KR20120013260A - Vaccines for Cervical Cancer - Google Patents

Abstract

PURPOSE: A vaccine pharmaceutical composition for human cervical cancer is provided to ensure excellent effect in Th1 type immune response and Th2 type immune response. CONSTITUTION: A method for manufacturing human papilloma virus(HPV) L1 virus-like particles(VLP) comprises: a step of culturing yeast containing a HPV L1-coding nucleotide sequence; a step of lysing the yeast; a step of performing ammonium sulfate precipitation of the yeast lysate to remove impurities; and a step of performing heparin chromatography or cation exchange chromatography. The nucleic acid sequence of VLP contains a sequence of sequence number 1.

Description

Vaccines for Cervical Cancer

The present invention relates to a human cervical cancer vaccine pharmaceutical composition, a method for preventing human cervical cancer, and a method for preparing human papilloma virus (HPV) L1 virus-like particles (VLP).

More than 80 types of human papillomavirus (HPV) are currently known, about 30 types of sexual contact cause cervix infections, and about half of them are known to be related to cervical cancer (Zur Hausen H, (1988) Mol. Carcinogenesis 8: 147-150). In addition, among HPV, the incidence of cancer in women infected with HPV types 16 and 18 is 500 times higher than that of non-infected people, and these are known to be infected through epithelial cells of the genital organs and then malignant transformation into cervical cancer. (Zur Hausen H, (1988) Mol. Carcinogenesis 8: 147-150; Hausen, Biochemica. Biophysica. Acta. 1288: 55-78, 1996).

The currently developed vaccine for cervical cancer has been developed using high-risk types of HPV virus-like particles (VLPs). VLP is the main capsid protein of HPV (L1 protein, about 55 kDa), and has the property of self-aligning to VLP without other viral gene products, which is almost similar to natural HPV virion. In addition, immunity lasts a long time and has the advantage of being able to expect high efficiency as a vaccine candidate because it is type-specific.

Merck’s “Gadasil” using these VLPs is a vaccine developed using “Alum,” an adjuvant commonly used for the antigens of each of the four most commonly occurring HPV types 6, 11, 16 and 18. GlaxoSmithCline's'Serberix' is a vaccine developed using'AS04', an adjuvant developed independently for HPV types 16 and 18, which are the most representative cervical cancer inducing factors.

Alum, an adjuvant used in Gardasil, is used in diphtheria, tetanus, and hepatitis B vaccines, and it has been reported that it increases the stability of the antigen and induces the release of cytokines, but it makes it impossible to freeze-dry or freeze the vaccine. It is not effective against all antigens and has a major disadvantage that its use is limited as it promotes only humoral immune responses.

AS04, included in GSK's Cervarix, was developed with the aim of inducing a strong and sustained immune response and consists of aluminum hydroxide and monophosphoryllipid A (MPL). MPL is an immune-promoting substance that can directly activate key immune mechanisms, and ultimately plays a role in strengthening the immune response to the antigens contained in the vaccine.

Recently, adjuvants are receiving new attention, and in addition to cervical cancer vaccines, flu vaccines are being used in various ways. Among these adjuvants, bacterial DNA has been attracting attention as an anticancer agent since the 1960s, and it has been studied until now, but it has not been used alone as an anticancer agent due to insufficient efficacy (Glick, JL The specificity of inhibition of tumor cell viability by DNA. Cancer Res. 27:2338, 1967). However, bacterial DNA is known to activate various immune cells without significant side effects, and it has been confirmed that it has many advantages, especially as an adjuvant (McCluskie MJ, etal ..CpG DNA is a potent enhancer of systemic and mucosal immune responses against hepatitis B. surface antigen with intranasal administration to mice JImmunol .Nov1; 161 (9):. 4463-6.1998).

Regarding this effect of bacterial DNA, Yamamoto et al. of Japan argued that sequences including CG play an important role, and it was widely known by research by Krieg et al. of the United States (Yamamoto S. et al. Unique palindromic sequences in synthetic oligonucleotides are required to .. induce IFN and augment IFN- mediated natural killer activity J. Immunol 148: 4072,1992; Krieg AM, Antitumor applications of stimulating toll-like receptor 9 with CpG oligodeoxynucleotides Curr Oncol Rep Mar; 6 (2)....: 88-95.2004). In particular, after the mid-1990s, as a result of research related to CPG, the possibility of synthetic DNA (CpG-ODN) including unmethylated CG was confirmed as a result of research related to CPG. S substitution and the like were studied. Through these efforts, CpG-ODN-related products have been able to be clinically tested not only as adjuvants but also as anticancer agents (http://www.coleypharma.com).

However, the immunogenicity problem for the S substitution of the diester bond of CpG-ODN and the still insufficient anticancer effect remain a problem to be solved. CPG 7909 currently in clinical use is a phosphorothioate oligonucleotide that induces anti-DNA Ab (Clin Immunol. 2001 Aug;100(2):157-63), which is an autoimmune disease such as SLE (systemic lupus erythematosus). Is closely related to (J Clin Immunol. 1986 July;6(4):292-8). In addition, it is known that the phosphorothioate structure acts as TI Ag to disrupt the immune defense function against infection (Mol Immunol. 1998 Dec;35(18):1161-70).

In the case of LPS, which has been known for its anticancer effect since the 1950s, it has been difficult to use due to toxicity, which can lead to death due to sepsis even with ng level contamination. In particular, it was a general opinion that the binding of LPS and DNA exhibits serious toxicity, and the removal of LPS from DNA-related drugs was understood as a very important process (Gao JJ. et. al, Bacterial DNA and lipopolysaccharide induce synergistic production of TNF-alpha through a post-transcriptional mechanism . JImmunol 166(11):6855-60,2001). In terms of efficacy, the increase of immunity by LPS is much stronger than that by DNA, but it was thought that it was not suitable as an anticancer agent because it was not the Th1 type, which is important for anticancer, but the Th2 type (Lebman DA et al. al Interleukin 4 causes isotype switching to IgE in T cell-stimulated clonal B cell cultures. J Exp Med . Sep 1;168(3):853-62. 1988). In particular, because Th2 type immune activity suppresses Th1 type immune activity, these characteristics of LPS made it more difficult to use LPS as an anticancer agent (Rengarajan J et al. al . Transcriptional regulation of Th1/Th2 polarization. Immunol Today. Oct;21(10):479-83. 2000).

Attempts to attenuate LPS have been studied continuously, and in particular, it was possible to successfully reduce toxicity through removal of polysaccharide chains or deacylation of lipid A (Katz SS et al. al Deacylation of lipopolysaccharide in whole Escherichia coli during destruction by cellular and extracellular components of a rabbit peritoneal inflammatory exudate. J Biol Chem . Dec 17;274(51):36579-84 1999). In particular, in the case of monophosphoryl lipid A (MPL) obtained through phosphorylation of Leaf A obtained by removing the polysaccharide chain of LPS, it was developed as an immune anticancer drug that removed the toxicity of LPS, but its effect is known to be insignificant (http:/ /www.corixa.com).

Accordingly, the applicant of the present invention has already developed a novel adjuvant that compensates for the disadvantages of the adjuvants mentioned above (Korean Patent Registration No. 0740237 (2007.07.10)).

Throughout this specification, a number of papers and patent documents are referenced and citations are indicated. The disclosure contents of cited papers and patent documents are incorporated by reference in this specification as a whole, and the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly described.

The present inventors have tried to develop a novel vaccine that can solve the problems of the conventional cervical cancer vaccine described above. As a result, the present inventors found that in the case of using a non-toxic LOS (Lipopolysaccharide) from which some fatty acids from lipid A are removed as an adjuvant in the immunization process using L1 VLP of HPV 16 or 18, it is greater than that of a conventional VLP-based vaccine. By inducing an improved HPV immune response and confirming that it can be an excellent vaccine for preventing cervical cancer, the present invention has been completed.

Accordingly, an object of the present invention is to provide a human cervical cancer vaccine pharmaceutical composition.

Another object of the present invention is to provide a method for preventing human cervical cancer.

Another object of the present invention is to provide a method for preparing HPV L1 VLP.

Another object of the present invention is to provide a novel nucleic acid sequence encoding the L1 VLP of HPV 16.

Another object of the present invention is to provide a novel nucleic acid sequence encoding the L1 VLP of HPV 18.

Another object of the present invention is to provide a Saccharomyces cerevisiae transformed with the above novel nucleic acid sequence.

Other objects and advantages of the present invention will become more apparent by the following detailed description, claims and drawings.

According to one aspect of the present invention, the present invention provides (a) (i) human papilloma virus (HPV) type 16 L1 virus-like particles (VLP), HPV type 18 L1 VLPs, or a combination thereof; And (ii) deacylated non-toxic LOS (Lipooligosaccharide); And, (b) it provides a human cervical cancer vaccine pharmaceutical composition comprising a pharmaceutically acceptable carrier.

According to another aspect of the present invention, the present invention provides a method for preventing human cervical cancer comprising administering the pharmaceutical composition of the present invention to a human.

The present inventors have tried to develop a novel vaccine that can solve the problems of the conventional cervical cancer vaccine described above. As a result, the present inventors found that in the immunization process using L1 VLPs of HPV 16 or 18, when using a deacylated non-toxic LOS (Lipooligosaccharide) as an adjuvant as an adjuvant, the HPV immune response significantly improved compared to the conventional VLP-based vaccine. It was confirmed that it can be an excellent vaccine for preventing cervical cancer by induction.

The VLP used in the pharmaceutical composition of the present invention is derived from HPV type 16 L1 or HPV type 18 L1. HPV type 16 or HPV type 18 is known as high-risk HPV, and is a representative HPV that causes cervical cancer.

In the specification of the present invention, the term HVP L1 protein is expressed from the L1 gene of HPV and refers to a major protein constituting the capsid of HPV. The L1 protein has the property of self-assembling into virus-like particles (VLPs) under suitable conditions with the L2 protein, which is another minor protein constituting the capsid, or the L1 protein alone.

The VLP used in the present invention includes a naturally occurring total sequence of the L1 protein of HPV type 16 or HPV type 18 and a functional L1 protein derivative thereof. The term "HPV L1 functional derivative" refers to a derivative of the naturally-derived HPV L1 protein, which is not identical to the natural-derived HPV L1 total sequence, but is capable of forming VLPs and inducing an immune response. The L1 functional derivative is a truncated L1 protein from which the nuclear localization signal has been removed. For example, in the case of HPV type 16 L1, 34 amino acids at the C-terminus have been removed, and HPV type 18 L1 In this case, the form in which 35 amino acids at the C-terminus have been removed is a functional derivative of HPV L1 that can be used in the present invention. And shown in SEQ ID NO: 4.

As an antigen that induces an immune response in the cervical cancer vaccine pharmaceutical composition of the present invention, HPV 16 L1 VLP or HPV 18 L1 VLP is used, preferably both HPV 16 L1 VLP and HPV 18 L1 VLP are used.

Optionally, as antigens, in addition to HPV 16 L1 VLP and HPV 18 L1 VLP, HPV type 31, HPV type 45, HPV type 6a, HPV type 6b, HPV type 11, HPV type 33, HPV type 35, HPV type 39, HPV At least one L1 VLP selected from the group consisting of L1 VLPs of type 51, HPV type 52, HPV type 56, HPV type 58, and HPV type 68 may be additionally included.

Optionally, the L1 VLP used in the present invention may be fused with other proteins such as L2 protein.

The greatest feature of the vaccine composition of the present invention is the use of deacylated non-toxic LOS (Lipooligosaccharide) as an adjuvant. The term “Lipooligosaccharide (LOS)”, which is adopted for the first time in the present specification, is a variant of LPS (lipopolysaccharide), which means that it has a shorter sugar chain than that of naturally occurring LPS and thus has a smaller molecular weight. Prior to deacylation, the LOS preferably has a molecular weight of 5,000-10,000 Da. The term “deacylated LOS” refers to the removal of fatty acids bound by -C(O)O- bonds to glucosamine of lipid A in these LOSs, resulting in a significant reduction in toxicity compared to LPO. Fatty acids in glucosamine of lipid A are It is bonded through -C(O)O- bond and -C(O)NH- bond. The deacylated LOS of the present invention is a fatty acid bonded by -C(O)O- bond by deacylation of lipid A. It indicates that it has been removed.

Deacylated non-toxic LOS can be prepared through various methods, but Korean Patent Registration No. 0456681, which is a prior patent of the present inventors; WO 2004/039413; Republic of Korea Patent Registration No. 0740237; And according to the method disclosed in WO 2006/121232. For example, LPS is deacylated by treatment with a strong base (eg, 2 N NaOH) to remove some fatty acids from lipid A to detoxify it.

According to a preferred embodiment of the present invention, the deacylated non-toxic LOS used as an adjuvant in the present invention is non-toxic by deacylating LPS by alkali treatment. Preferred examples of the alkali include NaOH, KOH, Ba(OH) ₂ , CsOH, Sr(OH) ₂ , Ca(OH) ₂ , LiOH, RbOH and Mg(OH) ₂ , more preferably NaOH, KOH , Ba(OH) ₂ , Ca(OH) ₂ , LiOH and Mg(OH) ₂ , more preferably NaOH, KOH and Mg(OH) ₂ , and most preferably NaOH.

The degree of toxicity of LPS can be analyzed through various methods known in the art. For example, toxicity can be analyzed by measuring the amount of TNF-α (tumor necrosis factor-α) secreted in acute monocytic leukemia (THP-1) treated with LPS. The deacylated non-toxic LOS of the present invention induces a relatively small amount of TNF-α secretion compared to conventional LPS.

Another characteristic of the deacylated non-toxic LOS, which is an adjuvant used in the present invention, is that its molecular weight is generally small compared to conventionally used LPS. Preferably, the deacylated non-toxic LOS used in the present invention is 1,500-10,000 Da, more preferably 2,000-5,000 Da, even more preferably 2,000-4,000 Da, even more preferably 3,000-4,000 Da, Most preferably, it has a molecular weight of 3,200-3,7000 Da. Such molecular weight can be determined using conventional methods in the art, for example MALDI-MASS.

According to a preferred embodiment of the present invention, the immunoadjuvant deacylated non-toxic LOS of the present invention is derived from E. coli , and most preferably derived from E. coli EG0021 (KCCM 10374), which is our own bacterium. It was done.

The deacylated non-toxic LOS used in the present invention not only has excellent immunostimulatory efficacy compared to conventional adjuvants, but also has much reduced toxicity, and is therefore very suitable for the vaccine composition of the present invention. As demonstrated in the examples below, the deacylation ratio of the present invention is more than monophosphoryl lipid A (MPL) obtained through phosphorylation of Leaf A obtained by removing the polysaccharide chain of LPS in order to eliminate the toxicity of LPS. Toxicity LOS is much less toxic.

In addition, as shown in the following examples (see Figs. 12A and 12B), when the deacylated non-toxic LOS of the present invention is administered together with HPV L1 VLP, not only IgG2a related to Th1 type immune response (cellular immunity) but also Th2 type The level of IgG1 related to the immune response (humoral immunity) was also increased, indicating that the deacylated non-toxic LOS induces a synergistic immune response together with HPV L1 VLP.

The vaccine composition of the present invention can exert an effect of preventing cervical cancer by inducing a sufficient immune response only with its basic composition, that is, HPV 16 L1 VLP, HPV 18 L1 VLP, or a combination thereof and deacylated non-toxic LOS. Optionally, the vaccine composition of the present invention may additionally contain other adjuvants, for example, with elements of Group 2 selected from the group consisting of Mg, Ca, Sr, Ba and Ra, Ti, Zr, Hf and Rf. It may include a Group 4 element selected from the group consisting of or a salt of aluminum or a hydrate thereof. The salts are preferably formed with oxides, peroxides, hydroxides, carbonates, phosphates, pyrophosphates, hydrogenphosphates, dihydrogenphosphates, sulfates or silicates. For example, adjuvants that can be additionally used in the vaccine composition of the present invention include magnesium hydroxide, magnesium carbonate hydroxide pentahydrate, titadium didoxide, calcium carbonate, barium oxide, barium hydroxide, Barium peroxide, barium sulfate, calcium sulfate, calcium pyrophosphate, magnesium carbonate, magnesium oxide, aluminum hydroxide, aluminum phosphate and hydrated aluminum potassium sulfate (Alum). Most preferably, the adjuvant that can additionally be used in the vaccine composition of the present invention is aluminum hydroxide.

According to a preferred embodiment of the present invention, the HPV type 16 or 18 L1 VLP comprises the steps of: (i) culturing yeast containing the L1-coding nucleotide sequence of HPV type 16 or 18; (Ii) lysis of the cultured yeast; (Iii) removing impurities by performing ammonium sulfate precipitation on the yeast lysate; (Iv) It is obtained through a purification process including performing heparin chromatography or cation-exchange chromatography on the resultant product from which the impurities have been removed.

A detailed description of the above purification process is provided below.

Pharmaceutically acceptable carriers included in the vaccine pharmaceutical composition of the present invention are commonly used at the time of formulation, and are lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginate, gelatin. , Calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, etc. It is not limited. The pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like in addition to the above components. Suitable pharmaceutically acceptable carriers and formulations are Remington's Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention can be administered orally or parenterally, preferably parenteral, and in the case of parenteral administration, it can be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, etc. have.

A suitable dosage of the pharmaceutical composition of the present invention is formulated in various ways depending on factors such as the formulation method, the mode of administration, the patient's age, weight, sex, pathological condition, food, administration time, route of administration, excretion rate and response sensitivity. Can be. Meanwhile, the oral dosage of the pharmaceutical composition of the present invention is preferably 0.0001-1000 mg/kg (body weight) per day.

The pharmaceutical composition of the present invention is prepared in unit dosage form by formulating using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily carried out by a person having ordinary knowledge in the art. Or it can be prepared by placing it in a multi-dose container. In this case, the formulation may be in the form of a solution, suspension or emulsion in an oil or aqueous medium, or may be in the form of an extract, powder, granule, tablet or capsule, and may additionally include a dispersant or a stabilizer.

The cervical cancer vaccine composition of the present invention is a conventional cervical cancer vaccine Cervarix ^TM and Gardasil ^TM It induces a greater immune response to HPV, so that it is not only more effective in preventing cervical cancer, but also has little toxicity from deacylated non-toxic LOS, which is used as an adjuvant, so it is very excellent in terms of safety.

In particular, the cervical cancer vaccine composition of the present invention, as demonstrated in the following examples, when administered the vaccine composition of the present invention, the level of interferon-γ, IgG2a and IgG2b related to Th1 type immune response (cellular immunity) The Cervarix ^TM and Gardasil ^TM Higher, and the level of IgG1 related to Th2 type immune response (humoral immunity) was also Cervarix ^{TM and Gardasil TM} Higher than ^{That is, the cervical cancer vaccine composition of the present invention is Cervarix TM} and Gardasil ^TM in both Th1 type immune response (cellular immunity) and Th2 type immune response (humoral immunity) in immunity against HPV. Better than

According to another aspect of the present invention, the present invention provides a method for preparing human papilloma virus (HPV) L1 virus-like particles (VLP) comprising the following steps:

(i) culturing yeast containing the HPV L1-encoding nucleotide sequence;

(Ii) lysis of the cultured yeast;

(Iii) removing impurities by performing ammonium sulfate precipitation on the yeast lysate; And

(Iv) performing heparin chromatography or cation-exchange chromatography on the resultant product from which the impurities have been removed.

The present inventors studied to develop a purification method capable of simplifying the production process of HPV VLPs and increasing the yield in the production process using the yeast expression system of HPV VLPs for the manufacture of vaccines against HPV. As a result, when the yeast cell lysate is precipitated with ammonium sulfate, and heparin chromatography or cation-exchange chromatography is performed, the purification efficiency (yield and degree of purification) is greatly increased. It was confirmed that it can be improved.

Hereinafter, the present invention is described in detail according to each step:

(i) Transgenic yeast culture expressing HPV L1 protein

In the method of the present invention, the type of HPV from which the L1 protein is derived is not particularly limited, and HPV type 6a, HPV type 6b, HPV type 11, HPV type 16, HPV type 18, HPV type 31, HPV type 33, HPV type 35, HPV type 39, HPV type 45, HPV type 51, HPV type 52, HPV type 56, HPV type 58 and HPV type 68, but are not limited thereto. Preferably, the L1 protein of the present invention is derived from HPV selected from the group consisting of HPV type 6a, HPV type 6b, HPV type 11, HPV type 16, HPV type 18, HPV type 31, HPV type 33 and HPV type 45. And more preferably derived from HPV type 16 and type 18.

Cells used as host cells in the present invention are yeast, for example, baker's yeast, Saccharomyces cerevisiae ( Saccharomyces cerevisiae ), Saccharomyces pastorianus ), Saccharomyces sp., Schizosaccharomyces pombe ), etc. can be used, but is not limited thereto. Most preferably, the host yeast of the present invention is Saccharomyces cerevisiae .

The transformed yeast expressing the HPV L1 protein refers to a yeast cell transformed with an expression vector that successfully expresses the HPV L1 protein. Various promoters known in the art, such as GALl promoter, GALl0 promoter (Johnson, M., and Davies, RW, Mol. and Cell. Biol., 4: 1440-), are known in the art upstream of the HPV L1 gene to be expressed in the expression vector. 1448 (1984)), ADH2 promoter (Russell, D., et al., J. Biol. Chem., 258: 2674-2682, (1983)), PH05 (EMBO J. 6: 675-680, (1982)) )), a PH05 promoter (EMBO J. 6: 675-680, (1982)) or an MFal promoter may be bound, and a polyadenylation sequence such as ADHI, MFal or TPI-derived polyA sequence (Alber, T. and Kawasaki) , G., J. Mol. & Appl. Genet. 1: 419-434 (1982)). YEp6, YEpl3, YEp4, and YEGα as yeast expression vectors (SN Kim, et al., J. Virol. Methods 139 (2007) 24-30; R. Kirnbauer, et al., J Virol 67 (1993) 6929-6936 ) Is known. Transformed yeast expressing HPV L1 protein can be easily prepared using methods known in the art, and such methods are described in US Pat. Nos. US 7250170, US 6613557, US 5888516, US 5871998, US 5618536, US5437951, etc., the contents of these patent documents are incorporated herein by reference.

According to a preferred embodiment of the present invention, the transformed yeast is cultured in a medium to which at least one carbon source of glucose and galactose is added as a carbon source. According to another preferred embodiment of the present invention, the ratio of glucose and galactose is glucose: galactose = 0-1: 3-4 by weight, more preferably glucose: galactose = 0.5-1: 3.5 by weight. -4, most preferably glucose:galactose=1:3. In the method of the present invention, when the weight ratio of glucose and galactose contained in the medium is within the above range, the expression of the HPV L1 protein is maximized.

An exemplary medium used in the cultivation of the transformed yeast is YPDG medium (including yeast extract, peptone, glucose and galactose).

(Ii) dissolution of cultured yeast

The method for lysis of the yeast cells cultured in the present invention may be any method capable of obtaining a total lysate of yeast cells, and is not limited to a specific method. Dissolution methods that can be used in the present invention include, for example, sonication and crushing by glass beads, but are not limited thereto.

(Iii) Precipitation of ammonium sulfate to remove impurities from yeast melts

The expressed protein was precipitated by adding ammonium sulfate to the yeast lysate and impurities were removed.

According to a preferred embodiment of the present invention, the concentration of ammonium sulfate added is 20-60% by weight, more preferably 40-50% by weight, and most preferably 42-48% by weight. When the concentration of ammonium sulfate is less than 40% by weight, the precipitation efficiency of the HPV L1 protein expressed in the present invention decreases, and when the concentration of ammonium sulfate is more than 50% by weight, the effect of increasing L1 protein precipitation as much as the increase in ammonium sulfate cannot be obtained.

Optionally, after dissolving the protein pellet formed by ammonium sulfate precipitation, 0.5 M or more of NaCl is added and incubated at 4° C. for at least 12 hours or more.

(Iii-2) Removal of impurities in low-concentration salts (eg, NaCl)

The solution obtained in step (iii) is dialyzed and exchanged in a buffer solution (eg, phosphate buffer) containing a low-concentration salt (eg, NaCl), and then incubated at room temperature to make impurities insoluble and removed.

In one embodiment of the present invention, the concentration of the low-concentration salt is 0.001-0.3 M, more preferably 0.01-0.2 M, most preferably 0.1-0.8 M, and the salts used are NaCl, KCl, MgCl ₂ , CaCl ₂ , Na ₂ SO ₄ and (NH ₄ ) ₂ SO _{4 It} is selected from the group consisting of, most preferably NaCl.

In a preferred embodiment of the present invention, the low concentration salt treatment process is carried out in the presence of a nonionic surfactant, and the nonionic surfactant is, for example, polyoxyalkylene sorbitan fatty acid ester, sorbitan fatty acid ester, alkylene. Glycol fatty acid esters, polyoxyalkylene fatty acid esters, fatty acid esters, polyoxyalkylene fatty acid ethers, C _{16 -24} fatty acids, fatty acid mono-, di- or poly-glycerides, polyoxyalkylene alkyl phenols, alkyl phenyl ethers, poly Oxyethylene polyoxypropylene block copolymer, fatty amine oxide, fatty acid alkanolamide, alkyl cellulose, carboxyalkyl cellulose or polyoxyalkylene castor oil derivative, preferably polyoxyethylene (20) sorbitan monolau Rate (twin 20), polyoxyethylene (4) sorbitan monolaurate (twin 21), polyoxyethylene (20) sorbitan monopalmitate (twin 40), polyoxyethylene (20) sorbitan monostearate ( Tween 60), polyoxyethylene (20) sorbitan tristearate (twin 65), polyoxyethylene (20) sorbitan monooleate (twin 80 or polysorbate 80) or polyoxyethylene (20) sorbitan tree Polyoxyethylene sorbitan mono- or tri-lauryl, palmityl, stearyl or oleyl esters such as oleate (Tween 85). In addition, the non-ionic surfactant is preferably in a concentration of 0.001-0.01%.

This low-concentration NaCl treatment process is very efficient in removing impurities, and is a step that greatly contributes to shortening the process and improving the purification efficiency in the method of the present invention.

When the insoluble protein produced in this step is removed by centrifugation, the purity of the VLP eluted in the next step, heparin chromatography or cation-exchange chromatography, can be greatly increased. Therefore, this step shows the effect of greatly increasing the purification efficiency by removing impurities by a simple method.

(Iv) heparin chromatography or cation exchange chromatography

The solution from which the impurities have been removed is subjected to heparin chromatography or cation exchange chromatography.

When the solution from which impurities are removed through the above method is purified through heparin chromatography or cation exchange chromatography, residual impurities can be very effectively removed.

When performing heparin chromatography, first, before applying the result of step (iii) or (iii-2) to a heparin resin column, the heparin resin is equilibrated with a suitable binding buffer. The binding buffer preferably contains NaCl and a non-ionic surfactant (eg Tweeen 80), and the NaCl is preferably a low concentration (0.1-0.2 M). Then, after applying the sample to heparin chromatography, the protein bound to the resin is eluted. The elution method is preferably a NaCl linear concentration gradient, more preferably, an impurity is eluted in a 0.33-0.66 M NaCl linear concentration gradient, and then the desired L1 protein is eluted in a 0.66-2 M NaCl linear concentration gradient.

Cation exchange chromatography can be performed using a variety of resins, preferably sulfo, sulfoalkyl (e.g., sulfomethyl, sulfoethyl and sulfopropyl), phosphate or phosphate alkyl functional group bound cation exchanger. And, most preferably, it is carried out using a cation exchanger to which a phosphate functional group is bonded. When performing cation exchange chromatography, first, before applying the result of step (iii) or (iii-2) to a heparin resin column, the heparin resin is equilibrated with a suitable binding buffer. The binding buffer preferably contains NaCl and a non-ionic surfactant (eg, Tweeen 80), and a low concentration of NaCl (0.3-0.4 M) is preferred. Then, the sample is applied to cation exchange chromatography, and then the protein bound to the resin is eluted. The elution method is preferably a NaCl step concentration gradient, and more preferably, the desired L1 protein is eluted using an elution buffer containing 0.6 M, 0.7 M, 0.8 M and 1 M NaCl.

(v) concentration

According to a preferred embodiment of the present invention, the L1 protein fraction obtained through the chromatography process is concentrated with a membrane filter. Preferably, the chromatographic fraction is concentrated using a membrane capable of cut-off molecular weight of 50-100 kDa. VLPs having an average size of about 50 nm have a larger size than other proteins, so they cannot pass through the membrane and are concentrated, whereas most of the remaining impurities are removed through the membrane. Therefore, this step further increases the purity of HPV 16 L1 and shows the effect of increasing the concentration.

The production method of the present invention maximizes the purification efficiency of the L1 protein by using a method of removing impurities by treating the cell lysate before the chromatography step under specific conditions. The cell lysate is precipitated by adding ammonium sulfate to first remove impurities, and secondly, incubation under low-concentration salt conditions to remove impurities. As in the above case, when the impurities are removed by incubation under low-concentration salt conditions after precipitation of ammonium sulfate, 80% of the impurities can be removed, and more than 80% of the L1 protein can be recovered, so the purity can be increased with a simple method. Therefore, it is possible to purify high-purity L1 protein without going through several chromatographic steps, and if it is applied to production and purification on a pilot and industrial scale, the time required for protein production, manufacturing cost, and labor can be greatly reduced. have.

According to another aspect of the present invention, the present invention provides a nucleic acid sequence of human papilloma virus (HPV) type 16 L1 virus-like particles (VLP) comprising the nucleotide sequence of SEQ ID NO: 1. In addition, the present invention provides a nucleic acid sequence of human papilloma virus (HPV) type 18 L1 virus-like particles (VLP) comprising the nucleotide sequence of SEQ ID NO: 3.

According to another aspect of the present invention, the present invention is Saccharomyces cerevisiae transformed with the nucleic acid sequence of the Roman virus (HPV) type 16 L1 virus-like particle (VLP) (accession number: KCCM11036P) Provides. In addition, the present invention provides Saccharomyces cerevisiae (accession number: KCCM11037P) transformed with the nucleic acid sequence of the human papilloma virus (HPV) type 18 L1 virus-like particle (VLP).

The nucleotide sequence of Sequence Listing 1 and Sequence Listing 3 is optimized so that the L1 protein-encoding nucleic acid sequence of HPV can be expressed with high efficiency in yeast.

The features and advantages of the present invention are summarized as follows:

(a) The cervical cancer vaccine composition of the present invention uses HPV 16 L1 VLP, HPV 18 L1 VLP or HPV 16 L1 VLP and HPV 18 L1 VLP as an antigen and deacylated non-toxic LOS as an adjuvant.

(b) Deacylated non-toxic LOS, an adjuvant, not only exhibits very good immunity promoting effects in the immune response to HPV, but also has very little toxicity, so its safety is very excellent.

(c) In addition, deacylated non-toxic LOS induces a synergistic immune response against HPV by increasing both Th1 type immune response (cellular immunity) and Th2 type immune response in the immune response induced by HPV L1 VLP. .

^{(d) The cervical cancer vaccine composition of the present invention is Cervarix TM} and Gardasil ^TM in both Th1 type immune response (cellular immunity) and Th2 type immune response (humoral immunity) in immunity against HPV. It is more excellent and exhibits excellent efficacy as a cervical cancer vaccine.

(e) In the method for preparing HPV L1 VLP of the present invention, high purity L1 protein can be obtained through a single chromatography step, and if this is applied to production and purification on a pilot and industrial scale, the time required for protein production, There is an effect that can greatly reduce manufacturing cost and labor.

1A-1B are genetic maps for YEG (yeast expression vector) expressing HPV type 16 and 18 L1 VLP proteins. AmpR is an ampicillin resistance gene, AmpR is an ampicillin resistance gene, Ura3 is an orotidine 5-phosphate decarboxylase synthesis gene, 2-micron is an origin of replication, and the rest of the symbols indicate restriction enzyme sites.
1C is a schematic diagram of a purification process of HPV 16 L1 and HPV 18 L1. After precipitation of ammonium sulfate, a sample from which the protein changed to insoluble in a low concentration of NaCl was removed was purified through two types of methods. Heparin chromatography (method 1) or cation-exchange chromatography (method 2) was used in the chromatography method of the present invention.
2A-2C are results of purification of HPV 16 L1. 2A is a result of SDS-PAGE after heparin chromatography is performed without undergoing a process of removing insoluble impurities. FIG. 2B is an SDS-PAGE result of heparin chromatography in Method 1 of FIG. 1C. Figure 2c is a Western blotting result of the first heparin chromatography. Lanes 1 and 2 are the loading and unbound samples, and lanes 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 and 16 are fractions 9, 12, 15 and 18, respectively. , 21, 24, 27, 30, 33, 36, 39, 42, 45 and 48. Arrows indicate the molecular weight of the L1 protein.
3A-3B are results of purification of HPV 16 L1. 3A is an SDS-PAGE result of cation-exchange chromatography in Method 2 of FIG. 1C. 3B is a result of Western blotting of cation-exchange chromatography. Lanes 1 and 2 are the loading and unbound samples, and lane 3 is the wash. Lanes 4, 5, 6, 7, 8 and 9 represent fractions 1, 2, 3, 4, 5, 6. Fractions 1, 2, and 3 were eluted with a buffer containing 0.6, 0.7, and 0.8 M NaCl, respectively, and fractions 4, 5, and 6 were eluted with a buffer containing 1 M NaCl. Arrows indicate the molecular weight of the L1 protein.
4 is a result of collecting the L1 fractions of heparin chromatography and cation-exchange chromatography as a result of purification of HPV 16 L1 and then concentrating using a membrane filter capable of cutting-off 50-100 kDa. Panel A is the SDS-PAGE result and B is the Western blotting result. Lanes 1, 2, and 3 represent BSA, HPV 16 L1 purified by method 1, and HPV 16 L1 purified by method 2, respectively. Each sample was quantified by bradford assay, and the same amount of 200 ng was loaded.
5A and 5B are electron micrographs of HPV 16 L1 protein purified by Method 1 and Method 2 of FIG. 1C, respectively. The purified HPV 16 L1 protein was adsorbed on a carbon-coated copper grid, stained with phosphotungstic acid, and examined with a transmission electron microscope. The magnification is 41,000 X, and the bar represents 100 nm.
6A-6C are results of purification of HPV 18 L1, and FIG. 6A shows SDS-PAGE results after heparin chromatography is performed without removing insoluble impurities. 6B is an SDS-PAGE result of heparin chromatography in Method 1 of FIG. 1C. 6C is a Western blotting result of heparin chromatography. Lanes 1 and 2 are loading and unbound samples, and lanes 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 are fractions 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48 are shown. Arrows indicate the molecular weight of the L1 protein.
7A-7B are results of purification of HPV 18 L1. 7A is an SDS-PAGE result of cation-exchange chromatography in Method 2 of FIG. 1C. 7B is a result of Western blotting of cation-exchange chromatography. Lanes 1 and 2 are the loading and unbound samples, and lane 3 is the wash. Lanes 4, 5, 6, 7, 8 and 9 represent fractions 1, 2, 3, 4, 5, 6. Fractions 1, 2, and 3 were eluted with a buffer containing 0.6, 0.7, and 0.8 M NaCl, respectively, and fractions 4, 5, and 6 were eluted with a buffer containing 1 M NaCl. Arrows indicate the molecular weight of the L1 protein.
8 is a result of collecting the L1 fraction of heparin chromatography and cation-exchange chromatography as a result of purification of HPV 18 L1, and then concentrating it using a membrane filter capable of cutting-off 50-100 kDa. Panel A is the result of SDS-PAGE and panel B is the result of Western blotting. Lanes 1, 2, and 3 represent BSA, HPV 18 L1 purified by method 1, and HPV 18 L1 purified by method 2, respectively. Each sample was quantified by bradford assay, and the same amount of 200 ng was loaded.
9A and 9B are electron micrographs of HPV 18 L1 protein purified by Method 1 and Method 2 of FIG. 1C, respectively. The purified HPV 18 L1 protein was adsorbed on a carbon-coated copper grid, stained with phosphotungstic acid, and examined with a transmission electron microscope. The magnification is 41,000 X, and the bar represents 100 nm.
10A and 10B are results of measuring neutralization activities of HPV 16 and 18 L1 purified by heparin chromatography and cation-exchange chromatography, respectively. Each purified L1 protein was immunized to mice three times at intervals of three weeks, and the neutralizing antibody titer in the serum was measured by a SEAP-based neutralizing antibody activity assay. Control, heparin, and cation-exchange of the X-axis are the groups immunized only with adjuvants, L1 purified by heparin chromatography and the group immunized with adjuvants, L1 purified by cation-exchange chromatography and the group immunized with adjuvants, respectively. Show. The number on the Y-axis represents the dilution ratio of the serum.
11 is a graph showing that the novel adjuvant (CIA05) has lower cytotoxicity (TNF-α secretion) than MPL.
12A-12B are graphs showing the antibody titer (Fig. 12a) and the fold of change in the antibody titer (Fig. 12b) according to the concentration of novel immunological adjuvant CIA05, HPV 16 L1 VLP, and aluminum hydroxide (Alum). . Blank bars are for total IgG, oblique bars are for IgG1 and filled bars are for IgG2a.
12C is a result of analyzing the degree of interferon-γ secretion from splenocytes according to concentrations of novel immune adjuvants CIA05, HPV 16 L1 VLP, and aluminum hydroxide (Alum). The diagonal bars correspond to the HPV 16 L1 VLP treatment period for 2 days and the filled bars for 3 days.
13A-13B are results of analysis of serum total IgG titers according to concentrations of novel immunological adjuvant CIA05, HPV 16/18 L1 VLP and aluminum hydroxide (Alum), and FIG. 13A is HPV 16 L1 VLP and 13b is the result of analyzing the total IgG titer for HPV 18 L1 VLP.
Figures 14a-14b is a new immunological adjuvant CIA05, HPV 16/18 L1 VLP, and the concentration of aluminum hydroxide (Alum) according to the concentration of HPV 16 L1 VLP in serum IgG1 titer (Fig. 14a), IgG2a titer ( Figure 14b) is the result of analysis.
FIG. 15 is a result of analyzing the degree of interferon-γ secretion from splenocytes according to concentrations of novel immune adjuvants CIA05, HPV 16/18 L1 VLP, and aluminum hydroxide (Alum).

Hereinafter, the present invention will be described in more detail through examples. These examples are only for describing the present invention in more detail, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Example

Example 1: Human Papillomavirus Type 16 and 18 VLP L1 Protein Production and Purification

Construction of a recombinant yeast expression system producing L1 protein of HPV type 16 and 18

Yeast of the L1 DNA sequence of HPV types 16 and 18 ( Saccharomyces cerevisiae ), the HPV type 16 L1 DNA sequence of the sequence listing first sequence and the HPV type 18 L1 DNA sequence of the sequence listing sequence 3 were obtained, and the “Synthesis in BHB Standard Vectors” service (www .blueheronbio.com) was used to synthesize each DNA (see: Sequence Listing 1 and 3). At this time, Hind III (AAGCTT) and Cla I (ATCGAT) restriction enzyme sites were ligated before and after the optimized DNA sequence. Thereafter, through a cloning process, recombinant plasmid DNA capable of expressing the recombinant HPV16L1 and HPV18L1 proteins to be used in the present invention was constructed using a yeast expression vector (YEG) to obtain YEGα-HPV16L1-ORS and YEGα-HPV18L1-ORS (Fig. 1a). The cloning process is as follows: pUCminusMCS vector (Invitrogen) into which the HPV type 16 L1 DNA sequence of SEQ ID NO: 1 or HPV type 18 L1 DNA sequence of SEQ ID NO: 3 is inserted, and YEG-MCS vector, which is a yeast expression vector. It was cut with Hind III and Cla I. After each of the cut DNA was subjected to electrophoresis, it was extracted using a gel extraction kit (HiYield ^TM Gel/PCR DNA Extraction Kit (#YDF100, RBC)). The DNA of HPV types 16 and 18 and the YEG-MCS vector to be used as a vector were conjugated using a ligase (DAKARA, 2011a), and then transformed into dam(-) E.coli competent cells (TAKARA, 9129). Colonies formed after transformation were counted and cultured, and then the plasmid was extracted and digested with an enzyme to confirm the size. The plasmid of which the size was confirmed was transformed into yeast (Saccharomyces cerevisiae ) and spread on an SD(-ura) plate. When colonies were generated, 25 colonies were picked and inoculated. (280 rpm, 30°C, shaking, 2 days). Only those grown well in SD (-ura) medium were selected and inoculated on YPDG medium. When the culture was completed, the best strain was selected through ELISA and Westernblot analysis after OD value during culture, total protein amount during cell disruption, and purification (purified by the following method).

The strains transformed with YEGα-HPV16L1-ORS and YEGα-HPV18L1-ORS vectors into which recombinant HPV16L1 and HPV18L1 were introduced, respectively, were deposited with the Korean Microbiological Conservation Center on October 8, 2009, and the deposit numbers KCCM11036P and KCCM11037P respectively (KCCM11036P-Saccharomyces cerevisiae EG0216; KCCM11037P-Saccharomyces cerevisiae EG0218).

Cell culture

The yeast strain cells prepared above were inoculated into SD-ura (Clontech), a synthetic complete medium without uracil, in a flask with a baffle attached, and cultured with shaking at 30°C. To express the HPV16 L1 and HPV18 L1 proteins from the GAL10 promoter, YPDG medium was used. 1% yeast extract (DIFCO Laboratories, USA) and 2% peptone (DIFCO Laboratories, USA) were added to all media, and 1% glucose and 3% galactose were included. After inoculating the transformed yeast strain with the plasmid in 3 L of YPDG medium, shaking culture was performed at 30°C for 48 hours.

Preparation of Cell Lysate

The cultured cells were obtained as a pellet, and the obtained cell pellet was frozen at 70°C. All subsequent procedures were carried out at 4°C. The cell pellet was dissolved and resuspended in ice-cooled 100 ml break buffer (20 mM sodium phosphate, pH 7.2, 100 mM NaCl, 1.7 mM EDTA, 0.01% Tween 80), and protease inhibitor cocktail tablet (Roche, USA) was added. Cells were transferred to a chamber of a bead-beater (Biospec Products, USA) with glass beads (Sigma, USA), and after vortexing for 5 minutes, lysates were transferred at 10000 g for 10 minutes at 4°C. Clearly removed by centrifugation several times.

Ammonium sulfate precipitation

HPV L1 protein was recovered by adding ammonium sulfate (Sigma, USA) to the cleared cell lysate. In order to evaluate the recovery efficiency, ammonium sulfate was added at a concentration of 43% or 45%, and then stirred at 4° C. for 30 minutes, and centrifuged at 12,000 g for 10 minutes to obtain a protein pellet. The pellet precipitated with ammonium sulfate was resuspended in PBS + 0.01% Tween 80 buffer, and NaCl was additionally added to make the final NaCl concentration greater than or equal to 0.5 M. Thereafter, incubation was performed at 4° C. for at least 12 hours or longer.

Removal of impurities through low-concentration NaCl incubation

The solution incubated with high concentration NaCl was dialyzed with PBS + 0.01% Tween 80 buffer, and then diluted with a solution containing 0.15 M NaCl and 0.01% Tween 80 in sodium phosphate pH 7.2 buffer, and the final protein concentration was 2-5 mg/ ml. This solution was allowed to stand at room temperature for 2 hours or longer to produce a protein in a non-soluble state. The resulting insoluble protein was removed by centrifugation at 10000 g for 10 minutes.

Heparin chromatography

After centrifuging the sample incubated with the low concentration NaCl, the supernatant was dialyzed with a binding buffer (PBS + 0.2 M NaCl pH 7.0, 0.01% Tween 80). A column filled with heparin resin (5 ml or 20 ml) (GE healthcare, USA) was also equilibrated by flowing a binding buffer 5 times the volume of the resin. The dialysis-completed solution was combined by passing through a heparin column, followed by washing by flowing a binding buffer at 5 times the volume of the resin. In the elution method, impurities were eluted with a linear concentration gradient so that the NaCl concentration rose from 0.33 M to 0.66 M for 35 minutes, and then the L1 protein was eluted by giving a linear concentration gradient from 0.66 M to 2 M NaCl.

Cation-exchange chromatography

After centrifuging the sample incubated with the low concentration NaCl, the supernatant was dialyzed against a binding buffer (PBS + 0.37 M NaCl pH 7.2, 0.01% Tween 80). An 8 cm X 4 cm Poly-Prep column (Bio-Rad Lab., USA) filled with P-11 cellulose phosphate resin (Whatman, UK) maintained equilibrium by flowing the binding buffer at 5 times the volume of the resin. . The dialysis-completed solution was combined by passing through a P-11 column, followed by washing by flowing a binding buffer at 5 times the volume of the resin. After washing, 4-5 ml of elution buffer containing 0.6 M, 0.7 M, 0.8 M, and 1 M NaCl was poured into the binding buffer to elute the L1 protein.

VLP concentration using a membrane filter

After collecting fractions of HPV 16 L1 or HPV 18 L1 obtained by the heparin chromatography or cation-exchange chromatography process, PBS containing 0.1-0.325 M NaCl + 0.01% Tween 80 Dialysis was performed. The dialysis-completed sample was concentrated using amicon ultra YM-50 or YM-100 (Millipore, USA) according to the method provided by the manufacturer.

SDS - PAGE And Western Blotting

All samples were analyzed by 12.5% PAGE in the presence of sodium dodecyl sulfate according to Laemmli's method (MP McCarthy, WI White, F. Palmer-Hill, S. Koenig, JA Suzich, J Virol 72 (1998) 32 -41). Samples containing HPV 16 L1 or HPV 18 L1 were transferred to a PVDF membrane (Q-Biogene, USA) at 200 mA for 120 minutes, and the L1 protein as a primary antibody was rabbit anti-HPV 16 L1 or anti-HPV 18 L1 antibody. (PIERCE, USA), and detected using a goat anti-rabbit IgG-HRP conjugate (PIERCE, USA) as a secondary antibody. Proteins were visualized using Western Blotting Luminol Reagent (Santa Cruz Biotechnology, USA).

Electron microscope observation

The purified HPV L1 protein was adsorbed onto a carbon-coated grid, followed by negative staining with 2% phosphotungstic acid. Transmission electron micrographs were taken using TEM200CX at a final magnification of 41,000 X (SN Kim, et al., J. Virol. Methods 139 (2007) 24-30).

Pseudovirus making

293TT cells such as p16SheLL (National Cancer Institute distribution) and p18SheLL (National Cancer Institute distribution), which are plasmids expressing the L1 protein and L2 protein of HPV types 16 and 18, are plasmids containing the reporter gene, pYSEAP (National Cancer Institute). Each was co-transformed using Lipofectamine 2000 (Invitrogen, USA) in (distribution of National Cancer Institute). Transformed 293TT cells were cultured for 72 hours and then disrupted using a disruption buffer (PBS + 0.5% Brij58, 0.2% Benzonase, 0.2% plasmid-safe ATP-dependent Dnase). The cell debris of the disrupted cells was removed by centrifugation at 12000 g for 10 minutes, and the supernatant was used for the measurement of neutralizing antibody.

Mouse immunity

6-week-old magnetic Balb/c mice were immunized subcutaneously with 2 μg of L1 protein, respectively purified by heparin chromatography and cation-exchange chromatography, three times every three weeks. The first immunization was performed using Freund's complete adjuvant (Sigma), and the first and second boosting was performed using Freund's incomplete adjuvant (Sigma). The control group was administered by diluting the same ratio of PBS to the complete adjuvant and the incomplete adjuvant. For each group of mice, serum was collected through the tail vein 10 days after the second boosting, and the obtained serum was used for the measurement of neutralizing antibodies.

SEAP - based Neutralizing antibody is measured

293TT cells were seeded in a 96-well plate at 3 x 10 ⁴ cells/well and incubated in a 37°C, 5% CO ₂ incubator for 4 hours. Mouse serum immunized with HPV type 16 and 18 L1 protein was serially diluted from 1/100 to 10,000,000, and then diluted with HPV type 16 and 18 pseudoviruses prepared as described above at a ratio of 1:4. The diluted mixture was reacted at 4° C. for 1 hour, added to 293TT cells prepared in a 96-well plate, and cultured at _{37° C. and 5% CO 2 for 72 hours.} After 72 hours of culture, the expression level of SEAP in the culture medium was confirmed using p-nitrophyl phosphate disodium salt hexahydrate (Sigma, USA) as a substrate. The neutralizing antibody titer in mouse serum was determined as half of the OD value of the well to which only pseudovirus was applied.

Experiment result

One. HPV 16 L1 Refining results

Heparin chromatography ( heparin chromatography )

The resolution of HPV 16 L1 protein was investigated in heparin chromatography. This result is a heparin chromatography result of Method 1 in FIG. 1c. As shown in FIG. 2A, in the case of a sample that did not undergo the step of removing insoluble impurities by incubating in a low concentration NaCl condition, a large amount of impurities were included during heparin chromatography. On the other hand, in the sample from which the insoluble impurities were removed under the low concentration of NaCl, the purity of the L1 protein was greatly improved during heparin chromatography (FIG. 2b). The protein bound to the heparin column was first eluted with a gradient gradient from 0.325 M to 0.66 M NaCl and then second eluted with a concentration gradient from 0.66 M to 2 M NaCl. As a result, impurities are mostly eluted at a concentration of 0.325 M to 0.5 M NaCl. Was (Fig. 2b), HPV 16 L1 was eluted at a concentration of 0.5 M to 1.3 M NaCl (Fig. 2c). This result indicates that removal of insoluble impurities under low NaCl conditions significantly increases the purity of L1 protein in heparin chromatography.When a sample from which insoluble impurities are removed is applied to heparin chromatography, L1 protein can be effectively purified. Show that there is.

Cation-exchange chromatography ( cation - exchange chromatography )

The resolution of HPV 16 L1 protein was investigated through cation-exchange chromatography. This result is a result of cation-exchange chromatography of Method 2 in FIG. 1c. As shown in FIG. 3, most of the impurities were removed by attaching to the column (FIG. 3a), and when eluting with NaCl, HPV 16 L1 was eluted at a concentration of 0.7 M to 1 M NaCl (FIG. 3b). This result shows that high purity L1 can be obtained through cation-exchange chromatography when insoluble protein is removed under low concentration of NaCl.

Refined HPV 16 L1 Purity of

Fractions of the L1 protein purified above were collected and concentrated with a membrane capable of cut-off 50-100 kDa (YM-50 or YM-100). As shown in FIG. 4, it was confirmed that the purity of the L1 protein was similar to that of the commercially available BSA standard (purity of 96% or more) (FIG. 4 panel A). As a result of Western blocking, a 55 kDa monomer observed on SDS-PAGE was detected, and a band of BSA did not appear (Panel B of FIG. 4). This result shows that high purity HPV 16 L1 was purified through the above purification process.

Refined HPV 16 L1 Protein self-assembly ( self - assembly )

As shown in FIGS. 5A and 5B, the HPV 16 L1 protein purified by the above two processes is magnetic to VLPs having a diameter (average diameter of 49 nm) of 35 nm to 64 nm, which is the same size as that of HPV VLPs. Was assembled (KA Aires, et al., Appl. Environ. 368 Microbiol. 72 (2006) 745-752). Therefore, the above two purification methods had no effect on the self-assembly of the L1 protein, which means that both methods are suitable for purification of the HPV 16 L1 protein.

As a result of measuring the step-by-step recovery rate through sandwich ELISA, an average of 80% of impurities were removed from cell disruption to ammonium sulfate precipitation and the step of removing the insoluble protein under low-concentration NaCl conditions. Up to this step, the average recovery rate of L1 protein was 81%. (Table 1, Table 2). The purity of the L1 protein was 0.2% in the cell disruption step, but after the ammonium sulfate precipitation and the step of removing insoluble impurities, the purity was improved to 0.8-1%, resulting in a 4-5 fold increase in purity (Tables 1 and 2).

step Total protein
(mg) Total L1
(mg) Recovery rate
(%) Refinement
(%) Cell lysate 3199.7 7.9 100 0.2 Ammonium sulfate precipitation 1914 7.2 92 0.4 Insoluble protein removal 642.1 6.7 84 One Heparin chromatography 6 6.6 83 109 Concentration using membrane filtration 5 5 64 100

step Total protein
(mg) Total L1
(mg) Recovery rate
(%) Refinement
(%) Cell lysate 3199.7 7.9 100 0.2 Ammonium sulfate precipitation 1914 7.2 92 0.4 Insoluble protein removal 713.5 5.7 72 0.8 Cation-exchange chromatography 5.1 4.9 62 96 Concentration using membrane filtration 4.8 4.9 62 101

Meanwhile, the recovery rate of the L1 protein purified through heparin chromatography was 64% (Table 1), and the total recovery rate of the L1 protein purified through cation-exchange chromatography was 62% (Table 2). The above recovery rates of 64% and 62% are the highest values reported so far.

2. HPV 18 L1 Refining results

Heparin Chromatography ( heparin chromatography )

The resolution of the HPV 18 L1 protein was investigated by heparin chromatography. This result is a heparin chromatography result of Method 1 in FIG. 1c. As shown in FIG. 6A, it can be seen that in the case of a sample that has not been incubated in a low concentration NaCl condition to remove insoluble impurities, a large amount of impurities are included during heparin chromatography. On the other hand, it can be seen that the purity of the L1 protein was greatly improved in the sample from which the impurities changed to the insoluble state were removed under the low concentration of NaCl condition (FIG. 6B). Proteins attached to the heparin column were first eluted with a gradient gradient from 0.325 M to 0.66 M NaCl and then second eluted with a gradient gradient from 0.66 M to 2 M NaCl. As a result, most impurities were found at a concentration of 0.325 M to 0.5 M NaCl. It was eluted (FIG. 6b) and HPV 18 L1 was eluted at a concentration of 1.2 M to 1.3 M NaCl (FIG. 6c ). This result shows that HPV 18 L1 can be effectively purified through heparin chromatography, similar to HPV 16 L1.

Cation-exchange chromatography ( cation - exchange chromatography )

The resolution of HPV 18 L1 protein was investigated through cation-exchange chromatography. This result is a cation-exchange chromatography result of Method 2 in FIG. 1c. As shown in FIG. 7a, most of the impurities were removed by binding to the column, and when eluting with NaCl, HPV 18 L1 was eluted at a concentration of 0.8 M to 1 M NaCl (FIG. 7b). This result shows that high purity L1 can be obtained through cation-exchange chromatography when the insoluble protein is removed under a low concentration of NaCl.

Refined HPV 18 L1 Purity of

Fractions of the L1 protein purified above were collected and concentrated with a membrane capable of cut-off 50-100 kDa (YM-50 or YM-100). As shown in FIG. 8, it was confirmed that impurities having a small molecular weight were removed after the concentration was completed, and it was confirmed that the purity of the L1 protein was similar to that of a commercially available BSA standard (purity of 96% or more) (FIG. 8 Panel A. ). The 55 kDa L1 protein shown on SDS-PAGE was detected in the same manner in the Western blocking results, and no band was detected in the control group BSA (FIG. 8, panel B). This result shows that high purity HPV L1 protein can be obtained through the above two purification methods.

Refined HPV 18 L1 Protein self-assembly ( self - assembly )

As shown in FIGS. 9A and 9B, the HPV 18 L1 protein purified by the above two processes is magnetic to VLPs having a diameter (average diameter of 49 nm) of 35 nm to 64 nm, which is the same size as that of HPV VLPs. Assembled (KA Aires, et al., Appl. Environ. 368 Microbiol. 72 (2006) 745-752). Accordingly, the L1 protein purified by the above two methods was not affected by VLP formation, which means that both methods are suitable for purification of HPV 18 L1 protein.

In conclusion, it was experimentally confirmed that HPV 16 L1 and HPV 18 L1 proteins can be purified with high purity and high recovery rate without going through methods such as sucrose cushion or size-exclusion chromatography that require a lot of time and complicated steps. .

3. Refined HPV Of types 16 and 18 L1 Protein Neutralization activity

Neutralizing antibody titers were measured by immunizing mice with HPV type 16 and type 18 L1 proteins purified by the above method. As a result, L1 of HPV type 16 purified by heparin chromatography and cation-exchange chromatography showed a neutralizing antibody titer up to an average of 1/18,000 dilution ratio (FIG. 10A). In the case of L1 of HPV type 18, L1 purified by heparin chromatography showed an average of 1/5,000, and L1 purified by cation-exchange chromatography showed a neutralizing antibody titer up to an average of 1/170,000 dilution ratio (FIG. 10b). This result shows that the HPV type 16 and 18 L1 protein purified by the above method can induce neutralizing antibodies at high levels.

Example 2: Very short as a novel immune adjuvant Lipopolysaccharide ( LPS ) CIA05 Production of

The present inventors have already discovered a strain (E. coli EG0021) having an LPS with a very short lipopolysaccharide sugar chain from E. coli living in the intestine of healthy humans, and this strain was transferred to the Korean Microbiological Conservation Center in 2002. It was deposited on the 2nd of January and was assigned the deposit number KCCM 10374 (Reference: Korean Patent Registration No. 0456681; WO 2004/039413; Korean Patent Registration No. 0740237; WO 2006/121232). Purification of LPS from the strain is obtained from Korean Patent Registration No. 0456681; WO 2004/039413; Republic of Korea Patent Registration No. 0740237; And according to the method disclosed in WO 2006/121232. The molecular weight of the purified LPS was measured with MALDI-MASS (Shimadz, Axima-LNR V 2.3.5 (Mode Liner, Power: 106)), and as a result of the measurement, it was confirmed that it has a molecular weight of about 3,500 Da. Korean Patent Registration No. 0456681 for purified LPS; WO 2004/039413; Republic of Korea Patent Registration No. 0740237; And detoxification according to the disclosed protocol according to the method disclosed in WO 2006/121232. Briefly, after mixing 3 mg/ml of 2 N NaOH in a 1:1 volume of the purified LPS, shaking once every 10 minutes at 60° C. for 140 minutes and deacylation, about 1/5 of the initial 0.2 N NaOH volume About 1N acetic acid was added and the pH was titrated to 7.0. After pH titration, ethanol precipitation was performed to obtain non-toxic lipopolysaccharide (CIA05).

Example 3: New immune adjuvant CIA05 And conventional adjuvants MPL Toxicity comparison experiment with

The toxicity of CIA05 prepared in the above example, which is used as an adjuvant in the cervical cancer vaccine of the present invention, and MPL (Monophosphoryl lipid A), used in the existing cervical cancer vaccine, were compared and analyzed. Healthy human blood was provided, PBMC (Peripheral Blood Mononuclear Cell) was isolated, and this was seeded in a ^{24-well culture dish at a concentration of 5 x 10 5 cells/ml.} At this time, the culture medium was a mixture of RPMI 1640 (Gibco) and 10% FBS (Gibco), and the volume per well was equalized to 1 ml. The prepared dishes were each treated under the following conditions: 1) Negative control: BSS (Balanced salt solution) 100 μl; 2) Deacylated non-toxic LOS (CIA05) 10 μg/100 μl; 3) MPL ( E. coli F583 MPL) 10 μg/100 μl.

After 12 hours, the treated culture was collected, centrifuged, and the amount of TNF-α, an inflammatory protein secreted from THP-1 (Acute monocytic leukemia), was measured using an ELISA kit (R&D system, DY210) using enzyme immunoassay (ELISA). Each was measured. As a result of the experiment, as shown in FIG. 11, it was confirmed that the toxicity of about 1/3 is less than that of MPL used as an adjuvant for a conventional cervical cancer vaccine.

Example 4: Exploring the efficacy of the cervical cancer vaccine of the present invention

Immunization with mice

The protein antigen purified through the above experiment and the new immunological supplement CIA05 were used to induce an immune response in mice. Six-week-old male Balb/c mice (SLC, Japan) were used, and 5 mice were randomly assigned per experimental group. In the mouse, CIA05 alone, Alum (aluminum hydroxide; Brenntag, Germany) alone or with a mixture of both, HPV16 L1 VLP 2 μg (Figs. 12A-12C), HPV16/18 L1 VLP 1 μg or 4 μg ( Each type of 0.5 μg or 2 μg) (FIGS. 13A-15) was immunized by intramuscular injection three times at intervals of 2 weeks. Control mice were administered PBS. For comparative experiments, conventional commercial products Cervarix ^TM and Gardasil ^TM were administered to mice at 1/10 doses based on humans. Serum of mice was collected 5 weeks after the third inoculation, and anti-L1 VLP IgG titer was measured by the endpoint ELISA method.

Endpoint ELISA Analysis of immune efficacy using IgG Titer analysis)

A 96-well ELISA microplate (Nalge Nunc International) by diluting the HPV type 16 or 18 VLP L1 antigen prepared in the above example to a concentration of 0.5 μg/ml in 100 μl of a coating buffer (50 mM carbonate, pH 9.6, 4° C.). , USA) and then coated overnight at 4°C. The next day, each well was washed three times using a washing buffer (PBS in which 0.05% Tween20 was dissolved), and 300 μl of blocking buffer (PBS in which 1% BSA was dissolved) was added to each well, and then at 37°C. Blocked for an hour. Thereafter, each well was washed 3 times using a washing buffer (PBS in which 0.05% Tween 20 was dissolved), and the collected mouse sample was diluted using a 1/2 step dilution method, and then 100 μl each was added to the coated microplate 37 It was reacted at °C for two hours. After the reaction is over, the microplate is washed twice using a washing buffer, and goat anti-mouse IgG (Zymed, 81-6520) or goat anti-mouse IgG1 (Serotec, STAR81P) or goat with HRP (horseradish peroxidase) conjugated According to the experiment, anti-mouse IgG2a (Serotec, STAR82P) was put into a microplate at a time of 100 μl and reacted at 37° C. for two hours. After washing twice using a washing buffer, TMB (tetramethylbenzidine, BD Bioscience, 55555214), a reagent that reacts with HRP to develop color, was added to a microplate at 100 μl to develop color. After color development at room temperature and dark place for about 10 minutes, _{100 μl of 1N H 2} SO ₄ was immediately added to complete the color development reaction, and the 96-well microplate after the reaction was placed in a microplate reader (BioTek, USA) and 450 nm The degree of color development (concentration) of each well was measured at a wavelength of. The endpoint titer was defined as the highest serum dilution resulting in twice the absorbance of non-immune serum with a cut-off value of 0.1, expressed as mean±SD.

As can be seen in FIGS. 12A-12B, when CIA05 was added to the HPV16 L1 VLP antigen, all of the total IgG, IgG1 and IgG2a titers were significantly increased. The pattern is clear. In addition, CIA05 was more effective in promoting immunity than alum. Therefore, it can be seen that immunization against HPV can be achieved very excellently by the HPV L1 VLP antigen and CIA05, which are the basic compositions of the vaccine of the present invention.

In addition, as can be seen in Figures 13a-13b, HPV16/18 L1 VLP-specific total IgG titer was significantly higher than that of Alum alone when CIA05 adjuvant and Alum were administered in combination, and immunized ^{with conventional Cervarix TM} and Gardasil ^TM The figures were superior to those of the group.

On the other hand, as can be seen in Figures 14a-14b analyzed by the IgG subtype, HPV16 L1 VLP-specific IgG1 was higher than that of Alum alone when CIA05 adjuvant and Alum were administered in combination, and immunized ^{with conventional Cervarix TM} and Gardasil ^TM It was slightly higher than that of the group (Fig. 14a). In the case of IgG2a, when a combination of CIA05 adjuvant and Alum was administered, it was significantly higher than that of Alum alone, and was superior to that of the group immunized ^{with conventional Cervarix TM} and Gardasil ^{TM (FIG. 14B).}

mouse Splenocytes Interferon-γ concentration analysis using

The spleen was extracted as aseptically as possible from the mice immunized by the above method and washed with PBS. In the culture solution (50 μM β-mercaptoethanol, 1 M HEPES buffer, 10% FBS, 1 x RPMI1640 added with antibiotics), 10 holes were made in the spleen using a 5 ml syringe. The splenocytes were separated by sucking the culture solution into the syringe and repeatedly spraying it vigorously several times, and the separated splenocytes were collected and centrifuged together with the culture solution at 1500 rpm and 4° C. for 10 minutes. The culture medium was removed and resuspended in RPMI1640 medium containing 100 μg/ml streptomycin and 50 mM β-mercaptoethanol in 10% FBS. Subsequently, splenocytes were dispensed into each well of a 96-well plate, HPV16 L1 VLP was added at a concentration of 15 μg/ml, and then cultured at 37° C. for 48 hours for 72 hours. The concentration of interferon-γ secreted from mouse splenocytes was measured by ELISA (BD OptEIA mouse ELISA kit).

As can be seen in Fig. 12c, when CIA05 was added to the HPV16 L1 VLP antigen, the concentration of interferon-γ was significantly increased, and the immunostimulating effect of CIA05 was better than that of alum. Therefore, it can be seen that immunization against HPV can be achieved very well by the HPV L1 VLP antigen and CIA05, which are the basic compositions of the vaccine of the present invention.

In addition, as can be seen in Figure 15, the concentration of interferon-γ secreted by splenocytes was much higher than that of Alum alone when CIA05 adjuvant and Alum were administered in combination, and were treated ^{with conventional Cervarix TM} and Gardasil ^TM. The figures were superior to those of the group.

On the other hand, in Figures 13a-14b, the vaccine composition of the present invention contains HPV type 16 and 18 L1 VLPs, aluminum hydroxide and CIA05, which can be seen that CIA05 is substituted for MPL in Cervarix of GSK. That is, it can be seen that CIA05 used in the present invention is far superior to MPL in terms of toxicity as well as in immunity promoting ability against HPV.

In conclusion, as a result of the above experiment, it can be seen that both the HPV16 L1 VLP and the HPV18 L1 VLP show that the experimental group mixed with the adjuvant CIA05 developed by the present inventors showed superior immunity than the experimental group immunized with the conventional cervical cancer vaccine. Considering that the currently developed cervical cancer vaccine has controversy over the lasting effect, the present invention can be said to be an original and advanced invention capable of solving this problem in strengthening immunity through the addition of adjuvants.

Korea Microorganism Conservation Center (KCCM) KCCM11036P 20091007 Korea Microorganism Conservation Center (KCCM) KCCM11037P 20091007

<110> EyeGene, Inc. <120> Vaccines for Cervical Cancer <160> 4 <170> KopatentIn 1.71 <210> 1 <211> 1518 <212> DNA <213> Artificial Sequence <220> <223> YEG-HPV16-ROS <220> <221> CDS <222> (1)..(1515) <400> 1 atg tcg tta tgg tta cct tca gaa gct acc gtt tat ctt cct cca gta 48 Met Ser Leu Trp Leu Pro Ser Glu Ala Thr Val Tyr Leu Pro Pro Val 1 5 10 15 cca gtt tcc aaa gtc gtt tct aca gat gaa tac gtt gct aga act aac 96 Pro Val Ser Lys Val Val Ser Thr Asp Glu Tyr Val Ala Arg Thr Asn 20 25 30 att tac tac cat gca ggc act tcc agg tta tta gct gtc ggt cat ccc 144 Ile Tyr Tyr His Ala Gly Thr Ser Arg Leu Leu Ala Val Gly His Pro 35 40 45 tat ttc cca att aaa aag ccc aac aat aat aag ata tta gtt cca aaa 192 Tyr Phe Pro Ile Lys Lys Pro Asn Asn Asn Lys Ile Leu Val Pro Lys 50 55 60 gtt tcc ggc tta caa tac aga gtc ttt aga ata cat tta cca gat cct 240 Val Ser Gly Leu Gln Tyr Arg Val Phe Arg Ile His Leu Pro Asp Pro 65 70 75 80 aac aag ttc ggt ttt cca gat aca agt ttt tat aac cct gat aca caa 288 Asn Lys Phe Gly Phe Pro Asp Thr Ser Phe Tyr Asn Pro Asp Thr Gln 85 90 95 cgt tta gtt tgg gca tgt gtt ggt gtt gaa gtg ggc cgt ggt caa cct 336 Arg Leu Val Trp Ala Cys Val Gly Val Glu Val Gly Arg Gly Gln Pro 100 105 110 ctt ggc gtc ggg att tct ggt cac cca tta ttg aat aag tta gac gat 384 Leu Gly Val Gly Ile Ser Gly His Pro Leu Leu Asn Lys Leu Asp Asp 115 120 125 act gaa aac gcc tcc gcc tac gct gct aac gca gga gtc gac aat agg 432 Thr Glu Asn Ala Ser Ala Tyr Ala Ala Asn Ala Gly Val Asp Asn Arg 130 135 140 gaa tgc att tca atg gac tat aaa caa act caa cta tgc tta att ggt 480 Glu Cys Ile Ser Met Asp Tyr Lys Gln Thr Gln Leu Cys Leu Ile Gly 145 150 155 160 tgt aaa cct cca att ggt gaa cat tgg ggt aaa gga tct ccc tgc acg 528 Cys Lys Pro Pro Ile Gly Glu His Trp Gly Lys Gly Ser Pro Cys Thr 165 170 175 aat gtt gca gtt aat cct ggt gat tgt cca ccc tta gaa ttg att aat 576 Asn Val Ala Val Asn Pro Gly Asp Cys Pro Pro Leu Glu Leu Ile Asn 180 185 190 act gtt ata caa gat ggt gac atg gtc gat acc ggc ttc gga gct atg 624 Thr Val Ile Gln Asp Gly Asp Met Val Asp Thr Gly Phe Gly Ala Met 195 200 205 gac ttt act aca tta caa gcg aat aaa tca gaa gtt ccg cta gat att 672 Asp Phe Thr Thr Leu Gln Ala Asn Lys Ser Glu Val Pro Leu Asp Ile 210 215 220 tgt act tcc att tgt aaa tac cca gat tac att aag atg gtg tct gaa 720 Cys Thr Ser Ile Cys Lys Tyr Pro Asp Tyr Ile Lys Met Val Ser Glu 225 230 235 240 cct tat ggt gat tca cta ttt ttc tac ctg cgt cgt gaa caa atg ttt 768 Pro Tyr Gly Asp Ser Leu Phe Phe Tyr Leu Arg Arg Glu Gln Met Phe 245 250 255 gtt aga cat ctt ttc aat cgt gct gga gcc gta gga gaa aat gta cca 816 Val Arg His Leu Phe Asn Arg Ala Gly Ala Val Gly Glu Asn Val Pro 260 265 270 gat gat tta tat ata aaa ggt tcc ggt tct aca gca aat tta gcc tca 864 Asp Asp Leu Tyr Ile Lys Gly Ser Gly Ser Thr Ala Asn Leu Ala Ser 275 280 285 tca aat tat ttc cca acc cca tca ggt tca atg gtt act tca gac gcg 912 Ser Asn Tyr Phe Pro Thr Pro Ser Gly Ser Met Val Thr Ser Asp Ala 290 295 300 caa att ttt aat aaa ccc tat tgg ctt caa agg gct caa ggt cat aac 960 Gln Ile Phe Asn Lys Pro Tyr Trp Leu Gln Arg Ala Gln Gly His Asn 305 310 315 320 aat ggt att tgt tgg ggc aat cag tta ttc gtc aca gtc gtc gat act 1008 Asn Gly Ile Cys Trp Gly Asn Gln Leu Phe Val Thr Val Val Asp Thr 325 330 335 act aga tca act aat atg agt tta tgt gca gca ata tcc act tca gaa 1056 Thr Arg Ser Thr Asn Met Ser Leu Cys Ala Ala Ile Ser Thr Ser Glu 340 345 350 acc act tat aag aat acc aac ttt aaa gaa tac ttg aga cac ggt gaa 1104 Thr Thr Tyr Lys Asn Thr Asn Phe Lys Glu Tyr Leu Arg His Gly Glu 355 360 365 gaa tat gac ttg caa ttc atc ttt caa tta tgc aaa atc act tta aca 1152 Glu Tyr Asp Leu Gln Phe Ile Phe Gln Leu Cys Lys Ile Thr Leu Thr 370 375 380 gca gac gta atg aca tat att cat tca atg aac tct aca att tta gaa 1200 Ala Asp Val Met Thr Tyr Ile His Ser Met Asn Ser Thr Ile Leu Glu 385 390 395 400 gat tgg aac ttc gga cta caa cct cct cca ggc gga aca cta gaa gac 1248 Asp Trp Asn Phe Gly Leu Gln Pro Pro Pro Gly Gly Thr Leu Glu Asp 405 410 415 act tat aga ttt gtt acc tca cag gcc atc gca tgt caa aag cat aca 1296 Thr Tyr Arg Phe Val Thr Ser Gln Ala Ile Ala Cys Gln Lys His Thr 420 425 430 cca cca gcg ccg aaa gaa gac ccc ttg aaa aaa tac act ttc tgg gaa 1344 Pro Pro Ala Pro Lys Glu Asp Pro Leu Lys Lys Tyr Thr Phe Trp Glu 435 440 445 gtt aac ttg aaa gaa aaa ttc tct gct gat tta gat caa ttt cca tta 1392 Val Asn Leu Lys Glu Lys Phe Ser Ala Asp Leu Asp Gln Phe Pro Leu 450 455 460 gga cgt aaa ttt tta cta caa gca ggt tta aaa gct aag cca aaa ttc 1440 Gly Arg Lys Phe Leu Leu Gln Ala Gly Leu Lys Ala Lys Pro Lys Phe 465 470 475 480 acc cta ggc aaa aga aaa gca aca cct act act tct tcc aca tct aca 1488 Thr Leu Gly Lys Arg Lys Ala Thr Pro Thr Thr Ser Ser Thr Ser Thr 485 490 495 act gcc aaa aga aaa aag aga aaa ttg taa 1518 Thr Ala Lys Arg Lys Lys Arg Lys Leu 500 505 <210> 2 <211> 505 <212> PRT <213> Artificial Sequence <400> 2 Met Ser Leu Trp Leu Pro Ser Glu Ala Thr Val Tyr Leu Pro Pro Val 1 5 10 15 Pro Val Ser Lys Val Val Ser Thr Asp Glu Tyr Val Ala Arg Thr Asn 20 25 30 Ile Tyr Tyr His Ala Gly Thr Ser Arg Leu Leu Ala Val Gly His Pro 35 40 45 Tyr Phe Pro Ile Lys Lys Pro Asn Asn Asn Lys Ile Leu Val Pro Lys 50 55 60 Val Ser Gly Leu Gln Tyr Arg Val Phe Arg Ile His Leu Pro Asp Pro 65 70 75 80 Asn Lys Phe Gly Phe Pro Asp Thr Ser Phe Tyr Asn Pro Asp Thr Gln 85 90 95 Arg Leu Val Trp Ala Cys Val Gly Val Glu Val Gly Arg Gly Gln Pro 100 105 110 Leu Gly Val Gly Ile Ser Gly His Pro Leu Leu Asn Lys Leu Asp Asp 115 120 125 Thr Glu Asn Ala Ser Ala Tyr Ala Ala Asn Ala Gly Val Asp Asn Arg 130 135 140 Glu Cys Ile Ser Met Asp Tyr Lys Gln Thr Gln Leu Cys Leu Ile Gly 145 150 155 160 Cys Lys Pro Pro Ile Gly Glu His Trp Gly Lys Gly Ser Pro Cys Thr 165 170 175 Asn Val Ala Val Asn Pro Gly Asp Cys Pro Pro Leu Glu Leu Ile Asn 180 185 190 Thr Val Ile Gln Asp Gly Asp Met Val Asp Thr Gly Phe Gly Ala Met 195 200 205 Asp Phe Thr Thr Leu Gln Ala Asn Lys Ser Glu Val Pro Leu Asp Ile 210 215 220 Cys Thr Ser Ile Cys Lys Tyr Pro Asp Tyr Ile Lys Met Val Ser Glu 225 230 235 240 Pro Tyr Gly Asp Ser Leu Phe Phe Tyr Leu Arg Arg Glu Gln Met Phe 245 250 255 Val Arg His Leu Phe Asn Arg Ala Gly Ala Val Gly Glu Asn Val Pro 260 265 270 Asp Asp Leu Tyr Ile Lys Gly Ser Gly Ser Thr Ala Asn Leu Ala Ser 275 280 285 Ser Asn Tyr Phe Pro Thr Pro Ser Gly Ser Met Val Thr Ser Asp Ala 290 295 300 Gln Ile Phe Asn Lys Pro Tyr Trp Leu Gln Arg Ala Gln Gly His Asn 305 310 315 320 Asn Gly Ile Cys Trp Gly Asn Gln Leu Phe Val Thr Val Val Asp Thr 325 330 335 Thr Arg Ser Thr Asn Met Ser Leu Cys Ala Ala Ile Ser Thr Ser Glu 340 345 350 Thr Thr Tyr Lys Asn Thr Asn Phe Lys Glu Tyr Leu Arg His Gly Glu 355 360 365 Glu Tyr Asp Leu Gln Phe Ile Phe Gln Leu Cys Lys Ile Thr Leu Thr 370 375 380 Ala Asp Val Met Thr Tyr Ile His Ser Met Asn Ser Thr Ile Leu Glu 385 390 395 400 Asp Trp Asn Phe Gly Leu Gln Pro Pro Pro Gly Gly Thr Leu Glu Asp 405 410 415 Thr Tyr Arg Phe Val Thr Ser Gln Ala Ile Ala Cys Gln Lys His Thr 420 425 430 Pro Pro Ala Pro Lys Glu Asp Pro Leu Lys Lys Tyr Thr Phe Trp Glu 435 440 445 Val Asn Leu Lys Glu Lys Phe Ser Ala Asp Leu Asp Gln Phe Pro Leu 450 455 460 Gly Arg Lys Phe Leu Leu Gln Ala Gly Leu Lys Ala Lys Pro Lys Phe 465 470 475 480 Thr Leu Gly Lys Arg Lys Ala Thr Pro Thr Thr Ser Ser Thr Ser Thr 485 490 495 Thr Ala Lys Arg Lys Lys Arg Lys Leu 500 505 <210> 3 <211> 1524 <212> DNA <213> Artificial Sequence <220> <223> YEG-HPV18L1-ROS <220> <221> CDS <222> (1)..(1521) <400> 3 atg gcc ctt tgg aga cct tca gac aat act gtg tac ctt ccc cct cct 48 Met Ala Leu Trp Arg Pro Ser Asp Asn Thr Val Tyr Leu Pro Pro Pro 1 5 10 15 tct gtc gct aga gta gta aac aca gat gat tac gta act aga aca tct 96 Ser Val Ala Arg Val Val Asn Thr Asp Asp Tyr Val Thr Arg Thr Ser 20 25 30 att ttt tac cac gcc ggt agc tca aga tta tta aca gta ggt aat cct 144 Ile Phe Tyr His Ala Gly Ser Ser Arg Leu Leu Thr Val Gly Asn Pro 35 40 45 tat ttc cgt gtc cca gca ggc ggc ggt aat aaa caa gac att cct aaa 192 Tyr Phe Arg Val Pro Ala Gly Gly Gly Asn Lys Gln Asp Ile Pro Lys 50 55 60 gtc tca gcc tac caa tat aga gta ttc aga gtc caa ctt ccc gat ccc 240 Val Ser Ala Tyr Gln Tyr Arg Val Phe Arg Val Gln Leu Pro Asp Pro 65 70 75 80 aac aaa ttt gga ttg cca gac acg tcg ata tat aac cca gaa aca cag 288 Asn Lys Phe Gly Leu Pro Asp Thr Ser Ile Tyr Asn Pro Glu Thr Gln 85 90 95 aga cta gtt tgg gcc tgt gca gga gtt gaa ata gga aga gga caa ccc 336 Arg Leu Val Trp Ala Cys Ala Gly Val Glu Ile Gly Arg Gly Gln Pro 100 105 110 ttg ggc gta ggt tta tca ggt cac ccc ttc tac aat aaa tta gac gat 384 Leu Gly Val Gly Leu Ser Gly His Pro Phe Tyr Asn Lys Leu Asp Asp 115 120 125 aca gaa tct agc cat gca gcc aca agt aat gtt tca gag gat gta agg 432 Thr Glu Ser Ser His Ala Ala Thr Ser Asn Val Ser Glu Asp Val Arg 130 135 140 gac aat gtt tcc gta gac tat aaa cag acc caa cta tgt ata tta ggt 480 Asp Asn Val Ser Val Asp Tyr Lys Gln Thr Gln Leu Cys Ile Leu Gly 145 150 155 160 tgt gct cca gca att ggc gaa cac tgg gct aag gga act gcg tgc aaa 528 Cys Ala Pro Ala Ile Gly Glu His Trp Ala Lys Gly Thr Ala Cys Lys 165 170 175 tca agg ccc cta tca caa ggc gat tgt ccc ccc tta gaa tta aaa aac 576 Ser Arg Pro Leu Ser Gln Gly Asp Cys Pro Pro Leu Glu Leu Lys Asn 180 185 190 aca gtc tta gaa gat gga gat atg gtc gat acc ggt tat ggc gca atg 624 Thr Val Leu Glu Asp Gly Asp Met Val Asp Thr Gly Tyr Gly Ala Met 195 200 205 gat ttt tca aca tta caa gac acg aag tgt gaa gtg ccc cta gat att 672 Asp Phe Ser Thr Leu Gln Asp Thr Lys Cys Glu Val Pro Leu Asp Ile 210 215 220 tgt caa tca att tgt aaa tac cct gac tac tta caa atg tct gcc gac 720 Cys Gln Ser Ile Cys Lys Tyr Pro Asp Tyr Leu Gln Met Ser Ala Asp 225 230 235 240 cca tat ggc gac tcc atg ttt ttt tgc ttg aga aga gaa caa tta ttt 768 Pro Tyr Gly Asp Ser Met Phe Phe Cys Leu Arg Arg Glu Gln Leu Phe 245 250 255 gct cgt cat ttt tgg aac cgt gca gga aca atg ggt gat aca gtt cct 816 Ala Arg His Phe Trp Asn Arg Ala Gly Thr Met Gly Asp Thr Val Pro 260 265 270 caa tct tta tat ata aaa ggc aca ggc atg cgt gct tcc cct ggt tcg 864 Gln Ser Leu Tyr Ile Lys Gly Thr Gly Met Arg Ala Ser Pro Gly Ser 275 280 285 tgc gta tat tct cct tca ccg tca ggt tct att gtc acg tca gat agt 912 Cys Val Tyr Ser Pro Ser Pro Ser Gly Ser Ile Val Thr Ser Asp Ser 290 295 300 caa tta ttt aac aaa cca tat tgg tta cat aaa gct caa ggt cat aat 960 Gln Leu Phe Asn Lys Pro Tyr Trp Leu His Lys Ala Gln Gly His Asn 305 310 315 320 aat ggt gta tgt tgg cac aat caa tta ttc gta act gta gtc gat aca 1008 Asn Gly Val Cys Trp His Asn Gln Leu Phe Val Thr Val Val Asp Thr 325 330 335 acc aga tcc act aat tta aca ata tgt gca tca act cag tca ccc gtt 1056 Thr Arg Ser Thr Asn Leu Thr Ile Cys Ala Ser Thr Gln Ser Pro Val 340 345 350 cca ggt caa tat gac gca aca aaa ttc aaa caa tac tcc aga cat gta 1104 Pro Gly Gln Tyr Asp Ala Thr Lys Phe Lys Gln Tyr Ser Arg His Val 355 360 365 gaa gaa tac gac tta caa ttc att ttc cag cta tgt act atc acg tta 1152 Glu Glu Tyr Asp Leu Gln Phe Ile Phe Gln Leu Cys Thr Ile Thr Leu 370 375 380 aca gcc gat gtc atg tct tat att cac tct atg aac tcc tct att tta 1200 Thr Ala Asp Val Met Ser Tyr Ile His Ser Met Asn Ser Ser Ile Leu 385 390 395 400 gaa gat tgg aac ttt ggc gtt cca cca cct ccc act aca tca tta gtc 1248 Glu Asp Trp Asn Phe Gly Val Pro Pro Pro Pro Thr Thr Ser Leu Val 405 410 415 gat aca tac cgt ttc gtt caa tct gtt gcc atc act tgt caa aaa gac 1296 Asp Thr Tyr Arg Phe Val Gln Ser Val Ala Ile Thr Cys Gln Lys Asp 420 425 430 gca gcc ccc gca gaa aat aaa gac cca tac gat aaa tta aaa ttt tgg 1344 Ala Ala Pro Ala Glu Asn Lys Asp Pro Tyr Asp Lys Leu Lys Phe Trp 435 440 445 aat gta gat ctt aag gaa aaa ttt agt tta gac tta gat cag tat cct 1392 Asn Val Asp Leu Lys Glu Lys Phe Ser Leu Asp Leu Asp Gln Tyr Pro 450 455 460 tta gga aga aaa ttt ctt gtt caa gca ggt ctg aga aga aaa cca acc 1440 Leu Gly Arg Lys Phe Leu Val Gln Ala Gly Leu Arg Arg Lys Pro Thr 465 470 475 480 atc ggc cca aga aaa aga tcc gca cct tct gcc act aca tca tca aaa 1488 Ile Gly Pro Arg Lys Arg Ser Ala Pro Ser Ala Thr Thr Ser Ser Lys 485 490 495 cca gcc aaa aga gta aga gta aga gct aga aag taa 1524 Pro Ala Lys Arg Val Arg Val Arg Ala Arg Lys 500 505 <210> 4 <211> 507 <212> PRT <213> Artificial Sequence <400> 4 Met Ala Leu Trp Arg Pro Ser Asp Asn Thr Val Tyr Leu Pro Pro Pro 1 5 10 15 Ser Val Ala Arg Val Val Asn Thr Asp Asp Tyr Val Thr Arg Thr Ser 20 25 30 Ile Phe Tyr His Ala Gly Ser Ser Arg Leu Leu Thr Val Gly Asn Pro 35 40 45 Tyr Phe Arg Val Pro Ala Gly Gly Gly Asn Lys Gln Asp Ile Pro Lys 50 55 60 Val Ser Ala Tyr Gln Tyr Arg Val Phe Arg Val Gln Leu Pro Asp Pro 65 70 75 80 Asn Lys Phe Gly Leu Pro Asp Thr Ser Ile Tyr Asn Pro Glu Thr Gln 85 90 95 Arg Leu Val Trp Ala Cys Ala Gly Val Glu Ile Gly Arg Gly Gln Pro 100 105 110 Leu Gly Val Gly Leu Ser Gly His Pro Phe Tyr Asn Lys Leu Asp Asp 115 120 125 Thr Glu Ser Ser His Ala Ala Thr Ser Asn Val Ser Glu Asp Val Arg 130 135 140 Asp Asn Val Ser Val Asp Tyr Lys Gln Thr Gln Leu Cys Ile Leu Gly 145 150 155 160 Cys Ala Pro Ala Ile Gly Glu His Trp Ala Lys Gly Thr Ala Cys Lys 165 170 175 Ser Arg Pro Leu Ser Gln Gly Asp Cys Pro Pro Leu Glu Leu Lys Asn 180 185 190 Thr Val Leu Glu Asp Gly Asp Met Val Asp Thr Gly Tyr Gly Ala Met 195 200 205 Asp Phe Ser Thr Leu Gln Asp Thr Lys Cys Glu Val Pro Leu Asp Ile 210 215 220 Cys Gln Ser Ile Cys Lys Tyr Pro Asp Tyr Leu Gln Met Ser Ala Asp 225 230 235 240 Pro Tyr Gly Asp Ser Met Phe Phe Cys Leu Arg Arg Glu Gln Leu Phe 245 250 255 Ala Arg His Phe Trp Asn Arg Ala Gly Thr Met Gly Asp Thr Val Pro 260 265 270 Gln Ser Leu Tyr Ile Lys Gly Thr Gly Met Arg Ala Ser Pro Gly Ser 275 280 285 Cys Val Tyr Ser Pro Ser Pro Ser Gly Ser Ile Val Thr Ser Asp Ser 290 295 300 Gln Leu Phe Asn Lys Pro Tyr Trp Leu His Lys Ala Gln Gly His Asn 305 310 315 320 Asn Gly Val Cys Trp His Asn Gln Leu Phe Val Thr Val Val Asp Thr 325 330 335 Thr Arg Ser Thr Asn Leu Thr Ile Cys Ala Ser Thr Gln Ser Pro Val 340 345 350 Pro Gly Gln Tyr Asp Ala Thr Lys Phe Lys Gln Tyr Ser Arg His Val 355 360 365 Glu Glu Tyr Asp Leu Gln Phe Ile Phe Gln Leu Cys Thr Ile Thr Leu 370 375 380 Thr Ala Asp Val Met Ser Tyr Ile His Ser Met Asn Ser Ser Ile Leu 385 390 395 400 Glu Asp Trp Asn Phe Gly Val Pro Pro Pro Pro Thr Thr Ser Leu Val 405 410 415 Asp Thr Tyr Arg Phe Val Gln Ser Val Ala Ile Thr Cys Gln Lys Asp 420 425 430 Ala Ala Pro Ala Glu Asn Lys Asp Pro Tyr Asp Lys Leu Lys Phe Trp 435 440 445 Asn Val Asp Leu Lys Glu Lys Phe Ser Leu Asp Leu Asp Gln Tyr Pro 450 455 460 Leu Gly Arg Lys Phe Leu Val Gln Ala Gly Leu Arg Arg Lys Pro Thr 465 470 475 480 Ile Gly Pro Arg Lys Arg Ser Ala Pro Ser Ala Thr Thr Ser Ser Lys 485 490 495 Pro Ala Lys Arg Val Arg Val Arg Ala Arg Lys 500 505

Claims (8)

Method for preparing human papilloma virus (HPV) L1 virus-like particle (VLP) comprising the following steps:
(i) culturing a yeeast comprising the HPV L1-encoding nucleotide sequence;
(Ii) lysis of the cultured yeast;
(Iii) conducting ammonium sulphate precipitation on the yeast lysate to remove impurities; And
(Iii) performing heparin chromatography or cation-exchange chromatography on the resultant from which the impurities are removed.
The method of claim 1, wherein the HPV is HPV Type 16 or HPV Type 18.
The method of claim 1, wherein the resultant from which impurities are removed by performing the step (VII-2) ammonium sulfate precipitation between steps (iii) and (iii) is treated with 0.001-0.3 M NaCl to further remove impurities. Method further comprising a.
The method of claim 1, wherein the cation exchange chromatography is carried out using a cation exchanger having a phosphate group.
A nucleic acid sequence of an L1 virus-like particle (VLP) of human papilloma virus (HPV) type 16 comprising the nucleotide sequence of SEQ ID NO: 1.
A nucleic acid sequence of an L1 virus-like particle (VLP) of human papilloma virus (HPV) type 18 comprising the nucleotide sequence of SEQ ID NO: 3.
Saccharomyces cerevisiae (accession number: KCCM11036P) transformed with the nucleic acid sequence of the human papilloma virus (HPV) type 16 L1 virus-like particle (VLP) of claim 5.
Saccharomyces cerevisiae (accession number: KCCM11037P) transformed with the nucleic acid sequence of the human papilloma virus (HPV) type 18 L1 virus-like particle (VLP) of claim 6.

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