emulsions, syrups, etc. can be used, and various excipients, such as wetting agents, sweeteners, fragrances, preservatives, etc., can be used in addition to water and liquid paraffin, which are commonly used simple diluents. have.

A sterilized aqueous solution, a non-aqueous agent, a suspension agent, an emulsion, or a lyophilized agent may be used as a formulation for parenteral administration. Non-aqueous preparations and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.

Another aspect of the present invention is an influenza virus matrix protein 1 (M1) coding nucleotide sequence amplified by a primer pair consisting of the nucleotide sequences of SEQ ID NOs: 3 and 4; And it is to provide an expression vector for producing a virus-like particle of malaria comprising a nucleotide sequence encoding an inner membrane complex (IMC) of a malaria protozoan amplified by a primer pair consisting of the nucleotide sequences of SEQ ID NOs: 5 and 6.

Specifically, the influenza virus matrix protein 1 may be composed of the amino acid sequence of SEQ ID NO: 1, and the inner membrane complex (IMC) antigen-like protein of the malaria protozoa may be composed of the amino acid sequence of SEQ ID NO: 2.

In the present invention, the term "vector" refers to a nucleic acid molecule used to carry a linked nucleic acid fragment. As the vector, bacteria, plasmids, phages, cosmids, episomes, viruses, and insertable DNA fragments (ie, fragments that can be inserted into the host cell genome by homologous recombination) may be used, but are not limited thereto. The plasmid refers to a cyclic double-stranded DNA loop capable of connecting additional DNA fragments therein as a type of vector. In addition, viral vectors can link additional DNA into the viral genome.

In the present invention, the term “expression vector” refers to a vector capable of directing the expression of a gene encoding a protein of interest to which it is operably linked. In general, the expression vector in the use of recombinant DNA technology is in the form of a plasmid, so the terms plasmid and vector can be used interchangeably. However, other types of expression vectors that perform the same function as viral vectors may also be included.

Specifically, the expression vector is pET-3a-d, pET-9a-d, pET-11a-d, pET-12a-c, pET-14b, pET-15b, pET-16b, pET-17b, pET-17xb , pET-19b, pET-20b(+), pET-21a-d(+), pET-22b(+), pET-23a-d(+), pET-24a-d(+), pET-25b( +), pET-26b(+), pET-27b(+), pET-28a-c(+), pET-29a-c(+), pET-30a-c(+), pET-30 Ek/LIC , pET-30 Xa/LIC, pET-31b(+), pET-32a-c(+), pET-32 Ek/LIC, pET-32 Xa/LIC, pET-33b(+), pET-34b(+ ), pET-35b(+), pET-36b(+), pET-37b(+), pET-38b(+), pET-39b(+), pET-40b(+), pET-41a-c( +), pET-41 Ek/LIC, pET-42a-c(+), pET-43.1ac(+), pET-43.1 Ek/LIC, pET-44a-c(+), pRSETA, pRSETB, pRSETC, pESC -HIS, pESC-LEU, pESC-TRP, pESC-URA, Gateway pYES-DEST52, pAO815, pGAPZ A, pGAPZ B, pGAPZ C, pGAPα A, pGAPα B, pGAPα C, pPIC3.5K, pPIC6 A, pPIC6 B, pPIC6 C, pPIC6α A, pPIC6α B, pPIC6α C, pPIC9K, pYC2/CT, pYD1 Yeast Display Vector, pYES2, pYES2/CT, pYES2/NT A, pYES2/NT B, pYES2/NT C, pYES2/CT, pYES2. 1, pYES-DEST52, pTEF1/Zeo, pFLD1, PichiaPink ^TM , p427-TEF, p417-CYC, pGAL-MF, p427-TEF, p417-CYC, PTEF-MF, pBY011, pSGP47, pSGP46, pSGP36, pSGP 40, ZM552, pAG303GAL-ccdB, pAG414GAL-ccdB, pAS404, pBridge, pGAD-GH, pGAD T7, pGBK T7, pHIS-2, pOBD2, pRS408, pRS410, pRS418, pRS420, pRS428, yeast micron A form, pRS403, pRS404 , pRS405, pRS406, pYJ403, pYJ404, pYJ405, or pYJ406, but is not limited thereto.

Meanwhile, the expression vector is introduced into a host cell, and the host cell transformed with the introduced vector can produce the virus-like particles of malaria. In this case, the vector may include a promoter recognized by the host organism.

The promoters are SBE4, 3TP, PAI-1, p15, p21, CAGA12, hINS, A3, NFAT, NFKB, AP1, IFNG, IL4, IL17A, IL10, GPD, TEF, ADH, CYC, INU1, PGK1, PHO5, TRP1 , GAL1, GAL10, GUT2, tac, T7, T5, nmt, fbp1, AOX1, AOX2, MOX1 and FMD1 may be selected from the group consisting of promoters, but may be different in consideration of various variables such as host cells or expression conditions.

The nucleic acid sequence encoding the virus-like particle of malaria may be operably linked with the promoter sequence. The "operably linked" means that one nucleic acid fragment is associated with another nucleic acid fragment so that its function or expression is affected by the other nucleic acid fragment. That is, the gene encoding the virus-like particle of malaria may be operably linked to a promoter in the vector to regulate its expression.

The expression vector may further include additional regulatory sequences. The control sequence may be a Shine-Dalgano sequence of a replicaase gene of phage MS-2 and a Shine-Dalgano sequence of cII of a bacteriophage lambda, but is not limited thereto.

In addition, the expression vector may contain an appropriate marker gene required to select the transformed host cell. The marker gene may be an antibiotic resistance gene or a fluorescent protein gene, and the antibiotic resistance gene may be selected from the group consisting of a hygromycin resistance gene, a kanamycin resistance gene, a chloramphenicol resistance gene, and a tetracycline resistance gene, but is limited thereto. It is not. The fluorescent protein gene is a yeast-enhanced green fluorescent protein (yEGFP) gene, a green fluorescent protein (GFP) gene, a blue fluorescent protein (BFP) gene, and a red fluorescent protein. (red fluorescent protein, RFP) may be selected from the group consisting of genes, but is not limited thereto.

Another aspect of the present invention is to provide a host cell transformed with the expression vector for preparing virus-like particles of malaria of the present invention.

In the present invention, the term “host cell” refers to any organism that can be infected by a virus and can be immunized by a virus-like particle. The host cell can be metabolized by transformation.

The host cells may be microorganisms, animal cells, plant cells, cultured cells derived from animals, or cultured cells derived from plants. Such suitable host cells may be naturally occurring or wild type host cells, or may be altered host cells. The wide-type host cell may be a host cell that has not been genetically changed by a recombinant method.

The type of the host cell is not particularly limited as long as it can be transformed by an engineering method to efficiently express a specific gene, and specifically, it may be an insect cell.

The insect cells may be all cells developed or marketed as a host system for gene expression, such as Spodoptera frugiperda SF21, SF9, Trichoplusia ni , anti Anticarsa gemmitalis , Bombyx mori , Estigmene acrea , Heliothis virescens , Leucania separata , Limantria dispa ( Lymantria dispar ), Malacasoma disstria , Mammestra brassicae , Manduca sexta , Plutella zylostella , Stodoftera Exigua ( Spodoptera exigua ) and Spodoptera littorlis one or more may be selected from the group consisting of, but is not limited thereto.

Specifically, the insect cells may be SF9 cells derived from Spodoptera frugiperda, but are not limited thereto.

In the present invention, the term “metabolically engineered” or “metabolic engineering” refers to biosynthetic genes, genes associated with operons, and these nucleic acid sequences for the production of desired metabolites such as proteins or alcohols in microorganisms. Metabolic flux by controlling and optimizing transcription, translation, protein stability and protein functionality by manipulating control elements or using appropriate culture conditions. In this case, the biosynthetic gene is exogenous to the host, mutagenesis, recombination, or modified by association with a heterologous expression control sequence in an endogenous host cell. , Microorganisms) Suitable culture conditions are conditions such as culture medium pH, ionic strength, nutrient content, temperature, oxygen, carbon dioxide, nitrogen content, humidity, and the production of compounds by the metabolism of the microorganism. And other culture conditions that allow it to be included.

Thus, the “engineered” or “modified” host cell can be produced by introducing genetic material into a selected host or parental microorganism to modify or alter cell physiology and biochemistry. Through the introduction of genetic material, the parental microorganism may acquire new properties, such as the ability to produce new intracellular metabolites or larger amounts of intracellular metabolites.

For example, the introduction of genetic material into the parental microorganism can result in a new or modified ability to produce chemicals. The genetic material introduced into the parental microorganism comprises a gene or part of a gene encoding one or more enzymes involved in the biosynthetic pathway for the production of chemicals, and additional components for the expression or regulation of these genes, for example, It may also contain a promoter sequence.

The "altered host cell" means a genetically designed host cell, wherein the protein of interest is produced at a level of expression or at a level greater than that of expression, or wild-type grown under essentially the same growth conditions It can be expressed at a level of expression greater than that of the protein of interest in the host cell.

The "modified host cell" refers to a wild-type or modified host cell that is genetically designed to overexpress a gene encoding a protein of interest. The modified host cell can express the protein of interest at a higher level than the wild-type or changed parental host cell.

In the present invention, the term “transformation” refers to a method of transporting the vector into a microorganism or a specific cell, and when the cell to be transformed is a prokaryotic cell, it may be carried out by the CaCl ₂ method, one method, and electroporation method. I can.

If the target cell for transformation is a eukaryotic cell, it can be carried out using a microinjection method, a calcium phosphate precipitation method, an electroporation method, a liposome-mediated transfection method, a DEAE-dextran treatment method, and a gene bombadment. It is not limited.

In the case of transformation of fungi such as yeast, it may be generally carried out by a transformation method using lithium acetate and heat shock, and an electroporation method, but is not limited thereto.

Another aspect of the present invention, transforming the host cell with the expression vector; And culturing the host cell to express the virus-like particle. It provides a method for producing a virus-like particle of malaria comprising.

The transformed host cell may be cultured under batch, feed-batch or continuous fermentation conditions, and the host cell may express the virus-like particles of malaria by transformation, so that the virus from the cultured host cell -A similar particle protein can be obtained.

At this time, the batch fermentation method can use a closed system, the culture medium is prepared before the fermentation is carried out, the medium is inoculated with organisms, and the fermentation can take place without the addition of any components to the medium. In certain cases, the pH and oxygen content, not the carbon source content of the growth medium, can be varied during the batch process. The metabolites and cellular biomass of the batch system can constantly change until fermentation is stopped. In a batch system, cells can progress over the high-growth log phase on a stationary lag, and eventually reach a stationary phase when the growth rate is reduced or stopped. In a typical period, these cells on the log can make most of the protein.

The batch system can be transformed into a "feed-batch fermentation" system. In such systems, nutrients (eg, carbon source, nitrogen source, O ₂ and typically other nutrients) can be added when the concentration of their cultures falls below the limit. Feed-batch systems can be useful when catabolic product inhibition inhibits metabolism of cells, and it is desirable for the medium to have a limited amount of nutrients in the medium. It can be predicted based on changes in measurable factors such as dissolved oxygen and the partial pressure of waste gases such as CO ₂ . Batch and feed-batch fermentation is well known in the art as a general system.

Continuous fermentation is an open system in which a defined culture medium is continuously added to a bioreactor and an equal amount of conditioned medium is removed simultaneously during the process. Continuous fermentation is generally able to maintain a constant high density culture in which the cells are initially in log phase growth. Continuous fermentation may allow manipulation of one factor or any number of factors affecting cell growth or final product concentration.

For example, a limiting nutrient such as a carbon source or a nitrogen source can be kept at a fixed rate and all other parameters appropriately. In other systems, many factors that influence growth can continue to change while the cell concentration, as measured by medium turbidity, remains constant. The continuous system tries to maintain a steady state of growth conditions. Thus, cell loss due to escaping of the medium can be balanced against the rate of cell growth in fermentation.

Unlike traditional vaccines, the virus-like particles of malaria of the present invention can be developed without causative infectious agents and do not induce non-selective antibody production.

In addition, the virus-like particles of malaria can activate the body's immune system to provide immunity and preventive effects against malaria, so it is safe without inducing resistance, and side effects can be minimized.

The effects of the present invention are not limited to the above effects, and should be understood to include all effects that can be deduced from the configuration of the invention described in the detailed description or claims of the present invention.

1 shows a virus-like particle of malaria of the present invention (A: a model of a virus-like particle of malaria including the influenza virus matrix protein 1 (M1) of the present invention and the inner membrane complex of malaria protozoa (IMC), B: The appearance of observation of malaria virus-like particles with an electron microscope, C: Western blot results after reaction of malaria virus-like particles with protozoa infected serum and influenza M1 antibody).
Figure 2 shows the results of confirming the formation of malaria-specific antibodies IgG, IgG2a, IgG1 and IgG2b over time through ELISA analysis after inoculation of the virus-like particles of malaria of the present invention in mice (A: time after inoculation Concentration of IgG over time, B: concentration of IgG1 over time after inoculation, C: concentration of IgG2a over time after inoculation, D: concentration of IgG2b over time after inoculation).
3 is a mouse infected with malaria protozoa ( Plasmodium berghei , ANKA) intraperitoneally after induction of an immune response by inoculation of a virus-like particle of malaria of the present invention (Imm+cha) and a mouse infected without inoculation (Naive+cha) The degree of formation of IgG, IgG2a, IgG1, and IgG2b, which are malaria-specific antibodies, was compared (A: concentration of IgG over time after infection, B: concentration of IgG1 over time after infection, C: after infection) Concentration of IgG2a over time, D: concentration of IgG2b over time after infection).
Figure 4 is a result of comparing the infection rates for the non-infected group (Naive), the negative control group (Naive + Cha) and the experimental group (Imm + Cha) to confirm the protective immunity effect according to the virus-like particle inoculation of malaria of the present invention (A: Infection rate (M2) of the non-infected group is 0%, B: Infection rate (M2) of the non-inoculated negative control group (Naive+Cha) is 13.8%, C: The experimental group infected after inoculation (Imm+Cha) is Infection rate (M2) was 2.9%, D: Infection rate comparison graph between the experimental group and the control group (*P<0.05)).
Figure 5 is a comparison of the weight change for the non-infected group (Naive), negative control (Naive + Cha) and experimental group (Imm + Cha) in order to confirm the protective immunity effect by the virus-like particle inoculation of malaria of the present invention. It shows the results.
Figure 6 shows the results of comparing the immune response of the T-cell by the inoculation of the virus-like particles of malaria of the present invention in a non-infected group (Naive), a negative control group (Naive+Cha) and an experimental group (Imm+Cha). (A: CD3+CD4 immune response of the non-infected group was 23.2%, B: CD3+CD4 immune response of the negative control group was 6.8%, C: CD3+CD4 immune response of the experimental group was 27.7%, D: A to A bar graph showing the value of C, E: CD3+CD8 immune response of non-infected group was 10.1%, F: CD3+CD8 immune response of negative control group was 5.9%, G: CD3+CD8 immune response of experimental group was 13.2 %, H: a bar graph depicting the values of E to G).
Figure 7 shows the results of confirming the immune response of B-cell by inoculation of virus-like particles of malaria of the present invention in the non-infected group (Naive), negative control group (Naive+Cha), and experimental group (Imm+Cha). (A: B220+CD19 immune response of non-infected group was 11.8%, B: B220+CD19 immune response of negative control group was 5.1%, C: B220+CD19 immune response of experimental group was 13.0%, D: A to C A bar graph depicting the figures of).

Hereinafter, the present invention will be described in detail by examples. However, the following examples are only illustrative of the present invention, and the present invention is not limited by the following examples.

Preparation Example 1. Preparation of vector for preparation of virus-like particles (VLPs) of malaria

In order to produce virus-like particles containing the inner membrane complex (IMC) of the malaria protozoan, the inner membrane complex of the rodent malaria ( Plasmodium berghei ANKA) consisting of the nucleotide sequence of SEQ ID NO: 7 sub-compartment protein 3 (inner membrane complex) Part of the sub-compartment protein 3) was selected as the antigen gene.

Endometrial complex (IMC) antigen gene of the malaria parasite is a forward primer having the nucleotide sequence of SEQ ID NO: 5 (5'-aaa GGATCC atgggaaacagcttgtgctgc- 3 ') and SEQ ID NO: 6 DNA sequence reverse primer (5'-tta CTCGAG having the ttaagcagttaagcaatgctt-3') was amplified through PCR. In the primers, restriction enzyme sites (BamH I and Xho I) were respectively introduced in the underlined portions.

Thereafter, a sequence encoding an inner membrane complex (IMC) antigen amplified using the primer as a cDNA template was introduced into the pFastBac vector (Invitrogen).

On the other hand, the influenza matrix protein 1 (M1) gene is derived from the influenza A/PR/8/34 virus, and has a forward primer having a nucleotide sequence of SEQ ID NO: 3 (5'-TCC CCCGGG CCACCATGAGCCTTCTGACCGAGGTC-3') and SEQ ID NO: 4 It was amplified through RT-PCR using a reverse primer (5'-TTACT TCTAGA TTACTTGAACCGTTGCATCTG-3') having the nucleotide sequence of. In the primers, restriction enzyme sites (SmaI and XbaI) were respectively introduced into the underlined portions. Thereafter, A/PR/8/34 virus was inoculated into MDCK cells, and viral RNA was extracted through the RNeasy Mini kit.

The influenza matrix protein 1 (M1) gene used the same sequence as the M1 gene described in Korean Patent Application Laid-Open No. 10-2019-0009691 (GenBank accession number: EF467824, 1027bp).

A recombinant plasmid was constructed by introducing the influenza M1 protein gene amplified using the above primers into the pFastBac vector into which the inner membrane complex (IMC) coding sequence was introduced. At this time, it was confirmed by DNA sequencing determination method whether the M1 and the IMC genes of SEQ ID NO: 7 were properly introduced into the pFastBac vector.

In order to prepare recombinant baculoviruses (recombinant BaculoViruses, rBVs) expressing IMC and M1, DNA transfection was performed using cellfectin II (Invitrogen) and SF9 cells. Transformation using the pFastBac vector containing IMC and M1 was performed by white/blue screening. Recombinant baculovirus was prepared according to the manufacturer's manual through the Bac-to-Bac expression system (Invitrogen).

Preparation Example 2. Preparation of virus-like particles of malaria

Virus-like particles of malaria were produced in SF9 insect cells co-infected with recombinant baculoviruses (rBVs) expressing IMC and M1. Virus-like particles in the supernatant were pelleted using high-speed centrifugation (30 min, 45,000 x g).

Virus-like particles were resuspended in phosphate buffered saline (PBS) at 4° C. overnight, and harvested and purified at 4° C., 45,000×g for 1 hour through a discontinuous sucrose gradient (20-30-60%). Protein concentration was determined by QuantiPro BCA Assay Kit (Sigma-Aldrich).

The virus-like particles were identified using Western blot and electron microscopy. In Western blot analysis, mouse serum was used to probe for IMC protein. Serum was collected from BALB/c mice after 4 weeks of infection with the malaria ANKA strain. The content of M1 protein was determined by anti-M1 monoclonal antibody. Virus-like particles were stained as a medium for electron microscopy and size measurement, and observed through transmission electron microscopy (TEM) (CANADA, ALBERTA).

SF9 cells producing virus-like particles by electron microscopy were found to be larger than normal cells as a control, and the shape of virus-like particles was observed in the form of an atypical sphere with spikes formed on the surface (FIG. 1B).

In order to confirm whether the virus-like particles of IMC and M1 were properly introduced, Western blot was performed using an anti-malaria polyclonal antibody and an anti-M1 monoclonal antibody (Fig. 1C). Specifically, for SDS-PAGE, 100 μg, 50 μg, and 25 μg of virus-like particles of malaria were each loaded, and protozoa infected serum and anti-M1 monoclonal antibody were used as probes.

As a result, it was confirmed that SF9 cells infected with recombinant baculoviruses (rBVs) expressing IMC and M1 produced particles similar in shape and size to virions.

Experimental Example 1. Immune induction activity test in an animal model

The immune response induced after inoculation of the virus-like particles of Preparation Example 2 into mice was confirmed.

After intramuscular route of the virus-like particles of Preparation Example 2, blood was collected from mice at a predetermined time, and the degree of formation of malaria-specific IgG, IgG1, IgG2a, and IgG2b antibodies in serum was measured through ELISA method ( Fig. 2).

As a result, there was no change in the degree of antibody formation of IgG1 and IgG2b in the mice (Figs. 2B and 2D), whereas the total IgG and IgG2a reactions of mice for which immunity was formed by inoculation with the virus-like particles was 1 week after inoculation. And it was confirmed that it increased significantly at 4 weeks (Figs. 2A, 2C). From these results, it was confirmed that the malaria IMC-specific antibodies were gradually matured by inoculating the virus-like particles of malaria of the present invention.

In addition, in order to confirm the antibody response profile according to the challenge infection, mice inoculated with virus-like particles were infected with the intraperitoneal route with Plasmodium berghei ANKA, followed by malaria-specific IgG, IgG2a, IgG1 and IgG2b antibodies. The degree of formation of was measured (Fig. 3).

As a result, the degree of antibody formation of IgG1 and IgG2b of the mice still remained unchanged (Figs. 3B and 3D), whereas mice (Imm+cha) in which immunity was formed by inoculation of the virus-like particles were infected without inoculation. It was confirmed that significantly higher levels of IgG and IgG2a antibody reactions occurred in serum due to malaria infection than mice (Naive+cha) (FIGS. 3A and 3C ). These results mean that mice whose immunity was formed by the inoculation of the virus-like particles could very rapidly produce antibodies of malaria protozoa-specific IgG and IgG2a by malaria infection.

That is, the above experimental results suggest that the virus-like particles of Preparation Example 2 provide high immunity against malaria and can effectively induce an antibody response of the tissue system in response to malaria infection.

Experimental Example 2. Protective immune activity test according to infection (infection rate)

After inoculation of the virus-like particles of Preparation Example 2 in mice was confirmed the induced protective immune effect.

After the virus-like particle vaccine inoculation of Preparation Example 2, mice infected with Plasmodium berghi ANKA were sacrificed 2 weeks later, and the rate of infection of the protozoal malaria was compared by flow cytometry (FIGS. 4A to 4C).

As a result, it was confirmed that the mice (Imm+Cha) for which immunity was formed by the virus-like particle inoculation showed high protective immunity compared to the negative control group (Naive+Cha). In particular, it was confirmed that parasitemia, in which parasites exist in the blood, was significantly reduced (Fig. 4D, *P<0.05).

In addition, as a result of measuring the change in body weight due to malaria infection, the degree of weight loss was alleviated in mice (Imm+Cha) with immunity compared to mice infected with malaria (Naive+Cha) (FIG. 5).

The above results suggest that the virus-like particles of the present invention can induce a protective immune effect against malaria infection.

Experimental Example 3. Antibody-secreting cell (B cell) response, T cell response test

After inoculation of the virus-like particles of Preparation Example 2 in mice, the antibody-secreting cell reaction was confirmed.

Specifically, the mice were sacrificed 2 weeks after infection, and splenocytes infected by malaria were cultured in vitro, and the immune responses of T-cells and B-cells in the spleen were confirmed by flow cytometry.

As a result, mice (Imm+Cha) for which immunity was formed by inoculation of virus-like particles significantly increased CD4+ T cell levels (Figs. 6A to 6D) and CD8+ T compared to normal mice (Naive) and infected mice (Naive+Cha). It was confirmed that the cell level (FIGS. 6E to 6H) was shown.

In addition, it was confirmed that even at the level of Total B-cell, immunity was formed mice (Imm+Cha) showed increased results compared to normal mice (Naive) and infected mice (Naive+Cha) (FIG. 7).

That is, from the above experimental results, it was confirmed that mice having immunity formed by virus-like particles can acquire a very high level of immunity against malaria infection.

The above description of the present invention is for illustrative purposes only, and those of ordinary skill in the art to which the present invention pertains will be able to understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not limiting. For example, each component described as a single type may be implemented in a distributed manner, and similarly, components described as being distributed may also be implemented in a combined form.

The scope of the present invention is indicated by the claims to be described later, and all changes or modified forms derived from the meaning and scope of the claims and the concept of equivalents thereof should be construed as being included in the scope of the present invention.

<110> University-Industry Cooperation Group of Kyung Hee University <120> VIRUS-LIKE PARTICLES COMPRISING THE INNER MEMBRANE COMPLEX OF PLASMODIUM BERGHEI, AND VACCINE COMPOSITIONS USING THEREOF <130> 19PP30062 <160> 7 <170> KoPatentIn 3.0 <210> 1 <211> 252 <212> PRT <213> Artificial Sequence <220> <223> Influenza virus matrix protein, M1 <400> 1 Met Ser Leu Leu Thr Glu Val Glu Thr Tyr Val Leu Ser Ile Ile Pro 1 5 10 15 Ser Gly Pro Leu Lys Ala Glu Ile Ala Gln Arg Leu Glu Asp Val Phe 20 25 30 Ala Gly Lys Asn Thr Asp Leu Glu Val Leu Met Glu Trp Leu Lys Thr 35 40 45 Arg Pro Ile Leu Ser Pro Leu Thr Lys Gly Ile Leu Gly Phe Val Phe 50 55 60 Thr Leu Thr Val Pro Ser Glu Arg Gly Leu Gln Arg Arg Arg Phe Val 65 70 75 80 Gln Asn Ala Leu Asn Gly Asn Gly Asp Pro Asn Asn Met Asp Lys Ala 85 90 95 Val Lys Leu Tyr Arg Lys Leu Lys Arg Glu Ile Thr Phe His Gly Ala 100 105 110 Lys Glu Ile Ser Leu Ser Tyr Ser Ala Gly Ala Leu Ala Ser Cys Met 115 120 125 Gly Leu Ile Tyr Asn Arg Met Gly Ala Val Thr Thr Glu Val Ala Phe 130 135 140 Gly Leu Val Cys Ala Thr Cys Glu Gln Ile Ala Asp Ser Gln His Arg 145 150 155 160 Ser His Arg Gln Met Val Thr Thr Thr Asn Pro Leu Ile Arg His Glu 165 170 175 Asn Arg Met Val Leu Ala Ser Thr Thr Ala Lys Ala Met Glu Gln Met 180 185 190 Ala Gly Ser Ser Glu Gln Ala Ala Glu Ala Met Glu Val Ala Ser Gln 195 200 205 Ala Arg Gln Met Val Gln Ala Met Arg Thr Ile Gly Thr His Pro Ser 210 215 220 Ser Ser Ala Gly Leu Lys Asn Asp Leu Leu Glu Asn Leu Gln Ala Tyr 225 230 235 240 Gln Lys Arg Met Gly Val Gln Met Gln Arg Phe Lys 245 250 <210> 2 <211> 150 <212> PRT <213> Artificial Sequence <220> <223> Plasmodium berghei ANKA, inner membrane complex <400> 2 Met Gly Asn Ser Leu Cys Cys Ile Asn Asp Leu Lys Asn Asn Lys Ser 1 5 10 15 Asn Ile Asp Ile Tyr Ala Tyr Pro Ser Gln Glu Lys Tyr Asp Glu Tyr 20 25 30 Asp Asn Trp Thr Leu Glu Thr Trp Ile Asp Lys Tyr Lys Asn Gly Asn 35 40 45 Thr Ile Arg Val Ala Phe Pro Asp Gly Asn Glu Ile Gln Cys Tyr Phe 50 55 60 Lys Ile Phe Leu Asn Glu Lys Cys Phe Glu Leu Ser Leu Asp Asn Lys 65 70 75 80 Val Arg Ile Ile Lys Phe Asn Asp Ile Lys Cys Val Leu His Arg Asn 85 90 95 Ser Cys Glu Ser Leu Leu Glu Ser Glu Gln Asn Leu Leu Lys Ser Pro 100 105 110 Lys Val Ile Gly Ile Arg Leu Ile Ser Thr Leu Lys Ala Ile Ala Phe 115 120 125 Ala Met Asp Ser Pro Gly Glu Glu Arg Met Phe Tyr Glu Phe Ile Lys 130 135 140 Lys His Cys Leu Thr Ala 145 150 <210> 3 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Influenza M1_primer F <400> 3 tcccccgggc caccatgagc cttctgaccg aggtc 35 <210> 4 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Influenza M1_primer R <400> 4 ttacttctag attacttgaa ccgttgcatc tg 32 <210> 5 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> IMC_primer F <400> 5 aaaggatcca tgggaaacag cttgtgctgc 30 <210> 6 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> IMC_primer R <400> 6 ttactcgagt taagcagtta agcaatgctt 30 <210> 7 <211> 453 <212> DNA <213> Artificial Sequence <220> <223> Plasmodium berghei ANKA, IMC sub-compartment protein 3 <400> 7 atgggaaaca gcttgtgctg cattaacgac ttaaaaaaca acaaatccaa tatagatata 60 tatgcatatc ctagccaaga aaaatatgat gagtatgata attggacatt agaaacttgg 120 atagataaat ataagaatgg taatacaatt agagtagcat ttcctgatgg aaatgaaatt 180 caatgttatt ttaaaatttt tttaaatgaa aagtgctttg aactatcctt agataataaa 240 gttcgtatta taaaatttaa tgatattaag tgtgtactcc acagaaatag ttgtgaatca 300 ttattagaat cagaacaaaa tttattaaaa tcacctaagg ttataggaat tcgattgatt 360 agtacactta aagctattgc ctttgcaatg gatagtcctg gagaggaaag aatgttttat 420 gaatttataa aaaagcattg cttaactgct taa 453

Claims (10)

Influenza virus matrix protein (M1); And
Virus-like particles capable of inducing an immune response against malaria, including; the inner membrane complex (IMC) of the protozoal malaria. The method of claim 1,
The influenza virus matrix protein 1 (M1) is composed of the amino acid sequence of SEQ ID NO: 1, and the inner membrane complex antigen-like protein (IMC) of the malaria protozoa is composed of the amino acid sequence of SEQ ID NO: 2, inducing an immune response against malaria Possible virus-like particles. The method of claim 1,
The influenza virus matrix protein 1 (M1) is encoded with a gene amplified by a primer pair consisting of the nucleotide sequences of SEQ ID NOs: 3 and 4, and the inner membrane complex (IMC) of the malaria protozoan is nucleotide sequences of SEQ ID NOs: 5 and 6. A virus-like particle capable of inducing an immune response against malaria, which is encoded by a gene amplified by a pair of primers. A vaccine composition comprising a virus-like particle capable of inducing an immune response against malaria according to any one of claims 1 to 3. Influenza virus matrix protein 1 (M1) coding nucleotide sequence amplified by a primer pair consisting of the nucleotide sequences of SEQ ID NOs: 3 and 4; And
An expression vector for preparing a virus-like particle capable of inducing an immune response against malaria, comprising; an inner membrane complex (IMC) encoding nucleotide sequence of malaria protozoa amplified by a primer pair consisting of the nucleotide sequences of SEQ ID NOs: 5 and 6. The method of claim 5,
The influenza virus matrix protein 1 is composed of the amino acid sequence of SEQ ID NO: 1, and the inner membrane complex (IMC) antigen-like protein of the malaria protozoan is composed of the amino acid sequence of SEQ ID NO: 2, which can induce an immune response against malaria. Expression vector for preparing virus-like particles. A host cell transformed by the expression vector of claim 5 or 6. The method of claim 7,
Transformed host cells, which are microorganisms, animal cells, plant cells, cultured cells derived from animals, or cultured cells derived from plants. The method of claim 7,
The host cell is an insect-derived SF9 cell, a transformed host cell. Transforming the host cell with the expression vector of claim 5 or 6; And
A method for producing a virus-like particle capable of inducing an immune response against malaria comprising; culturing the host cell to express a virus-like particle.

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