# Patent

## Title
Genes encoding major capsid protein L1 of human papilloma virus

## Abstract

The present invention discloses a codon-optimized gene encoding major capsid protein L1 of human papilloma virus, which is capable, after transduced into a yeast cell, of efficiently expressing the major capsid protein L1 of human papilloma virus. The present invention also discloses an immunogenic macromolecule which is essentially produced by expression of said codon-optimized gene encoding the major capsid protein L1 of human papilloma virus in a yeast cell. The present invention further discloses the use of said immunogenic macromolecule and a composition comprising said immunogenic macromolecule.

## Background
FIELD OF THE INVENTION
The present invention relates to the field of biotechnology, particularly to the major capsid protein of human papilloma virus, gene coding the same, and preparation method and use of the same.
BACKGROUND OF THE INVENTION
Human papilloma virus (HPV) is a non-enveloped small double-stranded circular DNA virus belonging to polyomavirus subfamily of papova virus family. HPV can be spread through intimate contact among human beings, leading to such lesions in the infected persons as verruca vulgaris on the skin and condylomata acuminata around the anus and genitalia, which are ranked as sexually transmitted diseases. The results of investigation published by the International Agency for Research on Cancer in 1995 showed that HPV is closely responsible for cervical cancer. It is thus clear that HPV has become a pathogen that is severely harmful to human health. Therefore, it is much significant to develop highly efficient and inexpensive HPV vaccines for the prevention of cervical cancer in women and the sexually transmitted diseases caused by HPV infection.
More than 100 subtypes of HPV have been identified currently. Nearly 100% of the cervical cancer patients can be detected positive for the presence of high-risk HPV DNA in the pathologic tissues by using sensitive detection methods. In terms of the relationship between HPV subtypes and malignancies in genital tract of female patient, HPV can be classified into low-risk type and high-risk type. HPV6, 11, 34, 40 and 42 and the like are low-risk HPV subtypes, typically found in benign cervical lesions such as cervical condylomas and mild atypical hyperplasia of cervical epithelium; while HPV 16, 18, 31, 33, 35, 39, 45 are high-risk HPV subtypes, mostly found in severe atypical hyperplasia of cervical epithelium as well as cervical cancer. A series of studies on different human populations have substantiated that HPV 16 and 18 infections in genital tract are more highly associated with the occurrence of cervical cancer than other risk factors. Among cervical cancer patients, about 50 to 60% of the cases are caused by HPV 16 infection, about 14% by HPV 18, about 8% by HPV 45, and about 5% by HPV 31, with the remaining 23% of the cases caused by other HPV subtypes.
HPV is non-enveloped and globular in shape with a diameter of about 45 to 55 nm, having an icosahedrally symmetric deflective capsid consisting of 72 capsid particles. The virion capsid is essentially comprised of major capsid proteins (L1) and minor capsid proteins (L2). After being expressed in cells, major capsid proteins L1 can be self-assembled into capsid particles called virus-like particles (VLPs).
A normal woman has a life-time accumulative probability of about 40% for cervical infection by at least one subtype of HPV during her whole life. Therefore, it is of great importance to develop suitably-priced and advantageously protective vaccines against cervical cancer, especially vaccines against HPV 16 and HPV 18, for lowering the morbidity and mortality of cervical cancer in women.
Although some vaccines have been developed against HPV in the prior art, these vaccines generally have the problems of low expression efficiency of HPV protein, low activity of the expressed protein, inability of the protein to assemble into virus-like particles or undesirability of the immunological effect of the assembled particles. Consequently, there exists a need in the art for improved HPV vaccine products.
SUMMARY OF THE INVENTION
Objects of the present invention include providing a gene for the major capsid protein L1 of human papilloma virus, and preparation method and use of the same.
In a first aspect of the present invention, there is provided an isolated gene encoding the major capsid protein L1 of human papilloma virus, said gene having the codons preferred by Pichia yeast.
In another preferred embodiment, said gene encodes the major capsid protein L1 of human papilloma virus having an amino acid sequence set forth in SEQ ID NO: 10 or in positions 62 to 568 of SEQ ID NO: 10 (i.e., the major capsid protein L1 of human papilloma virus subtype 18). More preferably, said gene has a nucleotide sequence selected from those set forth in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3; or has a nucleotide sequence selected from those set forth in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6.
In another preferred embodiment, said gene encodes the major capsid protein L1 of human papilloma virus having an amino acid sequence set forth in SEQ ID NO: 11 (i.e., the major capsid protein L1 of human papilloma virus subtype 16). More preferably, said gene has a nucleotide sequence selected from those set forth in SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9.
In a second aspect of the present invention, there is provided an expression vector comprising the sequence of said gene.
In another preferred embodiment, said expression vector is a Pichia yeast expression vector.
In a third aspect of the present invention, there is provided a genetically engineered host cell, said cell comprising said expressing vector or having said gene integrated into its genome.
In another preferred embodiment, said cell is Pichia yeast cell. More preferably, said Pichia yeast is selected from Pichia yeast X-33, GS115, KM71 and SMD1168 strains. Most preferably, said Pichia yeast is Pichia yeast X-33 strain.
In a fourth aspect of the present invention, there is provided an immunogenic macromolecule (i.e., virus-like particle). Said macromolecule, 50-80 nm in diameter, is self-assembled from the major capsid protein L1 of human papilloma virus, which is expressed by Pichia yeast.
In another preferred embodiment, said immunogenic macromolecule is prepared by the following method:
(1) culturing said host cell to allow said major capsid protein L1 of human papilloma virus to be expressed and to be self-assembled into said immunogenic macromolecule in said host cell;
(2) separating said immunogenic macromolecule.
In a fifth aspect of the present invention, there is provided a method for preparing said immunogenic macromolecule, said method comprising:
(1) culturing said host cell to allow said major capsid protein L1 of human papilloma virus to be expressed and to be self-assembled into said immunogenic macromolecule in said host cell;
(2) separating said immunogenic macromolecule.
In another preferred embodiment, step (2) as described above includes:
(a) disrupting the host cells obtained from step (1) to obtain a supernatant containing said immunogenic macromolecule; and
(b) successively purifying the supernatant obtained from step (a) using POROS 50 HS column chromatography and CHT column chromatography to obtain said immunogenic macromolecule.
In another preferred embodiment, in step (b) as described above, purification using POROS 50 HS column chromatography is performed as follows: The supernatant obtain from step (a) is loaded onto a POROS 50HS column having been cleaned and equilibrated to allow said immunogenic macromolecule to bind to the column After being rinsed and equilibrated, the column is eluted with a linear gradient of 100% buffer A to 100% buffer B, and the chromatographic peaks at 70-100 ms/cm are collected, wherein said buffer A contains 50±20 mM MOPS, 0.75±0.3 M NaCl and 0.05±0.02% Tween-80 (pH 6.5±1), and said buffer B contains 50±20 mM MOPS, 1.5M NaCl and 0.05±0.02% Tween-80 (pH 6.5±1); or
Purification using CHT column chromatography is performed as follows: the product purified from POROS 50 HS column chromatography is loaded onto a CHT column having been cleaned and equilibrated. After being rinsed and equilibrated, the column is eluted with a linear gradient of buffer C to 100% buffer D, and the chromatographic peaks 50-70 ms/cm are collected, wherein said buffer C contains 50±20 mM MOPS, 0.5±0.2 M NaCl, 0.04±0.02 M PB and 0.05±0.02% Tween-80 (pH 6.5±1), and said buffer D contains 0.5±0.2M NaCl, 200 mM PB, and 0.05±0.02% Tween-80 (pH 6.5±1).
In a sixth aspect of the present invention, there is provided an immunogenic composition, said composition comprising:
(i) an effective amount of said immunogenic macromolecule; and
(ii) a pharmaceutically acceptable carrier.
In another preferred embodiment, said pharmaceutically acceptable carrier comprises at least one of immunostimulant or adjuvant.
In another preferred embodiment, said adjuvant is an aluminium adjuvant.
In another preferred embodiment, said composition is a vaccine.
In a seventh aspect of the present invention, there is provided the use of said immunogenic macromolecule in the prevention or treatment of diseases related to human papilloma virus infection.
In an eighth aspect of the present invention, there is provided a method for preventing or treating diseases related to human papilloma virus infection, said method comprising administering to a subject in need of prevention or treatment an effective amount of said immunogenic macromolecule or said immunogenic composition.
In another preferred embodiment, said diseases related to human papilloma virus infection are selected from malignancies (such as cervical cancer, vaginal cancer, anal or perianal cancer, oropharyngeal cancer, maxillary sinus cancer, lung cancer) or cervical intraepithelial neoplasia.
Other aspects of the present invention will be apparent to one skilled in the art in view of the present disclosure.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a schematic depiction of the construction of pPICZ-18L1 vector.
FIG. 2 is a diagram of PCR amplification of the gene for HPV 18L1.
FIG. 3 is a diagram of identification of pPICZ-18L1 vector, wherein lane 1 represents BstBI- and KpnI-digested pPICZαB; and lane 2 represents BstBI- and KpnI-digested pPICZ-18L1.
FIG. 4 is a diagram of Western blot identification of the expression of HPV 18 L1, wherein M represents Rainbow Marker (Fermentas Co., Ltd); 1 represents the positive control of expression; 2 represents the negative control of expression; and 3 represents the strain expressing HPV18L1. The arrow designates the expressed HPV 18 L1.
FIG. 5 is a diagram of PCR amplification of the gene for truncated HPV 18L1.
FIG. 6 is a diagram of identification of pPICZ-18L1′ vector, wherein lane 1 represents BstBI- and KpnI-digested pPICZαB, and lane 2 represents BstBI- and KpnI-digested pPICZ-18L1′.
FIG. 7 is a diagram of Western blot identification of the expression of truncated HPV 18 L1, wherein M represents Rainbow Marker (Fermentas Co., Ltd); 1 represents the positive control of expression; 2 represents the strain expressing truncated HPV18L1; and 3 represents the negative control of expression. The arrow designates the expressed truncated HPV 18 L1.
FIG. 8 is a reduced SDS-PAGE electropherogram of a chromatographically purified sample of HPV 18 L1, wherein 1 represents the collected peak 1 of POROS 50HS; 2 represents the collected peak 2 of POROS 50HS; 3 represents the collected peak 3 of POROS 50HS; 4 represents the collected peak 1 of CHT; 5 represents the concentrated sample of CHT peak 1; and 6 represents positive control.
FIG. 9 shows the immunoblotting of the purified product of HPV 18 L1, wherein the primary antibody is anti-HPV-18 L1 antibody available from Fitzgerald Corp. at 1:5000 dilution; the secondary antibody is goat anti-mouse antibody at 1:250 dilution; 1 represents positive control; and both 2 and 3 represent purified HPV 18 L1 protein.
FIG. 10 is a transmission electron micrograph (×105000) of the purified sample of HPV 18 L1 VLPs.
FIG. 11A is a diagram depicting infection of 293FT cell by HPV 18 pseudotype virus.
FIG. 11B is a diagram depicting neutralization of HPV 18 pseudotype virus by murine serum.
FIG. 12 is a schematic depiction of the construction of pPICZ-16 L1 vector.
FIG. 13 is a diagram of PCR amplification of the gene for HPV 16 L1.
FIG. 14 is a diagram of identification of pPICZ-16 L1 vector, wherein lane 1 represents BstBI- and KpnI-digested pPICZαB; and lane 2 represents BstBI- and KpnI-digested pPICZ-16L1.
FIG. 15 is a diagram of Western blot identification of the expression of HPV 16 L1, wherein M represents Rainbow Marker (Fermentas Co., Ltd); 1 represents the positive control of expression; 2 represents the strain expressing HPV 16 L1; and 3 represents the negative control of expression. The arrow designates the expressed HPV 16 L1.
FIG. 16 is a reduced SDS-PAGE electropherogram of a chromatographically purified sample of HPV 16 L1, wherein 1 represents the collected peak sample of POROS 50HS; 2 represents CHT flowthrough 1; 3 represents CHT flowthrough 2; 4 represents HPV 16 L1 positive control; 5 represents CHT elution peak 1; 6 represents CHT elution peak 2; 7 represents CHT elution peak 4; 8 represents CHT elution peak 8; and 9 represents CHT elution peak 12.
FIG. 17 shows the purity of HPV 16 L1 as determined by capillary electrophoresis.
FIG. 18 is a transmission electron micrograph (×105000) of the purified sample of HPV 16 L1 VLPs.
FIG. 19A is a diagram depicting infection of 293FT cell by HPV 16 L1 pseudotype virus.
FIG. 19B is a diagram depicting neutralization of HPV 16 L1 pseudotype virus by murine serum.



DETAILED DESCRIPTION OF THE INVENTION
Through intensive research, the present inventors have for the first time disclosed a codon-optimized gene encoding the major capsid protein L1 of human papilloma virus, which is capable, when introduced into a yeast cell, of efficiently expressing the major capsid protein L1 of human papilloma virus which then self-assembles into virus-like particles, in an expression amount that meets the requirements of industrial production. The present invention also for the first time discloses an immunogenic macromolecule which is essentially produced by expression of said codon-optimized gene encoding the major capsid protein L1 of human papilloma virus in a yeast cell.
As used herein, said major capsid protein L1 of human papilloma virus is referred to, for short, as HPV L1 protein; said major capsid protein L1 of human papilloma virus subtype 18 is referred to, for short, as HPV18 L1 protein; and said major capsid protein L1 of human papilloma virus subtype 16 is referred to, for short, as HPV16 L1 protein. HPV L1 protein includes HPV18 L1 protein and HPV16 L1 protein, or truncated forms thereof.
As used herein, the term “immunogenic macromolecule” refers to a polymer macromolecule comprising a number of monomeric major capsid proteins L1 of human papilloma virus, preferably polymerized or assembled from a number of major capsid proteins L1 of human papilloma virus; said major capsid proteins L1 of human papilloma virus are encoded by a codon-optimized gene encoding the major capsid protein L1 of human papilloma virus and are expressed in yeast cell (preferably Pichia yeast cell). Said immunogenic macromolecule is granular in shape.
As used herein, “operably linked together” or “operably linked to” refers to a status in which certain portions of a linear DNA sequence can influence the activities of other portions of the same linear DNA sequence. For example, if a promoter controls the transcription of an encoding sequence, then it is operably linked to the encoding sequence.
As used herein, the phrase “comprising”, “having” or “including” (or grammatical variants thereof) encompasses “containing”, “substantially consisting of . . . ”, “essentially consisting of . . . ” and “consisting of . . . ”. Concepts “substantially consisting of . . . ”, “essentially consisting of . . . ” and “consisting of . . . ” are subordinate concepts of “comprising”, “having” or “including”.
Gene Encoding the Major Capsid Protein L1 of Human Papilloma Virus
Based on the object of expressing HPV L1 protein using yeast cell, the present inventors have, after repeated investigation, found an optimized gene encoding HPV L1 protein which is suitable to be efficiently expressed in yeast cell, particularly in Pichia yeast cell. Said optimized gene encodes a full-length or truncated HPV18 L1 protein. Alternatively, said optimized gene encodes a full-length HPV16 L1 protein.
It is well known to those skilled in the art that, although there exist 64 genetic codons, most organisms tend to utilize some of these codons. For example, the genes of yeast cell have a different preference for codons than do human genes. Due to degeneracy of genetic codons, each amino acid may be encoded by more than one codon, with the codons for the same amino acid having different frequencies of usage in wild-type genes. Yeast cell's preference for codons may result in low translation efficiency and expression levels of recombinant proteins.
Codon optimization according to the present invention is essentially achieved as follows. Firstly, modifications are made on the naturally-occurring gene encoding HPV L1 by optimizing the codons for all the corresponding amino acids of the gene and repeatedly conducting gene expression experiments on the optimized gene sequences so as to find a novel set of HPV DNA sequences suitable for expression in yeast cell. The gene encoding full-length HPV 18 L1 is set forth in Genbank accession no. AAP20601; the gene encoding truncated HPV 18 L1 is set forth in Genbank accession no. AAQ92369; and the gene encoding full-length HPV 16 L1 is set forth in Genbank accession no. AAC09292.
Furthermore, in order to avoid the presence of a high GC ratio in the transcribed mRNAs, the influence of mRNA secondary structure on the efficiency of translation, and the occurrence of common restriction sites, the present inventors have made alterations to some of the preferable codons, such as, the codon for asparagine (Asn) being altered from AAC to AAT; the codon for lysine (Lys) being altered from AAG to AAA; the codon for aspartic acid (Asp) being altered from GAT to GAC, the codon for phenylalanine (Phe) being altered from TTT to TTC; the codon for tyrosine (Tyr) being altered from TAC to TAT; and the codon for glycine (Gly) being altered from GGT to GGA, thus obtaining a altered, novel HPV DNA sequences.
In a preferred embodiment of the present invention, said gene encodes the major capsid proteins L1 of human papilloma virus having an amino acid sequence set forth in SEQ ID NO: 10 or in positions 62 to 568 of SEQ ID NO: 10 (i.e., HPV18 L1). More preferably, said gene has a nucleotide sequence selected from those set forth in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3; or has a nucleotide sequence selected from those set forth in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6. Even more preferably, said gene has a nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 4. Most preferably, said gene has the nucleotide sequence set forth in SEQ ID NO: 1, which is a gene for truncated HPV 18 L1 corresponding to a truncated HPV 18 L1 protein with deletions of 61 amino acids at the N-terminus. Such a truncated gene is more favorably expressed in a recombinant vector without changing the activity of the protein.
In another preferred embodiment of the present invention, said gene encodes the major capsid proteins L1 of human papilloma virus having an amino acid sequence set forth in SEQ ID NO: 11 (i.e., HPV16 L1). More preferably, said gene has a nucleotide sequence selected from those set forth in SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9. Most preferably, said gene has the nucleotide sequence set forth in SEQ ID NO: 7.
The codon-optimized gene provided by the present invention has the following advantages: 1) this optimized gene is more suitable for efficiently expressing target protein in yeast host and meets the requirements of industrial production; and 2) low cost, high yield and more uniform and stable quality of products can be achieved with the use of Pichia yeast expression system.
The present invention also provides a vector comprising said gene encoding HPV L1. Said vector may further comprise regulatory sequence(s) for expression which are operably linked to the sequence of said coding gene so as to facilitate the expression of said HPV L1 protein. Any suitable vector can be used, especially those used for cloning and expression in yeast cell. More preferably, said expression vector is selected from currently commonly used yeast expression vectors, such as pPICZ, pPIC6, pGAPZ and pAO815. Such expression vectors are commercially available.
Moreover, recombinant cell comprising said gene encoding HPV L1 is also included in the present invention. Said recombinant cell is a yeast cell, particularly a Pichia yeast cell. More preferably, said Pichia yeast cell is selected from Pichia yeast X-33, GS115, KM71 and SMD1168 strains. These yeast cell strains are commercially available. Methods for introducing an exogenous gene into yeast cell are known in the art, for example, electrotransformation or protoplast transformation.
Method for producing HPV L1 is also included in the present invention. Said method comprises culturing said recombinant cell comprising said coding gene. Said method may include allowing the cells to express the encoded HPV L1 protein, and may further include separating and/or purifying the expressed protein. The HPV L1 protein obtained as above can be purified into essential homogeneity, for example, exhibiting a single band on SDS-PAGE electrophoresis.
The HPV L1 protein expressed in yeast cell according to the present invention can be used for preparing immunogenic macromolecule which can induce immune responses in vivo, particularly humoral immune responses.
As an embodiment of the present invention, the present inventors have designed, through optimization, several gene sequences suitable for expressing HPV L1 protein in Pichia yeast, which were then used for the total synthesis of full-length HPV L1 gene or its truncated form. The gene or its truncated form was cloned into existing Pichia yeast expression vector which was then used to construct recombinant Pichia yeast expression strain through homologous recombination and screening with high concentration antibiotics. The recombinant Pichia yeast was fermentatively cultured and then induced with methanol to highly express HPV L1 protein intracellularly, which is capable of concurrently self-assembling into virus-like particles (VLPs) intracellularly. The virus-like particles purified by chromatography of the supernatant obtained from disrupted cells had a purity of greater than 90% and were highly immunogenic when adsorbed to an aluminium adjuvant, permitting them to be used as a human vaccine against cervical cancer.
Protein Expression and Purification
HPV L1 protein can be efficiently expressed by culturing said recombinant cell comprising said gene encoding HPV L1, the expressed proteins being concurrently allowed to be self-assembled into immunogenic macromolecules. A method of expression and purification includes: (1) culturing said recombinant cell to allow said major capsid protein L1 of human papilloma virus to be expressed and to be concurrently self-assembled into immunogenic molecules in the recombinant cells; (2) disrupting the cells obtained from step (1) to obtain supernatant containing the immunogenic molecules; and (3) successively purifying the supernatant obtained from step (2) using POROS 50 HS column chromatography and CHT column chromatography to obtain said immunogenic macromolecules.
Preferably, cell culturing and protein expression are conducted as follows. The genetically engineered yeast according to the present invention is inoculated into activation media (YPD or LB or SOC) and cultured at 25 to 37° C. overnight. Then the activated fluid is inoculated into seed culture media (YPD or LB or SOC) and cultured at 25 to 37° C. overnight. Fermentation is conducted using basal salt medium (BSM1 or BSM2 or BSM3) added with trace amounts of salts (PTM1, PTM2, PTM3) at a temperature of 20 to 37° C. and an initial pH of 3 to 8. After the initial proliferation stage for about 15 to 30 hours, the dissolved oxygen value is maintained at 20 to 80% by adjusting the speed of stirring, air flow, and tank pressure. When the carbon source is completely consumed, the wet weight of yeast would reach about 50 to 150 g/L. At this time, glycerol or glucose solution is supplemented and the dissolved oxygen value is maintained at 20 to 80%. After a period of time of supplementation, the wet weight of yeast would reach about 50 to 500 g/L, at which time supplementation is stopped and methanol is added for induction while the pH value is maintained at pH 3 to 8. The dissolved oxygen value is maintained at higher than 20 to 80%, the temperature is maintained at 20 to 37° C., and the pH value is maintained at pH 3 to 8. Samples are taken at an interval of 2 to 10 hours and subjected to Western blot detection. Fermentation is stopped 5 to 90 hours after induction, and the fermentation broth is discharged. The fermentation broth is centrifuged using a refrigerated centrifuge, and then the cells are collected and stored at −20° C. The inducibly expressed HPV L1 protein can be self-assembled into virus-like particles (HPV L1 VLPs) inside Pichia yeast cell.
As said HPV L1 protein is expressed inside Pichia yeast cell, purer proteins can be obtained by performing cell disruption and protein purification and then allowed to be self-assembled into virus-like particles. Purification is generally performed as follows. The cells are washed to remove the attached medium components (salts, pigments, etc) in order to reduce their influence on subsequent purification. The washed cells are mixed into a suitable cell disruption buffer containing salt and surfactant components at certain concentrations. The salt components that can be used are for example NaCl and KCl at a concentration in the range of about 0.4 to 0.8 mol/l. The surfactant components that can be used are for example Tween-80, Tween-20 and Triton-X 100 at a concentration in the range of about 0.005 to 0.05% (w/v). The buffer systems that can be used are for example phosphate buffer, Tris buffer, MOPS buffer, and HEPES buffer at a concentration in the range of about 0.02 to 0.2 mol/l. The mixed cells can be disrupted using for example a high pressure homogenizer operating in a pressure range of about 800 to 2000 bars, achieving a disruption rate of greater than 90% in 2 to 4 disruption cycles, or a bead mill homogenizer using beads having diameters in the range of 0.2 to 0.4 mm which are loaded in an amount of about 70 to 90%, achieving a disruption rate of greater than 80% in 1 to 2 disruption cycles. The solution containing disrupted cells is subjected to high speed centrifugation at 6,000 to 10,000 rpm (SORVALL, HITACHI, etc.) for 20 to 60 minutes, or to tangential flow microfiltration using 0.45 to 0.65 μm membrane module (Millipore, PALL, etc.), in order to separate the supernatant and the precipitate, obtaining supernatant for further purification. The supernatant obtained can be subjected to anion exchange chromatography using such as Q Sepharose Flast Flow (GE) or DEAE SephroseFast Flow (GE) to remove some of the impurities in the supernatant, such as DNA, RNA and impurity proteins prior to conducting further purification. Alternatively, the supernatant obtained can be directly used to conduct further purification. For the further purification, the supernatant samples are loaded onto HPV L1 VLPs-absorbable chromatographic media, such as SP Sepharose FF, Heparin SepharoseCL-6B (GE), Poros 50HS (Merck) and Fractogel@EMD TMAE-650 (Merck) to allow HPV L1 VLPs protein to efficiently bind to the chromatographic media. Then the media is washed using a salt concentration gradient (such as 0.5 to 1.0 M NaCl or KCl buffer solution) to separate impurities from HPV L1 VLPs protein. Subsequently, high concentration salt-containing buffer solution (such as 1.0 to 2.0 M NaCl or KCl buffer solution) is used to elute the bound HPV L1 VLPs protein, and the eluted preliminarily-purified HPV L1 VLPs protein is collected. Thus obtained preliminarily-purified HPV L1 VLPs protein is loaded onto chromatographic media for fine purification, such as CHT (BIO-RAD Type II), to which HPV L1 VLPs protein can efficiently bind under the conditions of a certain range of salt concentration (such as 0.3 to 1.5 M NaCl or KCl buffer solution). Then the media is eluted using a phosphate concentration gradient (such as a phosphate concentration in the range of 20 to 400 mM) to separate impurities from HPV L1 VLPs. Alternatively, the preliminarily-purified HPV L1 VLPs protein can also be loaded onto such chromatographic media for fine purification as Sephacryl S-1000 (GE) and HW-75 (TSK) to achieve the separation of impurities from HPV L1 VLPs protein through gel chromatography. The eluted HPV L1 VLPs protein after fine purification is collected as the final purified sample.
The purification method according to the present invention can remove most of the contaminating biomolecules (including DNAs, lipids and proteins). Detection using reduced SDS-PAGE electrophoresis or capillary electrophoresis (Beckman Coulter) revealed that the sample obtained by purification using POROS 50HS chromatography media had a purity of 75% to 80%, and the final HPV L1 VLPs protein sample purified using hydroxyapatite media had a purity of 90% to 95%. Western blot (Bio-RAD) detection showed specific staining reaction between target protein band and monoclonal or polyclonal antibody to HPV L1 VLPs. Dynamic light scattering detection (Malvern Instruments Zetasizer Nano ZS) showed that the purified sample had particles in a size range of about 50 to 80 nm, and transmission electron microscopy observation (Philips) revealed virus-like particles (VLPs) in the sample, with the particle size in the range of about 50 to 80 nm. In the present experiment, the hydroxyapatite media used is most preferably ceramic hydroxyapatite media filler with a particle size in the range of about 20 to 50 nm and a pore size of about 800 Å. The buffers used in chromatography have a pH in the range of 6 to 9, the preferable buffering system being 50 mM MOPS.
In comparison with the prior art, the present invention optimally designed a gene for HPV 18 L1 (full-length gene, or preferably truncated gene corresponding to HPV 18 L1 protein with deletions of 61 amino acids at the N-terminus) and cloned it into Pichia yeast such that a markedly increased expression of HPV 18 L1 protein was achieved compared with other expression systems (such as mammal cell, baculovirus, Saccharomyces cerevisiae). Following the expression, the virus-like particles obtained through purification were observed by electron microscopy to be 50-80 nm in size, similar to wild-type HPV particles. The virus-like particles assembled from recombinant HPV 18 L1 protein were adsorbed to an aluminium adjuvant and used to immune mice, generating high titers of anti-HPV 18 L1 antibodies. Neutralization experiment using pseudotype virus showed that the antibodies had very good neutralizing activity (that is, capable of inhibiting entry of pseudotype virus into cells). In addition, the optimally designed gene for HPV 16 L1 according to the present invention, after being cloned into Pichia yeast, resulted in a very high level of expression of HPV 16 L1 protein.
Immunogenic Macromolecule
The present invention also provides an immunogenic macromolecule having a diameter of 50 to 80 nm, which is a poly-molecular polymer essentially self-assembled from major capsid proteins L1 of human papilloma virus, said major capsid proteins L1 of human papilloma virus being expressed by Pichia yeast.
Preferably, the immunogenic macromolecule according to the present invention is prepared by the following method: (1) culturing said recombinant cell to allow said major capsid protein L1 of human papilloma virus to be expressed and to concurrently self-assemble into immunogenic molecules in the recombinant cells; (2) disrupting the cells obtained from step (1) to obtain supernatant containing the immunogenic molecules; and (3) successively purifying the supernatant obtained from step (2) using POROS 50 HS column chromatography and CHT column chromatography to obtain said immunogenic macromolecules.
The present invention also provides the use of said immunogenic macromolecule in the manufacture of a composition for the prevention or treatment of diseases related to human papilloma virus (HPV) infection. Said diseases are selected from, but not limited to, malignancies (such as cervical cancer, vaginal cancer, anal or perianal cancer, oropharyngeal cancer, maxillary sinus cancer, lung cancer) and cervical intraepithelial neoplasia.
Composition
The present invention also provides an immunogenic composition (such as a preventive or therapeutic vaccine) which comprises an effective amount of said immunogenic macromolecule according to the present invention and a pharmaceutically acceptable carrier.
The present invention also provides a method for preparing a vaccine against human papilloma virus, comprising preparing virus-like particles of recombinant human papilloma virus protein L1 using the method as described above, and then adding pharmaceutically acceptable vaccine adjuvant. Said vaccine adjuvant can be an aluminium adjuvant or other adjuvant. The virus-like particles formed from purified human papilloma virus protein (HPV L1), when adsorbed to an adjuvant, can be used as a vaccine.
As used herein, the term “pharmaceutically acceptable” component refers to a substance suitable for use in human and/or mammal subjects without undue adverse side effects (such as toxicity), commensurate with a reasonable benefit/risk ratio. The term “pharmaceutically acceptable carrier” refers to carriers for the administration of therapeutic agents, including various excipients and diluents. This term refers to such carriers for therapeutic agents as are not per se the essential active components and are not unduly toxic after application. Suitable carriers are well-known to those of ordinary skill in the art. A full description of pharmaceutically acceptable carriers can be found in Reminton's Pharmaceutical Sciences (Mack Pub. Co., N.J. 1991). The pharmaceutically acceptable carriers for the composition may comprise liquids, such as water, saline, glycerol and sorbitol. Moreover, these carriers may have auxiliary agents present, such as for example lubricating agent, glidant, wetting agent or emulsifying agent, pH buffering substance and stabilizing agent, e.g., albumin, or the like.
Said composition can be formulated into various dosage forms suitable for administration to mammals, including but not limited to injections, capsules, tablets, emulsions, and suppositories.
Animal experiments have showed that immunization of the animals with the vaccine prepared using the immunogenic macromolecule according to the present invention induced strong immune responses in the animals.
When intended for use, the immunogenic macromolecule according to the present invention is administered to the mammal subject (such as human subject) in a safe and effective amount, wherein said safe and effective amount is typically at least about 1 μg/kg body weight and is in most cases not more than about 10 mg/kg body weight, preferably in the range of about 1 μg/kg body weight to about 1 mg/kg body weight. The particular dosage administered is, of course, dependent on such considerations as the route of administration, the general health of the patient and the like, which are within the skill of medical practitioners.
The present invention is further described in conjunction with the following specific examples which should be understood to be illustrative of the present invention rather than limitative of the scope of the present invention. The experiment procedures in the following examples for which no particular conditions are specified generally follow those conditions such as described in Sambrook et. al., Molecular Cloning: A Laboratory Manual, New York, Cold Spring Harbor Laboratory Press, 1989), or those conditions recommended by the manufacturers. Unless otherwise indicated, the percentages and parts are by weight.
In the examples of the present invention, heat activated Pfu enzyme purchased from Stratagene Co., Ltd was used for DNA extension and PCR amplification.
In the examples of the present invention, pUC18 plasmid was purchased from Generay Biotech (Shanghai) Co., Ltd (Shanghai, China), and pPICZαB plasmid was purchased from Invitrogen Corp.
In the examples of the present invention, rabbit polyclonal antibodies to HPV18 L1 and HPV16 L1 proteins were prepared by Shanghai PrimeGene Bio-tech, LTD (Shanghai, China), MAB885 murine monoclonal antibody was purchased from CHEMICON Co., Ltd, and HRP labeled sheep anti-mouse IgG was purchased from Being Dingguo Changsheng Biotechnology Co., Ltd (Beijing, China).
In the examples of the present invention, BALB/c mice were purchased from Shanghai SLAC laboratory Animal Co., Ltd. (Shanghai, China).
In the examples of the present invention, the washing buffer used in the purification step comprises: 100 mM PB, 0.15 M NaCl, pH 7.0; The buffer used in cell disruption: 200 mM MOPS, 0.4 M NaCl, 0.05% Tween-80, pH 7.0;
Buffer E: 50 mM MOPS, 0.5 M NaCl, 0.05% Tween-80, pH 6.5;
Buffer F: 50 mM MOPS, 1.5 M NaCl, 0.05% Tween-80, pH 6.5;
Buffer G: 50 mM MOPS, 0.6M NaCl, 0.05 M PB, 0.05% Tween-80, pH 6.5;
Buffer H: 0.6M NaCl, 200 mM PB, 0.05% Tween-80, pH 6.5.
Example 1
Design and Synthesis of Codon-Optimized Gene for HPV 18 L1
1.1. Design of Codon-Optimized Gene for HPV 18 L1
The present invention relates to DNA molecules encoding the major capsid protein L1 of human papilloma virus subtype 18 (HPV 18), which have been codon-optimized with codons preferred by Pichia yeast. Three DNA sequences, which are as set forth in SEQ ID NO: 4, 5 and 6, respectively, were obtained through optimization of codons and alteration of optimized codons, as detailed below.
Firstly, through repeated experiments, modifications were made on the naturally-occurring gene encoding HPV 18L1 by optimizing the codons for all the corresponding amino acids of the gene to design a novel HPV DNA sequence, that is, SEQ ID NO:4.
Then, in order to avoid the presence of a high GC ratio in the transcribed mRNAs, the influence of mRNA secondary structure on the efficiency of translation, and the occurrence of common restriction sites, alterations were made to the optimized codons, such as, the codon for asparagine (Asn) being altered from AAC to AAT, the codon for lysine (Lys) being altered from AAG to AAA, the codon for aspartic acid (Asp) being altered from GAT to GAC, the codon for phenylalanine (Phe) being altered from TTT to TTC, the codon for tyrosine (Tyr) being altered from TAC to TAT, and the codon for glycine (Gly) being altered from GGT to GGA, thus obtaining two altered, novel HPV DNA sequences: SEQ ID NO: 5, which was altered from SEQ ID NO: 4 through alterations to the codons for asparagine (Asn), lysine (Lys) and aspartic acid (Asp); and SEQ ID NO: 6, which was altered from SEQ ID NO: 4 through alterations to the codons for phenylalanine (Phe), tyrosine (Tyr) and glycine (Gly).
1.2. Synthesis of Codon-Optimized gene for HPV 18 L1
The codon-optimized gene for HPV 18 L1 as set forth in SEQ ID NO: 4 was synthesized and used as template to perform PCR amplification using primers a1 and a2 which have the following sequences:







(SEQ ID NO: 12)








a1:
5′-ATAGAATTCAAGATGTGTTTGTACACTAGAGTTT-3′;


 







(SEQ ID NO: 13)








a2:
5′-AATGGTACCCTATTACTTTCTAGCTCTAACT-3′.






The PCR products obtained were subjected to separation by agarose gel electrophoresis and the target sequence was recovered from the gel, obtaining a fragment about 1.7 kb in size. This fragment was sequenced, with the result of sequencing as shown in SEQ ID NO: 4, and was demonstrated from the result to be the full-length codon-optimized gene for HPV 18L1. Thus obtained gene for HPV 18L1, by virtue of the EcoRI and KpnI restriction sites on its ends, was ligated into pUC18 plasmid (available from Generay Biotech (Shanghai) Co., Ltd (Shanghai, China)). The resulting construct was verified by sequencing to be correct, and was named as pUC-18L1.
Codon-optimized genes for HPV 18L1 as shown in SEQ ID NO: 5 and 6 were obtained in a similar procedure as above mentioned.
In order to verify the feasibility of the optimized sequences, one of the codon-optimized genes for HPV 16L1 obtained in this Example 1 (SEQ ID NO: 4) was exemplarily used to construct an expression plasmid to assess for its expression, as detailed in Example 2.
Example 2
Construction of Expression Vector of HPV 18L1 Gene
The optimized sequence of SEQ ID NO: 4 was cloned into Pichia yeast expression vector, as depicted in FIG. 1 and as detailed in the following steps.
2.1. The Forward and Reverse Primers Having the Following Sequences, which were Required for Amplifying HPV 18L1 Gene, were Synthesized:









forward primer:



(SEQ ID NO: 14)



5′-TCCCAATCTTCGAAACGATGTGTTTGTACACTAGAGTTT-3′;



 



reverse primer:



(SEQ ID NO: 13)



5′-AATGGTACCCTATTACTTTCTAGCTCTAACT-3′;






Wherein the forward primer comprises a BstBI restriction site, and the reverse primer comprises a KpnI restriction site flanking the termination codon, said restriction sites respectively as shown in the underlined portions of the primer sequences above.
2.2. PCR amplification was performed using the above primers and using the pUC-18L1 obtained in Example 1 as template. The amplified product was detected by electrophoresis, with the result being shown in FIG. 2, demonstrating that a full-length codon-optimized gene for HPV 18L1 was obtained. The PCR amplified fragment was digested with BstBI and KpnI (restriction endonucleases) and then ligated with pPICZαB (Invitrogen Corp.) which was also digested with BstBI and KpnI. Then, the ligated construct was used to transform competent cells of E. coli Top10 strain (available from Generay Biotech (Shanghai) Co., Ltd (Shanghai, China)), and the transformed cells were plated onto LB agar containing 25 μg/ml of zeocin.
The transformed cells were able to grow on LB media containing zeocin as the pPICZαB vector carried zeocin-resistance gene. Single colonies of the transformed cells were separated to prepare plasmid DNA. HPV 18L1 gene and vector sequence were detected through restriction mapping (see FIG. 3) and nucleotide sequence analysis to identify the correct construct comprising HPV 18L1 gene, which was named pPICZ-18L1. As secretion signal (a-factor signal) has been cleaved from the constructed plasmid, HPV 18L1 protein should be intracellularly expressed protein.
Example 3
Construction and Expression of HPV 18L1 Gene-Expressing Strain
3.1. Construction of HPV 18L1 Gene-Expressing Strain
pPICZ-18L1 plasmid was linearized with a restriction endonuclease enzyme SacI, and empty plasmid pPICZαB was likewise digested with Sad to serve as negative control. The enzyme digestion solution was added with absolute ethanol to obtain DNA precipitate. The linearized pPICZ-18L1 fragment was dissolved with a small amount of double-distilled water and used to transform Pichia yeast strain X-33 (Invitrogen Corp.) by electroporation under the following conditions: DNA fragment, 5 μg; voltage, 1,500 volts; resistance, 25 omhs; electroporation time, 5 milliseconds. The electroporated cells were plated onto YPDS agar containing 200 μg/ml zeocin. As the pPICZαB vector carried zeocin-resistance gene, the transformed cells were able to grow on YPDS media containing zeocin. Single colonies of transformed cells were isolated, thus obtaining HPV 18L1 gene-expressing Pichia yeast strain.
3.2. Expression of HPV 18L1 Gene-Expressing Pichia Yeast Strain
The obtained HPV 18L1 gene-expressing Pichia yeast strain was plated onto resistance plates containing 1,000 μg/ml or 1,500 μg/ml zeocin. Clones obtained from plates having high concentration of resistance were separately cultured in 4 mL of YPD liquid culture media for 24 hours followed by induction of gene expression in BMMY culture media for 48 hours. Then cells were harvested by centrifugation and some of the cells were disrupted to obtain supernatant for performing Western blotting identification. The result of identification is shown in FIG. 4, which indicated the presence of HPV 18L1 protein in the supernatant.
Although SEQ ID NO: 4 sequence was used for cloning and expression in Examples 2 and 3 of the present invention, it will be obvious to those skilled in the art that similar results can be obtained when SEQ ID NO: 5 and 6 sequences are used for cloning and expression, and therefore these two sequences are also within the scope of the present invention. Moreover, those skilled in the art will, in light of the spirit of the present invention, readily construct similar sequences and clone and express the constructed sequences in Pichia yeast to obtain similar or better results, and therefore these sequences are also deemed to be within the scope of the present invention.
Example 4
Purification of HPV 18L1 Protein
The cells prepared in Example 3 were disrupted and centrifuged, and the resulting supernatant was subjected to purification by chromatography to obtain HPV 18L1 protein which has self-assembled into virus-like particles, as detailed below.
HPV 18L1 gene-expressing Pichia yeast cells were mixed with washing buffer in a ratio of 1:3 and shaken well, and the mixture was centrifuged at 8,000 rpm for 5 minutes to collect the cells. The above procedure was repeated twice.
The washed cells were mixed with cell disruption buffer in a ratio of 1:5 and shaken well, and the cells in the resulting suspension were disrupted under high pressure. The above procedure was repeated so that 90% of the cells were disrupted. The resulting disrupted cell solution was centrifuged at 9,000 rpm at 10° C. for 30 minutes and the supernatant was collected.
The supernatant obtained was preliminarily purified on POROS 50HS (Applied Biosystems Co., Ltd) chromatography column by eluting with a linear gradient of 100% buffer E to 100% buffer F and collecting the elution fractions. Sample was taken from the purified elution fractions and reduced to form monomeric proteins for detection by SDS-PAGE and Western bloting.
The elution fractions containing HPV 18L1 protein were combined and subjected to fine purification on CHT (BIO-RAD Type II) chromatography column eluted with a linear gradient of 100% buffer G to 100% buffer H. The elution fractions were collected, from which sample was taken and reduced to form monomeric proteins for detection by SDS-PAGE and Western bloting. The elution fractions containing HPV 18L1 protein were combined, thereby obtaining the final purified sample with a purity of greater than 90%.
Example 5
Preparation of HPV 18L1 Vaccine
The purified HPV 18L1 protein obtained in Example 4 above was adsorbed to an aluminium adjuvant to prepare an immunogenic HPV 18L1 vaccine according to the method described in Chinese Pharmacopoeia (2005 edition).
Example 6
Determination of Immunogenicity of the Expression Product of HPV 18L1 Gene
Twenty-four 6 to 8 weeks old SPF BALB/c mice were divided into 4 groups, with 6 mice in each group. Mice in the first group (as the negative control group) were immunized by cutaneous injection of 0.1 mL of aluminium adjuvant-containing buffer (0.32 M sodium chloride, 0.35 mM sodium borate, 0.01% Tween-80, 0.01 M histidine, pH 6.5) for three times on days 0, 7 and 21 respectively, and mice in the other three groups (as the test groups) were similarly immunized with 0.1 mL of aluminium adjuvant-adsorbed VLPs at a concentration of 5 μg/mL, 0.5 μg/mL and 0.05 μg/mL respectively. Blood samples were collected two weeks after the third immunization. The blood samples collected were stood at 37° C. for 2 hours and then centrifuged at 4,000 g for 10 minutes. The supernatant, which was obtained as mouse polyclonal antiserum, was aspirated and stored at −20° C. Additionally, the supernatant was assayed for seroconversion rate and titer, as detailed below.
6.1. Detection of Seroconversion Rate
The purified HPV 18L1 protein expressed by Pichia yeast was diluted with coating solution to 1 μg/mL. An aliquot of 0.1 mL of the dilution was added into each well of an ELISA plate, and the plate was incubated at 4° C. overnight. The coating solution was removed from each well, which was then washed with 0.3 mL of PB ST. Then, each well was blocked by incubating with 0.3 mL of blocking solution (5% skimmed milk powder+PBST) at 37° C. for 2 hours. Each well was added with 0.1 mL of test sera (serum obtained from mice immunized with aluminium adjuvant-adsorbed HPV 18L1 protein and serum obtained from mice immunized with aluminium adjuvant alone) solution obtained by diluting the sera with dilution buffer (2% skimmed milk powder+PBST) in a ratio of 1:400, and then incubated at 37° C. for 1 hour. Then the test sera solution was removed and each well was washed with 0.3 mL of washing buffer. Subsequently, each well was added with 0.1 mL of HRP labeled goat anti-mouse IgG obtained by diluting the IgG with dilution buffer in a ratio of 1:5,000 and incubated at 37° C. for 0.5 hour. Then the ELISA solution was removed and each well was washed with 0.3 mL of washing buffer. Then, each well was added with 0.1 mL DAB color development solution, and interaction was allowed to occur in dark at room temperature for 20 minutes. Afterwards, 0.05 mL of 2 M H2SO4 stop solution was added to each well to stop the reaction, and OD450 value was determined using an ELISA reader, with the readings shown in Table 2 below.







TABLE 2







OD450 readings














Mouse 1
Mouse 2
Mouse 3
Mouse 4
Mouse 5
Mouse 6
















5 μg immu-
1.231
1.208
1.229
1.208
1.106
1.666


nization group








0.5 μg immu-
1.150
1.244
1.227
1.158
1.252
1.171


nization group








0.05 μg immu-
1.424
0.357
1.182
1.101
1.164
2.448


nization group








Aluminium 
0.087
0.204
0.155
0.113
0.143
0.100


adjuvant group








Cutoff value
0.263









The Cutoff value is the sum of the average of the OD450 values of antibody to test serum of negative control (mouse serum obtained from immunization with aluminium adjuvant) plus three times the standard deviation. Mouse with an OD450 value greater than the Cutoff value was regarded as positive, and mouse with an OD450 value lower than the Cutoff value was regarded as negative. The results of seroconversion rate for the three test groups are shown in Table 3 below.







TABLE 3







Results of seroconversion rate











5 μg/mL
0.5 μg/mL
0.05 μg/mL



immunization
immunization
immunization



group
group
group














Seroconversion
100%
100%
100%


rate









6.2. Determination of Sera Titers

The purified HPV 18L1 protein was diluted with coating solution to 1 μg/mL. An aliquot of 0.1 mL of the dilution was added into each well of an ELISA plate, and the plate was incubated at 4° C. overnight. The coating solution was removed from each well, which was then washed with 0.3 mL of PB ST. Then, each well was blocked by incubating with 0.3 mL of blocking solution (5% skimmed milk powder+PBST) at 37° C. for 2 hours. The test sera (sera obtained from mice immunized with HPV 18L1 protein) was serially double diluted with dilution buffer (2% skimmed milk powder+PBST) from 1:500 dilution to 1:32,000 dilution, while the negative control serum (serum obtained from mice immunized with aluminium adjuvant) was diluted in a ratio of 1:10,000. Each well was added with an aliquot of 0.1 mL of diluted serum (test serum or negative control serum) and incubated at 37° C. for 1 hour. Then the test serum solution was removed and each well was washed with 0.3 mL of washing buffer. Subsequently, each well was added with 0.1 mL of HRP labeled sheep anti-mouse IgG obtained by diluting the IgG with dilution buffer in a ratio of 1:5,000, and incubated at 37° C. for 0.5 hour. Then the ELISA solution was removed and each well was washed with 0.3 mL of washing buffer. Then, each well was added with 0.1 mL DAB color development solution, and interaction was allowed to occur at room temperature for 20 minutes. Afterwards, 0.05 mL of 2 M H2SO4 stop solution was added to each well to stop the reaction, and OD450 value was determined using an ELISA reader.
Sera titers were calculated by end point titration method, with the results shown in Table 4 below.







TABLE 4







Results of titer determination














Mouse 1
Mouse 2
Mouse 3
Mouse 4
Mouse 5
Mouse 6





5 μg immu-
  8383
1315490
896331
2029474
 43088
 23781


nization group








0.5 μg immu-
1154285
 342344
415340
 149924
1003880
103784


nization group








0.05 μg immu-
 24237
  1000
306880
 341405
 564089
 27950


nization group









In summary, as shown in Examples 1-6, the gene for major capsid protein L1 of human papilloma virus subtype 18, as provided by the present invention, is an optimized HPV 18L1 gene, which has the advantages of being more suitable for efficiently expressing target protein in yeast host and meeting the requirements of industrial production. Moreover, the HPV 18L1 vaccine as provided by the present invention, which was prepared from adjuvant-adsorbed purified VLPs (virus-like particles self-assembled from HPV 18L1 protein), was demonstrated to be strongly immunogenic in mice as determined by seroconversion rate and serum titer. Further, said method has the advantages of low cost, high yield and more uniform and stable quality of products due to the use of Pichia yeast expression system.
Example 7
Design and Synthesis of Codon-Optimized Gene for Truncated HPV 18 L1
7.1. Design of Codon-Optimized Gene for Truncated HPV 18 L1
The present invention relates to DNA molecules encoding the truncated major capsid protein L1 of human papilloma virus subtype 18 (HPV18), which have been codon-optimized with codons preferred by Pichia yeast. Three DNA sequences, which are as set forth in SEQ ID NO: 1, 2 and 3, respectively, were obtained through optimization of codons, alteration of optimized codons and truncation of 61 amino acids from the N terminus, as detailed below.
Firstly, modifications were made on the naturally-occurring gene encoding HPV 18L1 in a similar manner as in Example 1 above to design a novel HPV DNA sequence.
Then, in order to avoid the presence of a high GC ratio in the transcribed mRNAs, the influence of mRNA secondary structure on the efficiency of translation, and the occurrence of common restriction sites, alterations were made to the optimized codons, such as, the codon for asparagine (Asn) being altered from AAC to AAT, the codon for lysine (Lys) being altered from AAG to AAA, the codon for aspartic acid (Asp) being altered from GAT to GAC, the codon for phenylalanine (Phe) being altered from TTT to TTC, the codon for tyrosine (Tyr) being altered from TAC to TAT, and also 61 amino acids were truncated from the N terminus, thus obtaining three novel truncated HPV DNA sequences, wherein:
SEQ ID NO: 1 is the DNA sequence without codon alteration;
SEQ ID NO: 2 is the DNA sequence obtained from SEQ ID NO: 1 by altering the codons for asparagine (Asn), lysine (Lys) and aspartic acid (Asp);
SEQ ID NO: 3 is the DNA sequence obtained from SEQ ID NO: 1 by altering the codons for phenylalanine (Phe), tyrosine (Tyr) and glycine (Gly).
7.2. Synthesis of Codon-Optimized Gene for Truncated HPV 18 L1
The full-length codon-optimized gene for truncated HPV 18 L1, as set forth in SEQ ID NO: 1 was obtained using essentially the same method as described in Example 1 above mentioned. Thus obtained gene for truncated HPV 18L1, by virtue of the EcoRI and KpnI restriction sites on its ends, was ligated into pUC18 plasmid (available from Generay Biotech (Shanghai) Co., Ltd (Shanghai, China)). The resulting construct was verified by sequencing to be correct, and was named as pUC-18L1′.
The codon-optimized genes for truncated HPV 18 L1, as set forth in SEQ ID NO: 2 and 3, were obtained in a similar method.
In order to verify the feasibility of the optimized sequences, one of the codon-optimized genes for truncated HPV 18L1 obtained in this Example 7 (SEQ ID NO: 1) was exemplarily used to construct an expression plasmid to assess for its expression, as detailed in Example 8.
Example 8
Construction of Expression Vector of Truncated HPV 18L1 Gene
The optimized sequence of SEQ ID NO: 1 was cloned into Pichia yeast expression vector, as detailed in the following steps.
8.1. The Forward and Reverse Primers Having the Following Sequences, which Were Required for Amplifying HPV 18L1 Gene, Were Synthesized:









forward primer:



(SEQ ID NO: 15)



5′-TCCCAATCTTCGAAACGATGGCTTTGTGGA-3′;



 



reverse primer:



(SEQ ID NO: 13)



5′-AATGGTACCCTATTACTTTCTAGCTCTAACT-3′.






Wherein the forward primer comprises a BstBI restriction site, and the reverse primer comprises a KpnI restriction site flanking the termination codon, said restriction sites respectively as shown in the underlined portions of the primer sequences above.
8.2. PCR amplification was performed using the above primers and using the pUC-18L1′ obtained in Example 7 as template. The amplified product was detected by electrophoresis, with the result being shown in FIG. 5, demonstrating that a full-length codon-optimized gene for truncated HPV 18L1 was obtained. The PCR amplified fragment was digested with BstBI and KpnI (restriction endonucleases) and then ligated with pPICZαB (Invitrogen Corp.) which was also digested with BstBI and KpnI. Then, the ligated construct was used to transform competent cells of E. coli Top10 strain (available from Generay Biotech (Shanghai) Co., Ltd (Shanghai, China)), and the transformed cells were plated onto LB agar containing 25 μg/ml of zeocin.
The transformed cells were able to grow on LB media containing zeocin as the pPICZαB vector carried zeocin-resistance gene. Single colonies of the transformed cells were separated to prepare plasmid DNA. HPV 18L1 gene and vector sequence were detected through restriction mapping (see FIG. 6) and nucleotide sequence analysis to identify the correct construct comprising truncated HPV 18L1 gene, which was named pPICZ-18L1′. As secretion signal (a-factor signal) has been cleaved from the constructed plasmid, therefore the HPV 18L1 protein expressed should be intracellularly expressed protein.
Example 9
Construction and Expression of Truncated HPV 18L1 Gene-Expressing Strain
9.1. Construction of Truncated HPV 18L1 Gene-Expressing Strain
pPICZ-18L1′ plasmid was linearized with a restriction endonuclease enzyme SacI, and empty plasmid pPICZαB was likewise digested with Sad to serve as negative control. The enzyme digestion solution was added with absolute ethanol to obtain DNA precipitate. The linearized pPICZ-18L1′ fragment was dissolved with a small amount of double-distilled water and used to transform Pichia yeast strain X-33 (Invitrogen Corp.) by electroporation under the following conditions: DNA fragment, 5 μg; voltage, 1,500 volts; resistance, 25 omhs; electroporation time, 5 milliseconds. The electroporated cells were plated onto YPDS agar containing 200 μg/ml zeocin (from Zeocin Co.). As the pPICZαB vector carried zeocin-resistance gene, the transformed cells were able to grow on YPDS media containing zeocin. Single colonies of transformed cells were isolated, thus obtaining truncated HPV 18L1 gene-expressing Pichia yeast strain.
9.2. Expression of Truncated HPV 18L1 Gene-Expressing Pichia Yeast Strain
The obtained truncated HPV 18L1 gene-expressing Pichia yeast strain was plated onto resistance plates containing 1,000 μg/ml or 1,500 μg/ml zeocin. Clones obtained from plates having high concentration of resistance were separately cultured in 4 mL of YPD liquid culture media for 24 hours followed by induction of gene expression in BMMY culture media for 48 hours. Then cells were harvested by centrifugation and some of the cells were disrupted to obtain supernatant for performing Western blotting identification. The result of identification is shown in FIG. 7, which indicated the presence of truncated HPV 18L1 protein in the supernatant.
Although SEQ ID NO: 1 sequence was used for cloning and expression in Examples 8 and 9 of the present invention, it will be obvious to those skilled in the art that similar results can be obtained when SEQ ID NO: 2 and 3 sequences are used for cloning and expression, and therefore these two sequences are also within the scope of the present invention. Moreover, those skilled in the art will, in light of the spirit of the present invention, readily construct similar sequences and clone and express the constructed sequences in Pichia yeast to obtain similar or better results, and therefore these sequences are also deemed to be within the scope of the present invention.
Example 10
Large-Scale Expression and Detection of Truncated HPV 18L1 Protein
1. Large-Scale Expression of Recombinant Truncated HPV 18L1 Protein
Preparation of inoculum solution: One glycerol freezing tube of recombinant yeast stock was taken from working cell bank. After thawing, 100 μL of the stock was inoculated into 5 mL of YPD media and cultured at 280 rpm at 30° C. for 20 hours, at which time OD600 was detected to be 1 to 2 and no contamination from other microorganisms was microscopically observed. 1 mL of the activation fluid (which has been tested to be satisfactory) was inoculated into 500 mL of YPD media and cultured at 280 rpm at 30° C. for 20 hours, at which time OD600 was detected to be 2 to 6 and no contamination from other microorganisms was microscopically observed.
Process of fermentation: Basal salt medium for fermentation was prepared using deionized water: BSM1 (K2SO4 273 g, MgSO4 109 g, CaSO4.2H2O 17.6 g, H3PO4 400.5 mL, KOH 62 g, glycerol 600 g, PTM1 60 mL, defoamer 1 mL; add deionized water to a volume of 15 L). The prepared medium, which contained no antibiotics, was transferred into a 30 L fermentation tank (Bioengineering Co., Ltd) and sterilized in the tank at 121° C. for 30 minutes, followed by cooling to 30° C. The inoculum solution previously prepared was used to inoculate the sterilized medium in a ratio of 1:15. Fermentation was started at a temperature of 30.0±0.5° C., an initial pH of 5.00±0.05, an initial stirring speed of 300 rpm, an aeration volume of 0.5 vvm and a dissolved oxygen (DO) of 100%, with addition of trace salts PTM1 (CuSO4.5H2O 6.0 g, NaI 0.008 g, MnSO4 3.0 g, NaMoO4 0.2 g, H3BO3 0.02 g, ZnSO4 20.0 g, CoCl2 0.5 g, FeSO4.7H2O 65.0 g, biotin 0.2 g, and H2SO4 5.0 mL; add deionized water to a volume of 1 L). The initial proliferation stage lasted for about 20 hours, during which time the dissolved oxygen was maintained at no lower than 30%. When the carbon source was completely consumed, the dissolved oxygen increased rapidly, at which time the wet cell weight was up to about 100 g/L. In the initial two hours, the fermentation was supplemented with 50% (v/v) glycerol solution (added with 12 mL of PTM1 per liter) at a rate of 200 mL/h Then the supplementation was continued at a rate of 300 mL/h for about 8 hours, during which time the dissolved oxygen was maintained at higher than 20% by adjusting the stirring speed, air flow and tank pressure (<0.8 bar). When the wet cell weight reached about 350 g/L, supplementation was stopped, and the dissolved oxygen increased accordingly. Meanwhile, the pH value was adjusted to 6.00±0.05, and methanol (added with 12 mL of PTM1 per liter) was added at an initial addition rate of 30 mL/h to induce gene expression. The addition rate of methanol was increased slowly until a set rate of 90 mL/h after 4 hours. During induction, the dissolved oxygen was maintained at higher than 20% (v/v), the temperature at 30° C. and the pH at 6.00±0.05. Sample was taken every eight hours and subjected to Western blotting detection. Forty-eight hours after induction, the fermentation completed and the fermentation broth was discharged.
Harvest of cells: The fermentation broth was centrifuged using a cryo-centrifuge to collect the cells, which were then weighed. The batch number, date and weight for the recovered cells were recorded, and the cells were sent for purification or stored at −20° C.
2. Detection
The purified (according to the method described in Example 11 below) truncated HPV 18L1 protein was used to prepare a protein concentration standard curve. With cells before induction as negative control, the expression amount of HPV 18L1 gene in Pichia yeast during fermentation was detected using sandwich ELISA method, as detailed below.
Rabbit polyclonal antibody to HPV 18L1 protein was diluted 2,000 fold with coating solution. An aliquot of 0.1 mL of the diluted rabbit polyclonal antibody was added to each well of an ELISA plate, and the plate was incubated at 4° C. overnight. Then the coating solution was removed from each well, which was then washed with 0.3 mL of PB ST. Then, each well was blocked by incubating with 0.3 mL of blocking solution at 37° C. for 2 hours.
The purified HPV 18L1 protein was gradiently diluted with dilution buffer from a concentration of 2 μg/mL to 0.0625 μg/mL. Meanwhile, the supernatant obtained from disrupted fermentation broth was diluted 200 fold. Then, for each of the HPV 18 L1 protein solutions of different concentrations obtained by the serial dilution and for the diluted supernatant, an aliquot of 0.1 mL was taken and added to a separate well of the above-treated ELISA plate, and the plate was incubated at 37° C. for 1 hour. After incubation, the aliquot of antigen solution was removed from each well, which was then washed with 0.3 mL of washing buffer. Then, MAB885 mouse monoclonal antibody (purchased from CHEMICON Co., Ltd) was diluted 1,000 fold with dilution buffer, and an aliquot of 0.1 mL of the dilution was added to each well and incubated at 37° C. for 1 hour. Following incubation, the monoclonal antibody solution was removed from each well, which was then washed with 0.3 mL of washing buffer. Subsequently, each well was added with 0.1 mL of HRP labeled goat anti-mouse IgG obtained by diluting the IgG 5,000-fold with dilution buffer, and incubated at 37° C. for 0.5 hour. Then the ELISA solution was removed and each well was washed with 0.3 mL of washing buffer. Then, each well was added with 0.1 mL DAB color development solution, and interaction was allowed to occur at room temperature for 20 minutes. Afterwards, 0.05 mL of 2 M H2SO4 stop solution was added to each well to stop the reaction, and OD450 value was determined using an ELISA reader.
The OD450 values for the gradiently diluted solutions of HPV 18L1 protein were used to prepare a protein concentration standard curve. Using this standard curve, the expression amount of truncated HPV 18L1 protein during fermentation was obtained, as shown in Table 5 below.
Particularly speaking, the purified target protein stock solution with known concentrations was serially diluted into solutions of a range of concentrations, such as 2 μg/mL, 1 μg/mL, 0.5 μg/mL, 0.25 μg/mL and 0.125 μg/mL, which were used as standard concentrations. ELISA detection was conducted on these solutions to obtain the corresponding OD450 values. Then a standard linear regression equation was generated, with the ordinate representing the concentration and the abscissa representing the OD450 value.
The supernatant obtained from the disrupted fermentation broth was serially diluted, such as being diluted 50 fold, 100 fold, 200 fold and 400 fold. OD450 values were detected for these serial dilutions, and the corresponding concentrations (μg/mL) were found from the standard linear regression equation. The concentration of the target protein in the supernatant (μg/mL) was then calculated by multiplying the found concentration with the corresponding dilution factor. As the disrupted broth was prepared from wet cell weight:cell disruption buffer=1:5, therefore the expression amount of the target protein in the wet cells (μg/g wet cells) was 5× the concentration of the target protein in the supernatant (μg/mL). And the concentration of expression of the target protein in the fermentation broth (μg/L fermentation broth) was obtained by multiplying the above-obtained expression amount of the target protein in the wet cells with the density of cells in the fermentation broth (g wet cells/L fermentation broth).

Concentration of the target protein in the supernatant obtained from the disrupted fermentation broth(μg/mL)=Dilution factor×Concentration of standard target protein concentration(μg/mL)×OD450(supernatant obtained from the disrupted fermentation broth)/OD450(standard target protein concentration);

Concentration of the expressed target protein in the fermentation broth(μg/L fermentation broth)=5×Concentration of the target protein in the supernatant obtained from the disrupted fermentation broth(μg/mL)×Density of cells in the fermentation broth(g wet cells/L fermentation broth).








TABLE 5







Detection of expression amount of truncated HPV 18 L1 gene in Pichia yeast during


fermentation

















Concentration



Expression





of stock
Average


amount



Dilution

solution
concentration
Converted
Fermentation
during


Sample
factor
OD450
(μg/mL)
(μg/mL)
concentration
density
fermentation

















Disrupted
200
0.455
161.4
176
880 μg/g 
438 g/L
385 mg/L


fermentation




wet cells

fermentation


broth






broth


Supernatant 1
100
0.718
185.6






Disrupted
200
0.365
153.0
152
760 μg/g 
413 g/L
314 mg/L


fermentation




wet cells

fermentation


broth






broth


Supernatant 2
100
0.612
160.5






Negative
100
0.091







control









Blank
—
0.083







control
















It can be seen from the results shown in Table 5 that, the optimized sequence of the gene for HPV 18L1 protein according to the present invention not only can be expressed into HPV 18L1 protein in Pichia yeast, but also has a high expression amount, meeting the requirements of industrial production.
Example 11
Purification of Truncated HPV 18L1 Protein
Cleaning:
The above-obtained Pichia yeast cells expressing truncated HPV 18 L1 protein, which had been stored at −20° C., was thawed at room temperature. Then the thawed cells were mixed with washing buffer (100 mM PB, pH 7.0, 0.15 M NaCl) or water for purification in a ratio of 1:3 (g/mL), and completely homogenized with the buffer or water in a homogenizer (FLUKO). Subsequently, the homogenate was subjected to high speed centrifugation (SORVALLRC6PLUS) at 8000 rpm for 5 minutes to separate the cells, the supernatant being decanted. The above procedure was repeated twice, thereby achieving the cleaning of the cells.
Disruption:
The cleaned cells were added and mixed into cell disruption buffer (100 mM MOPS, 0.75M NaCl, 0.05% Tween-80, pH 7.0) in a ratio of 1:5 (g/mL), and completely homogenized with the buffer in a homogenizer (FLUKO). The cells suspended in the homogenate were subjected to disruption using a high-pressure homogenizer (ATSAH110B) operating at a pressure in the range of 1200 to 1300 bars. This disruption was repeated four times, and then the disrupted cell solution was discharged at 4 to 8° C., with 90% of the cells being disrupted.
Clarification:
The disrupted cell solution obtained by high-pressure disruption as above was poured into a centrifugation cup and centrifuged at high speed to remove cell debris, obtaining supernatant for subsequent column chromatography separation. The centrifuge used is SORVALLRC6PLUS, rotor model: FIBERLITEF10-6x500y, centrifugation setting: 9,000 rpm, 30 min, 10° C.
Preliminary Purification:
Chromatography media POROS 50HS (Applied Biosystems) was loaded into a chromatography column (diameter 26 mm, height 10, volume 50 mL). Preliminary purification was conducted in the following steps: (1) washing and disinfection: the column was washed with two column volumes of 0.5 M NaOH; (2) regeneration and equilibration: the column was washed with two column volumes of Buffer F and then equilibrated with Buffer E; (3) loading sample: the supernatant obtained by centrifuging the disrupted cell solution as above was loaded onto the column; (4) rinsing: the column was rinsed with five column volumes of Buffer E until the baseline stabilized; (5) elution: the column was eluted with a linear gradient of 100% Buffer E to 100% Buffer F in a total elution volume of six column volumes; and (6) collection: the chromatographic peaks having a conductivity in the range of 70 to 100 ms/cm were collected and stored at 4° C.
Fine Purification:
Chromatography media CHT (BIO-RAD, Type II, 40 μm) was loaded into a chromatography column (diameter 26 mm, height 10 cm, volume 50 mL). Fine purification was conducted in the following steps: (1) washing and disinfection: the column was washed with two column volumes of 0.5 M NaOH; (2) regeneration and equilibration: the column was washed with two column volumes of Buffer H and then equilibrated with Buffer G; (3) loading sample: the sample collected after preliminary purification was added with PB to a final concentration of 30 mM and then loaded onto the column; and (4) rinsing: the column was rinsed with five column volumes of Buffer G until the baseline stabilized; (5) elution: the column was eluted with a linear gradient of 100% Buffer G to 100% Buffer H; and (6) collection: the elution fractions were collected and the fractions containing truncated HPV 18L1 protein were combined to obtain the final purified sample. The purified sample was reduced to monomeric proteins which were then subjected to reduced SDS-PAGE (Bio-RAD), demonstrating a purity of greater than 90% (see FIG. 8).
Western blot (Bio-RAD) detection showed specific staining reaction between target electrophoresis band and monoclonal or polyclonal antibody to HPV 18 L1 protein (see FIG. 9). Dynamic light scattering detection (Malvern Instruments Zetasizer Nano ZS) showed that the purified sample had particles in a size range of about 50 to 80 nm, and electron microscopy observation (Philips Tecnai-12Biotwin transmission electron microscope, Electron Microscope Laboratory, Science and Technology Center, Shanghai University of Traditional Chinese Medicine, China) revealed virus-like particles (VLPs) in the purified sample, with the particle size in the range of about 50 to 80 nm (see FIG. 10). The full-length HPV 18L1 protein expressed as described hereinabove, when purified in the same manner, also afforded purified VLPs which were revealed by dynamic light scattering and electron microscopy detection to have the same particle size as truncated HPV 18L1 protein particles, ranging from 50 to 80 nm
Example 12
Preparation of Truncated HPV 18L1 Vaccine
The purified HPV 18L1 protein obtained in Example 11 above was adsorbed to an aluminium adjuvant to prepare an immunogenic HPV 18L1 vaccine according to the method described in Chinese Pharmacopoeia (2005 edition).
Example 13
Determination of Immunogenicity of the Expression Product of Truncated HPV 18L1 Gene
Twenty-four 6 to 8 weeks old SPF BALB/c mice were divided into 4 groups, with 6 mice in each group. Mice in the first group (as the negative control group) were immunized by hypodermic injection of 0.1 mL of aluminium adjuvant-containing buffer (0.32 M sodium chloride, 0.35 mM sodium borate, 0.01% Tween-80, 0.01 M histidine, pH 6.5) for three times on days 0, 7 and 21 respectively, and mice in the other three groups (as the test groups) were similarly immunized with 0.1 mL of aluminium adjuvant-adsorbed VLPs at a concentration of 5 μg/mL, 0.5 μg/mL and 0.05 μg/mL respectively. Blood samples were collected two weeks after the third immunization. The blood samples collected were stood at 37° C. for 2 hours and then centrifuged at 4,000 g for 10 minutes. The supernatant, which was obtained as mouse polyclonal antiserum, was aspirated and stored at −20° C. Additionally, the supernatant was assayed for seroconversion rate and titer, as detailed below.
13.1. Detection of Seroconversion Rate
The purified truncated HPV 18L1 VLPs expressed by Pichia yeast were diluted with coating solution to 1 μg/mL. An aliquot of 0.1 mL of the dilution was added into each well of an ELISA plate, and the plate was incubated at 4° C. overnight. The coating solution was removed from each well, which was then washed with 0.3 mL of PBST. Then, each well was blocked by incubating with 0.3 mL of blocking solution (5% skimmed milk powder+PBST) at 37° C. for 2 hours. Each well was added with 0.1 mL of test serum (sera obtained from mice immunized with aluminium adjuvant-adsorbed HPV 18L1 protein and serum obtained from mice immunized with aluminium adjuvant alone) solution obtained by diluting the serum with dilution buffer (2% skimmed milk powder+PBST) in a ratio of 1:400, and then incubated at 37° C. for 1 hour. Then the test serum solution was removed and each well was washed with 0.3 mL of washing buffer. Subsequently, each well was added with 0.1 mL of HRP labeled goat anti-mouse IgG obtained by diluting the IgG with dilution buffer in a ratio of 1:5,000 and incubated at 37° C. for 0.5 hour. Then the ELISA solution was removed and each well was washed with 0.3 mL of washing buffer. Then, each well was added with 0.1 mL DAB color development solution and allowed to react in dark at room temperature for 20 minutes. After reaction, 0.05 mL of 2 M H2SO4 stop solution was added to each well to stop the reaction, and OD450 value was determined using an ELISA reader, with the readings shown in Table 6 below.







TABLE 6







OD450 readings














Mouse 1
Mouse 2
Mouse 3
Mouse 4
Mouse 5
Mouse 6





5 μg immu-
0.964
0.990
0.901
0.820
1.040
1.193


nization group








0.5 μg immu-
0.769
0.898
0.830
1.706
0.763
0.982


nization group








0.05 μg immu-
0.933
0.953
1.422
1.502
0.572
1.489


nization group








Aluminium 
0.097
0.146
0.114
0.176
0.110
0.158


adjuvant group








Cutoff value
0.226









The Cutoff value is the sum of the average of the OD450 values of antibody to test serum of negative control (mouse serum obtained from immunization with aluminium adjuvant) plus three times the standard deviation. Mouse with an OD450 value greater than the Cutoff value was regarded as positive, and mouse with an OD450 value lower than the Cutoff value was regarded as negative. The results of seroconversion rate for the three test groups are shown in Table 7 below.







TABLE 7







Results of seroconversion rate











5 μg/mL
0.5 μg/mL
0.05 μg/mL



immunization
immunization
immunization



group
group
group














Seroconversion
100%
100%
100%


rate









13.2. Determination of Serum Titer

The purified truncated HPV 18L1 protein was diluted with coating solution to 1 μg/mL. An aliquot of 0.1 mL of the dilution was added into each well of an ELISA plate, and the plate was incubated at 4° C. overnight. The coating solution was removed from each well, which was then washed with 0.3 mL of PBST. Then, each well was blocked by incubating with 0.3 mL of blocking solution (5% skimmed milk powder+PBST) at 37° C. for 2 hours. The test sera (sera obtained from mice immunized with HPV 18L1 protein) was serially double diluted with dilution buffer (2% skimmed milk powder+PBST) from 1:500 dilution to 1:32,000 dilution, while the negative control serum (serum obtained from mice immunized with aluminium adjuvant) was diluted in a ratio of 1:10,000. Each well was added with an aliquot of 0.1 mL of diluted serum (test serum or negative control serum) and incubated at 37° C. for 1 hour. Then the test serum solution was removed and each well was washed with 0.3 mL of washing buffer. Subsequently, each well was added with 0.1 mL of HRP labeled goat anti-mouse IgG obtained by diluting the IgG with dilution buffer in a ratio of 1:5,000, and incubated at 37° C. for 0.5 hour. Then the ELISA solution was removed and each well was washed with 0.3 mL of washing buffer. Then, each well was added with 0.1 mL DAB color development solution and allowed to react at room temperature for 20 minutes. After reaction, 0.05 mL of 2 M H2SO4 stop solution was added to each well to stop the reaction, and OD450 value was determined using an ELISA reader. Serum titers were calculated by end point titration method, with the results shown in Table 8 below.







TABLE 8







Results of titer determination














Mouse 1
Mouse 2
Mouse 3
Mouse 4
Mouse 5
Mouse 6
















5 μg immu-
 418416
83602
252997
192334
470324
215223


nization group








0.5 μg immu-
1073694
13854
189584
 18371
226400
192297


nization group








0.05 μg immu-
 79083
68971
 61310
 11819
 2000
 14099


nization group









In summary, as shown in Examples 1-13, the gene for major capsid protein L1 of human papilloma virus subtype 18 or the gene for truncated major capsid protein L1 of human papilloma virus subtype 18, as provided by the present invention, is an optimized L1 gene, which has the advantages of being more suitable for efficiently expressing target protein in yeast host and meeting the requirements of industrial production. Moreover, the HPV 18L1 vaccine as provided by the present invention can be self-assembled into the structure of VLPs. The vaccine was demonstrated to be strongly immunogenic in mice as determined by seroconversion rate and serum titer with the adjuvant-adsorbed purified VLPs. Further, the method has the advantages of low cost, high yield, and more uniform and stable quality of products due to the use of Pichia yeast expression system.
Example 14
Neutralization Activity of Truncated HPV 18 L1 VLPs
Mice were immunized with the same procedure as described above and blood was collected two weeks after immunization. The collected blood was stood at 37° C. for 2 hours, then centrifuged at 4,000 g for 10 minutes. The supernatant, which was obtained as mouse polyclonal antiserum, was aspirated and stored at −20° C.
293FT cells (Invitrogen) were initially plated onto 15 cm cell culture dish (1.1×107 cells/dish). Twenty-four hours later, the cells were cotransfected with plasmids p18L1h, p18L2h (Buck C B, Pastrana D V, Lowy D R et al. Efficient Intracellular Assembly of Papillomaviral Vectors. J Virol, 2004, 78(2):751-757) and green fluorescent gene-carrying pIRES2-EGFP (purchased from BD Biosciences Clontech) respectively in an amount of 20 μg, 10 μg and 10 μg using calcium phosphate transfection method. Forty-eight hours later, cells were observed, collected and lysed. Cell lysis supernatant was immediately subjected to purification or stored at −80° C.
Purification was performed by centrifuging the cell lysis supernatant in a 5 mL centrifugation tube (Beckman) using 30% OptiPrep density gradient centrifugation at 100,000 g at 16° C. for 4 hours (MLS-50 centrifuge rotor, Beckman ultra-speed centrifuge). The centrifugation fractions were collected, each fraction being about 500 μL, and assayed for the content of HPV 18 L1 protein using Western blotting. The fractions having highest concentration of L1 protein were combined as pseudotype virus solution, which was then aliquoted and stored at −80° C.
293FT cells were plated onto 24-well cell culture plate (1.5×105 cells/well). Twenty-four hours later, neutralization experiment was done as follows. Different serum samples were serially diluted 100-fold with DMEM medium. An aliquot of 50 μL of each dilution was mixed with 50 μL of the pseudotype virus solution diluted in DMEM medium, and each mixture was incubated at 4° C. for 1 hour. Then each incubated mixture was added into the 24-well cell culture plate pre-plated with 293FT cells and incubated at 37° C. for 48 hours. After incubation, the cells were observed for expression of green fluorescent protein under an OLUMPUSCKX41F32FL inverted fluorescence microscope, and fluorescence images were collected, as shown in FIGS. 11a and 11b. Fluorescence was seen in FIG. 11a, which indicates that the HPV 18 pseudotype virus had infected the 293FT cells. Little fluorescence remained in FIG. 11b, which indicates that mouse serum had neutralized the HPV 18 pseudotype virus, thereby reducing the viral infection of the 293FT cells. It is therefore clear that mice immunized with recombinant HPV 18L1 VLPs vaccine could generate neutralizing antibody which would inhibit entry of the pseudotype virus into cells.
In the present invention, nucleotide sequence SEQ ID NO: 2 or 3 and nucleotide sequence SEQ ID NO: 5 or 6 can respectively be substituted for SEQ ID NO: 1 and SEQ ID NO: 4 in the above examples to prepare HPV 18 L1 protein and truncated form thereof of the present invention using the methods as described in the above examples. In addition, the above-said genes can also be cloned, in appropriate cloning manner, into other existing Pichia yeast expression vector, such as pPIC6, pGAPZ or pAO815. Then the recombinant expression vectors obtained can be used to transform other Pichia yeast strains, such as Pichia pastoris GS115, KM71 or SMD1168 strains, so as to construct genetically engineered strains. Using conventional methods of culturing and fermentation, and separation and purification (which are not detailed herein), HPV 18 L1 protein (HPV 18 L1 VLPs) or truncated form thereof of the present invention can be obtained from the genetically engineered strains. Thus prepared HPV 18 L1 protein or truncated form thereof will have similar immunogenicity to that of the HPV 18 L1 protein or truncated form thereof prepared in the above examples.
Example 15
Design and Synthesis of Codon-Optimized Gene for HPV 16 L1
15.1. Design of Codon-Optimized Gene for HPV 16 L1
The present invention relates to DNA molecules encoding the major capsid protein L1 of human papilloma virus subtype 16 (HPV 16), which have been codon-optimized with codons preferred by Pichia yeast. Three DNA sequences, which are as set forth in SEQ ID NO: 7, 8 and 9, respectively, were obtained through optimization of codons and alteration of optimized codons, as detailed below.
Firstly, through repeated experiments, modifications were made on the naturally-occurring gene encoding HPV 16L1 by optimizing the codons for all the corresponding amino acids of the gene to design a novel HPV DNA sequence, that is, SEQ ID NO:7.
Then, in order to avoid the presence of a high GC ratio in the transcribed mRNAs, the influence of mRNA secondary structure on the efficiency of translation, and the occurrence of common restriction sites, alterations were made to the optimized codons, such as, the codon for asparagine (Asn) being altered from AAC to AAT, the codon for lysine (Lys) being altered from AAG to AAA, the codon for aspartic acid (Asp) being altered from GAT to GAC, the codon for phenylalanine (Phe) being altered from TTT to TTC, the codon for tyrosine (Tyr) being altered from TAC to TAT, and the codon for glycine (Gly) being altered from GGT to GGA, thus obtaining two novel HPV DNA sequences, wherein:
SEQ ID NO: 8 is the DNA sequence obtained from SEQ ID NO: 7 by altering the codons for asparagine (Asn), lysine (Lys) and aspartic acid (Asp); SEQ ID NO: 9 is the DNA sequence obtained from SEQ ID NO: 7 by altering the codons for phenylalanine (Phe), tyrosine (Tyr) and glycine (Gly).
15.2. Synthesis of Codon-Optimized Gene for HPV 16 L1
The codon-optimized gene for HPV 16 L1 as set forth in SEQ ID NO: 7 was synthesized and used as template to perform PCR amplification using primers a1 and a2 which have the following sequences:









(SEQ ID NO: 16)










a1:
5′-ATAGAATTCATGTCTTTGTGGTTGCCATC-3′;



 









(SEQ ID NO: 17)










a2:
5′-ATAGGTACCCTATTACAACTTTCTCTTCTTT-3′.






The PCR products obtained were subjected to separation by agarose gel electrophoresis and the target sequence was recovered from the gel, obtaining a fragment about 1.5 kb in size. This fragment was sequenced, with the result of sequencing as shown in SEQ ID NO: 7, and was demonstrated from the result to be the full-length codon-optimized gene for HPV 16L1. Thus obtained gene for HPV 16L1, by virtue of the EcoRI and KpnI restriction sites on its ends, was ligated into pUC18 plasmid (available from Generay Biotech (Shanghai) Co., Ltd (Shanghai, China)). The resulting construct was verified by sequencing to be correct, and was named as pUC-16L1.
Codon-optimized genes for HPV 16L1 as shown in SEQ ID NO: 8 and 9 were obtained in a similar procedure as above.
In order to verify the feasibility of the optimized sequences, one of the codon-optimized genes for HPV 16L1 obtained in this Example 15 (SEQ ID NO: 7) was exemplarily used to construct an expression plasmid to assess for its expression as follows.
Example 16
Construction of Expression Vector of HPV 16L1 Gene
The optimized sequence of SEQ ID NO: 7 was cloned into Pichia yeast expression vector as shown in FIG. 12 and as detailed in the following steps.
16.1. The Forward and Reverse Primers Having the Following Sequences, which were Required for Amplifying HPV 16L1 Gene, Were Synthesized:









forward primer:



(SEQ ID NO: 18)



5′-ACTAATTATTCGAAACGATGTCTTTGTGG-3′;



 



reverse primer:



(SEQ ID NO: 19)



5′-AGCGGTACCCTATTACAACTTTCTCTTCTTTC-3′.






Wherein the forward primer comprises a BstBI restriction site, and the reverse primer comprises a KpnI restriction site flanking the termination codon, said restriction sites respectively as shown in the underlined portions of the primer sequences above.
16.2. PCR amplification was performed using the above primers and using the pUC-16L1 obtained in Example 15 as template. The amplified product was detected by electrophoresis, with the result being shown in FIG. 13, demonstrating that a full-length codon-optimized gene for HPV 16L1 was obtained. The PCR amplified fragment was digested with BstBI and KpnI (restriction endonucleases) and then ligated with pPICZαB (Invitrogen Corp.) which was also digested with BstBI and KpnI. Then, the ligated construct was used to transform competent cells of E. coli Top10 strain (available from Generay Biotech (Shanghai) Co., Ltd (Shanghai, China)), and the transformed cells were plated onto LB agar containing 25 μg/ml of zeocin.
The transformed cells were able to grow on LB media containing zeocin as the pPICZαB vector carried zeocin-resistance gene. Single colonies of the transformed cells were separated to prepare plasmid DNA. HPV 16L1 gene and vector sequence were detected through restriction mapping (see FIG. 14) and nucleotide sequence analysis to identify the correct construct comprising HPV 16L1 gene, which was named pPICZ-16L1. As secretion signal (a-factor signal) has been cleaved from the constructed plasmid, therefore the HPV 16L1 protein expressed should be intracellularly expressed protein.
Example 17
Construction and Expression of HPV 16L1 Gene-Expressing Pichia Yeast Strain
17.1. Construction of HPV 16L1 Gene-Expressing Pichia Yeast Strain
pPICZ-16L1 plasmid was linearized with a restriction endonuclease enzyme SacI, and empty plasmid pPICZαB was likewise digested with Sad to serve as negative control. The enzyme digestion solution was added with absolute ethanol to obtain DNA precipitate. The linearized pPICZ-16L1 fragment was dissolved with a small amount of double-distilled water and used to transform Pichia yeast strain X-33 (Invitrogen Corp.) by electroporation under the following conditions: DNA fragment, 5 μg; voltage, 1,500 volts; resistance, 25 omhs; electroporation time, 5 milliseconds. The electroporated cells were plated onto YPDS agar containing 200 μg/ml zeocin (from Zeocin). As the pPICZαB vector carried zeocin-resistance gene, the transformed cells were able to grow on YPDS media containing zeocin. Single colonies of transformed cells were isolated, thus obtaining HPV 16L1 gene-expressing Pichia yeast strain.
17.2. Expression of HPV 16L1 Gene-Expressing Pichia Yeast Strain
The obtained HPV 16L1 gene-expressing Pichia yeast strain was plated onto resistance plates containing 1,000 μg/ml or 1,500 μg/ml zeocin. Clones obtained from plates having high concentration of resistance were separately cultured in 4 mL of YPD liquid culture media for 24 hours followed by induction of gene expression in BMMY culture media for 48 hours. Then cells were harvested by centrifugation and some of the cells were disrupted to obtain supernatant for performing Western blotting identification. The result of identification is shown in FIG. 15, which indicated the presence of HPV 16L1 protein in the supernatant.
Although SEQ ID NO: 7 sequence was used for cloning and expression in Examples 16 and 17 of the present invention, it will be obvious to those skilled in the art that similar results can be obtained when SEQ ID NO: 8 and 9 sequences are used for cloning and expression, and therefore these two sequences are also within the scope of the present invention. Moreover, those skilled in the art will, in light of the spirit of the present invention, readily construct similar sequences and clone and express the constructed sequences in Pichia yeast to obtain similar or better results, and therefore these sequences are also deemed to be within the scope of the present invention.
Example 18
Large-Scale Expression and Detection of HPV 16L1 Protein
1. Large-scale expression of recombinant HPV 16L1 protein
Preparation of inoculum solution: One glycerol freezing tube of recombinant yeast stock was taken from working cell bank. After thawing, 100 μL of the stock was inoculated into 5 mL of YPD media and cultured at 280 rpm at 30° C. for 20 hours, at which time OD600 was detected to be 1 to 2 and no contamination from other microorganisms was microscopically observed. 1 mL of the activation fluid (which has been inspected to be satisfactory) was inoculated into 500 mL of YPD media and cultured at 280 rpm at 30° C. for 20 hours, at which time OD600 was detected to be 2 to 6 and no contamination from other microorganisms was microscopically observed.
Process of fermentation: Basal salt medium for fermentation was prepared using deionized water: BSM1 (K2SO4 273 g, MgSO4 109 g, CaSO4 2H2O 17.6 g, H3PO4 400.5 mL, KOH 62 g, glycerol 600 g, PTM1 60 mL, defoamer 1 mL; add deionized water to a volume of 15 L). The prepared medium, which contained no antibiotics, was transferred into a 30 L fermentation tank (Bioengineering Co., Ltd) and sterilized in the tank at 121° C. for 30 minutes, followed by cooling to 30° C. The inoculum solution previously prepared was used to inoculate the sterilized medium in a ratio of 1:15. Fermentation was started at a temperature of 30.0±0.5° C., an initial pH of 5.00±0.05, an initial stirring speed of 300 rpm, an aeration volume of 0.5 vvm and a dissolved oxygen (DO) of 100%, with addition of trace salts PTM1 (CuSO4 5H2O 6.0 g, NaI 0.008 g, MnSO4 3.0 g, NaMoO4 0.2 g, H3BO3 0.02 g, ZnSO4 20.0 g, CoCl2 0.5 g, FeSO4.7H2O 65.0 g, biotin 0.2 g, and H2SO4 5.0 mL; add deionized water to a volume of 1 L). The initial proliferation stage lasted for about 20 hours, during which time the dissolved oxygen was maintained at no lower than 30%. When the carbon source was completely consumed, the dissolved oxygen increased rapidly, at which time the wet cell weight was up to about 100 g/L. In the subsequent two hours, the fermentation was supplemented with 50% (v/v) glycerol solution (added with 12 mL of PTM1 per liter) at a rate of 200 mL/h Then the supplementation was continued at a rate of 300 mL/h for about 8 hours, during which time the dissolved oxygen was maintained at higher than 20% by adjusting the stirring speed, air flow and tank pressure (<0.8 bar). When the wet cell weight reached about 350 g/L, supplementation was stopped, and the dissolved oxygen increased accordingly. Meanwhile, the pH value was adjusted to 6.00±0.05, and methanol (added with 12 mL of PTM1 per liter) was added at an initial addition rate of 30 mL/h to induce gene expression. The addition rate of methanol was increased slowly until a set rate of 90 mL/h after 4 hours. During induction, the dissolved oxygen was maintained at higher than 20% (v/v), the temperature at 30° C. and the pH at 6.00±0.05. Sample was taken every eight hours and subjected to Western blotting detection. Twenty-four hours after induction, the fermentation completed and the fermentation broth was discharged.
Harvest of cells: The fermentation broth was centrifuged using a refrigerated centrifuge to collect the cells, which were then weighed. The batch number, date and weight for the recovered cells were recorded, and the cells were sent for purification or stored at −20° C.
2. Detection
The purified (according to the method described in Example 19 below) HPV 16L1 protein was used to prepare a protein concentration standard curve. With cells before induction as negative control, the expression amount of HPV 16L1 gene in Pichia yeast during fermentation was detected using sandwich ELISA method, as detailed below.
Rabbit polyclonal antibody to HPV16L1 protein was diluted 2,000 fold with coating solution. An aliquot of 0.1 mL of the diluted rabbit polyclonal antibody was added to each well of an ELISA plate, and the plate was incubated at 4° C. overnight. The coating solution was removed from each well, which was then washed with 0.3 mL of PBST. Then, each well was blocked by incubating with 0.3 mL of blocking solution at 37° C. for 2 hours.
The purified HPV 16L1 protein was gradiently diluted with dilution buffer from a concentration of 2 μg/mL to 0.0625 μg/mL. Meanwhile, the supernatant obtained from disrupted fermentation broth was diluted 200 fold. Then, for each of the HPV 16 L1 protein solutions of different concentrations obtained by the serial dilution and for the diluted supernatant, an aliquot of 0.1 mL was taken and added to a separate well of the ELISA plate, and the plate was incubated at 37° C. for 1 hour. After incubation, the aliquot of antigen solution was removed from each well, which was then washed with 0.3 mL of washing buffer. Then, MAB885 mouse monoclonal antibody (purchased from CHEMICON Co., Ltd) was diluted 1,000 fold with dilution buffer, and an aliquot of 0.1 mL of the dilution was added to each well and incubated at 37° C. for 1 hour. Following incubation, the monoclonal antibody solution was removed from each well, which was then washed with 0.3 mL of washing buffer. Subsequently, each well was added with 0.1 mL of HRP labeled goat anti-mouse IgG obtained by diluting the IgG 5,000-fold with dilution buffer, and incubated at 37° C. for 0.5 hour. Then the ELISA solution was removed and each well was washed with 0.3 mL of washing buffer. Then, each well was added with 0.1 mL DAB color development solution, and interaction was allowed to occur at room temperature for 20 minutes. Afterwards, 0.05 mL of 2 M H2SO4 stop solution was added to each well to stop the reaction, and OD450 value was determined using an ELISA reader.
The OD450 values for the gradiently diluted solutions of HPV 16L1 protein were used to prepare a protein concentration standard curve. Using this standard curve, the expression amount of HPV 16L1 protein during fermentation was obtained, as shown in Table 9 below. Particularly speaking, the purified target protein stock solution with known concentration was serially diluted into solutions of a range of concentrations, such as 2 μg/mL, 1 μg/mL, 0.5 μg/mL, 0.25 μg/mL and 0.125 μg/mL, which were used as standard concentrations. ELISA detection was conducted on these solutions to obtain the corresponding OD450 values. Then a standard linear regression equation was generated, with the ordinate representing the concentration and the abscissa representing the OD450 value.
The supernatant obtained from the disrupted fermentation broth was serially diluted, such as being diluted 50 fold, 100 fold, 200 fold and 400 fold. OD450 values were detected for these serial dilutions, and the corresponding concentrations (μg/mL) were found from the standard linear regression equation. The concentration of the target protein in the supernatant (μg/mL) was then calculated by multiplying the found concentration with the corresponding dilution factor. As the disrupted broth was prepared from wet cell weight:cell disruption buffer=1:5, therefore the expression amount of the target protein in the wet cells (μg/g wet cells) was 5× the concentration of the target protein in the supernatant (μg/mL). And the concentration of expression of the target protein in the fermentation broth (μg/L fermentation broth) was obtained by multiplying the above-obtained expression amount of the target protein in the wet cells with the density of cells in the fermentation broth (g wet cells/L fermentation broth).

Concentration of the target protein in the supernatant obtained from the disrupted fermentation broth(μg/mL)=Dilution factor×Concentration of standard target protein(μg/mL)×OD450(supernatant obtained from the disrupted fermentation broth)/OD450(standard target protein concentration);

Concentration of expression of the target protein in the fermentation broth(μg/L fermentation broth)=5×Concentration of the target protein in the supernatant obtained from the disrupted fermentation broth(μg/mL)×Density of cells in the fermentation broth(g wet cells/L fermentation broth).








TABLE 9







Detection of expression of HPV 16L1 protein during fermentation

















Concentration



Expression





of stock
Average


amount



Dilution 

solution
concentration
Converted
Fermentation
during


Sample
factor
OD450
(μg/mL)
(μg/mL) 
concentration
density
fermentation

















Disrupted
200
0.601
382
381
1904 μg/g 
472 g/L
899 mg/L


fermentation




wet cells

fermentation


broth






broth


Supernatant 1
100
1.034
379.5






Disrupted
200
0.826
578.1
542
2708 μg/g
347 g/L
940 mg/L


fermentation




wet cells 

fermentation


broth






broth


Supernatant 2
100
1.323
505.1






Negative
100
0.085







control









Blank control
—
0.077














It can be seen from the results shown in Table 9 that, the optimized sequence of the gene for HPV 16L1 protein according to the present invention not only can be expressed into HPV 16L1 protein in Pichia yeast, but also has a high expression amount, meeting the requirements of industrial production.
Example 19
Purification of HPV 16L1 Protein
Cleaning:
The above-obtained Pichia yeast cells expressing HPV 16 L1 protein, which had been stored at −20° C., was thawed at room temperature. Then the thawed cells were mixed with washing buffer (100 mM PB, pH 7.0, 0.15 M NaCl) or water for purification in a ratio of 1:3 (g/mL), and completely homogenized with the buffer or water in a homogenizer (FLUKO). Subsequently, the homogenate was subjected to high speed centrifugation (SORVALLRC6PLUS) at 8000 rpm for 5 minutes to separate the cells, the supernatant being decanted. The above procedure was repeated twice, thereby achieving the cleaning of the cells.
Disruption:
The cleaned cells were added and mixed into cell disruption buffer (200 mM MOPS, pH 7.0, 0.4 M NaCl, 0.05% Tween-80) in a ratio of 1:5 (g/mL), and completely homogenized with the buffer in a homogenizer (FLUKO). The cells suspended in the homogenate were subjected to disruption using a high-pressure homogenizer (ATSAH110B) operating at a pressure in the range of 1200 to 1300 bars. This disruption was repeated four times, and then the disrupted cell solution was discharged at 4 to 8° C., with 90% of the cells being disrupted.
Clarification:
The disrupted cell solution obtained by high-pressure disruption as above was poured into a centrifugation cup and centrifuged at high speed to precipitate cell debris, obtaining supernatant for subsequent column chromatography separation. The centrifuge used is SORVALLRC6PLUS, rotor model: FIBERLITEF10-6×500y, centrifugation setting: 9,000 rpm, 30 min, 10° C.
Preliminary Purification:
Chromatography media POROS 50HS (Applied Biosystems) was loaded into a chromatography column (diameter 26 mm, height 10 cm, volume 50 mL). Preliminary purification was conducted in the following steps: (1) washing and disinfection: the column was washed with two column volumes of 0.5 M NaOH; (2) regeneration and equilibration: the column was washed with two column volumes of Buffer F and then equilibrated with Buffer E; (3) loading sample: the supernatant obtained by centrifuging the disrupted cell solution as above was loaded onto the column; (4) rinsing: the column was rinsed with five column volumes of Buffer E until the baseline stabilized; (5) elution: the column was eluted with a linear gradient of 100% Buffer E to 100% Buffer F; and (6) collection: the elution fractions were collected, and the fractions containing HPV 16 L1 protein (detected by SDS-PAGE and Western blotting) were combined and used for fine purification.
Fine Purification:
Chromatography media CHT (BIO-RAD, Type II, 40 μm) was loaded into a chromatography column (diameter 26 mm, height 10 cm, volume 50 mL). Fine purification was conducted in the following steps: (1) washing and disinfection: the column was washed with two column volumes of 0.5 M NaOH; (2) regeneration and equilibration: the column was washed with two column volumes of Buffer H and then equilibrated with Buffer G; (3) loading sample: the sample collected after preliminary purification was added with PB to a final concentration of 20 mM and then loaded onto the column; and (4) rinsing: the column was rinsed with five column volumes of Buffer G until the baseline stabilized; (5) elution: the column was eluted with a linear gradient of 100% Buffer G to 100% Buffer H; and (6) collection: the elution fractions were collected and the fractions containing HPV 16L1 protein were combined to obtain the final purified sample. The purified sample was reduced to monomeric proteins which were then subjected to reduced SDS-PAGE (Bio-RAD) (see FIG. 16) and capillary electrophoresis (see. FIG. 17), demonstrating a purity of greater than 90%. The parameters of capillary electrophoresis for HPV 16L1 is as following:










Results of detection at 220 nm wavelength















Proportion of



Retention
Peak
Peak area after
peak area after


Peak
time
height
calibration
calibration














1
18.417
1530
1175.79185520
6.30041539


2
19.583
14269
17486.34042553
93.69958461


Total

15799
18662.13228074
100.00000000









Western blot (Bio-RAD) detection showed specific staining reaction between target electrophoresis band and monoclonal or polyclonal antibody to HPV 16 L1 protein. Dynamic light scattering detection (Malvern Instruments Zetasizer Nano ZS) showed that the purified sample had particles in a size range of about 50 to 80 nm, and electron microscopy observation (Philips Tecnai-12Biotwin transmission electron microscope, Electron Microscope Laboratory, Science and Technology Center, Shanghai University of Traditional Chinese Medicine, China) revealed the structure of virus-like particles (VLPs) in the purified sample, with the particle size in the range of about 50 to 80 nm (see FIG. 18).
Example 20
Preparation of HPV 16L1 Vaccine
The purified HPV 16L1 protein obtained in Example 19 above was adsorbed to an aluminium adjuvant to prepare an immunogenic HPV 16L1 vaccine according to the method described in Chinese Pharmacopoeia (2005 edition).
Example 21
Determination of Immunogenicity of the expression Product of HPV 16L1 Gene
Twenty-four 6 to 8 weeks old SPF BALB/c mice were divided into 4 groups, with 6 mice in each group. Mice in the first group (as the negative control group) were immunized by hypodermic injection of 0.1 mL of aluminium adjuvant-containing buffer (0.32 M sodium chloride, 0.35 mM sodium borate, 0.01% Tween-80, 0.01 M histidine, pH 6.5) for three times on days 0, 7 and 21 respectively, and mice in the other three groups (as the test groups) were similarly immunized with 0.1 mL of aluminium adjuvant-adsorbed VLPs at a concentration of 5 μg/mL, 0.5 μg/mL and 0.05 μg/mL respectively. Blood samples were collected two weeks after the third immunization. The blood samples collected were stood at 37° C. for 2 hours and then centrifuged at 4,000 g for 10 minutes. The supernatant, which was obtained as mouse polyclonal antiserum, was aspirated and stored at −20° C. Additionally, the supernatant was assayed for seroconversion rate and titer, as detailed below.
21.1. Detection of Seroconversion Rate
The purified HPV 16L1 VLPs expressed by Pichia yeast were diluted with coating solution to 1 μg/mL. An aliquot of 0.1 mL of the dilution was added into each well of an ELISA plate, and the plate was incubated at 4° C. overnight. The coating solution was removed from each well, which was then washed with 0.3 mL of PB ST. Then, each well was blocked by incubating with 0.3 mL of blocking solution (5% skimmed milk powder+PBST) at 37° C. for 2 hours. Each well was added with 0.1 mL of test serum (serum obtained from mice immunized with aluminium adjuvant-adsorbed HPV 16L1 protein and serum obtained from mice immunized with aluminium adjuvant alone) solution obtained by diluting the serum with dilution buffer (2% skimmed milk powder+PBST) in a ratio of 1:400, and then incubated at 37° C. for 1 hour. Then the test serum solution was removed and each well was washed with 0.3 mL of washing buffer. Subsequently, each well was added with 0.1 mL of HRP labeled goat anti-mouse IgG obtained by diluting the IgG with dilution buffer in a ratio of 1:5,000 and incubated at 37° C. for 0.5 hour. Then the ELISA solution was removed and each well was washed with 0.3 mL of washing buffer. Then, each well was added with 0.1 mL DAB color development solution and allowed to react in dark at room temperature for 20 minutes. After reaction, 0.05 mL of 2 M H2SO4 stop solution was added to each well to stop the reaction, and OD450 value was determined using an ELISA reader, with the readings shown in Table 10 below.







TABLE 10







OD450 readings














Mouse 1
Mouse 2
Mouse 3
Mouse 4
Mouse 5
Mouse 6





5 μg immu-
1.306
1.961
1.840
1.280
0.992
0.889


nization group








0.5 μg immu-
0.194
1.526
0.795
0.220
0.263
1.056


nization group








0.05 μg immu-
0.786
1.481
0.648
0.377
0.287
0.161


nization group








Aluminium 
0.100
0.137
0.092
0.119
0.099
0.128


adjuvant group








Cutoff value
0.167









The Cutoff value is the sum of the average of the OD450 values of antibody to test serum of negative control (mouse serum obtained from immunization with aluminium adjuvant) plus three times the standard deviation. Mouse with an OD450 value greater than the Cutoff value was regarded as positive, and mouse with an OD450 value lower than the Cutoff value was regarded as negative. The results of seroconversion rate for the three test groups are shown in Table 11 below.







TABLE 11







Results of seroconversion rate











5 μg/mL
0.5 μg/mL
0.05 μg/mL



immunization
immunization
immunization



group
group
group














Seroconversion
100%
100%
83%


rate









21.2. Determination of Serum Titer

The purified HPV 16L1 protein was diluted with coating solution to 1 μg/mL. An aliquot of 0.1 mL of the dilution was added into each well of an ELISA plate, and the plate was incubated at 4° C. overnight. The coating solution was removed from each well, which was then washed with 0.3 mL of PB ST. Then, each well was blocked by incubating with 0.3 mL of blocking solution (5% skimmed milk powder+PBST) at 37° C. for 2 hours. The test serum (serum obtained from mice immunized with HPV 16L1 protein) was serially double diluted with dilution buffer (2% skimmed milk powder+PBST) from 1:500 dilution to 1:32,000 dilution, while the negative control serum (serum obtained from mice immunized with aluminium adjuvant) was diluted in a ratio of 1:10,000. Each well was added with an aliquot of 0.1 mL of diluted serum (test serum or negative control serum) and incubated at 37° C. for 1 hour. Then the test serum solution was removed and each well was washed with 0.3 mL of washing buffer. Subsequently, each well was added with 0.1 mL of HRP labeled goat anti-mouse IgG obtained by diluting the IgG with dilution buffer in a ratio of 1:5,000, and incubated at 37° C. for 0.5 hour. Then the ELISA solution was removed and each well was washed with 0.3 mL of washing buffer. Then, each well was added with 0.1 mL DAB color development solution and allowed to react at room temperature for 20 minutes. After reaction, 0.05 mL of 2 M H2SO4 stop solution was added to each well to stop the reaction, and OD450 value was determined using an ELISA reader.
Serum titers were calculated by end point titration method, with the results shown in Table 12 below.







TABLE 12







Results of titer determination














Mouse 1
Mouse 2
Mouse 3
Mouse 4
Mouse 5
Mouse 6
















5 μg immu-
71727
48969
30700
64289
118279
4598178


nization group








0.5 μg immu-
7309
52706
335668
4593
47049
85822


nization group








0.05 μg immu-
24330
42552
8421
5751
7108
<400


nization group









In summary, as shown in Examples 15-21, the gene for major capsid protein L1 of human papilloma virus subtype 16, as provided by the present invention, is an optimized HPV 16L1 gene, which has the advantages of being more suitable for efficiently expressing target protein in yeast host and meeting the requirements of industrial production. Moreover, the HPV 16L1 vaccine as provided by the present invention, which was prepared from adjuvant-adsorbed purified VLPs (virus-like particles self-assembled from HPV 16L1 protein), was demonstrated to be strongly immunogenic in mice as determined by seroconversion rate and serum titer. Further, the method has the advantages of low cost, high yield and more uniform and stable quality of products can be achieved due to the use of Pichia yeast expression system.
Example 22
Neutralization Activity of HPV 16 L1 VLPs
Mice were immunized in the same procedure as described above and blood was collected two weeks after immunization. The collected blood was stood at 37° C. for 2 hours, then centrifuged at 4,000 g for 10 minutes. The supernatant, which was obtained as mouse polyclonal antiserum, was aspirated and stored at −20° C.
293FT cells (Invitrogen) were initially plated onto 15 cm cell culture dish (1.1×107 cells/dish). Twenty-four hours later, the cells were cotransfected with plasmids p16L1h, p16L2h (Buck C B, Pastrana D V, Lowy D R et al. Efficient Intracellular Assembly of Papillomaviral Vectors. J Virol, 2004, 78(2):751-757) and green fluorescent gene-carrying pIRES2-EGFP (purchased from BD Biosciences Clontech) respectively in an amount of 20 μg, 10 μg and 10 μg using calcium phosphate transfection method. Forty-eight hours later, cells were observed, collected and lysed. Cell lysis supernatant was immediately subjected to purification or stored at −80° C.
Purification was performed by centrifuging the cell lysis supernatant in a 5 mL centrifugation tube (Beckman) using 30% OptiPrep density gradient centrifugation at 100,000 g at 16° C. for 4 hours (MLS-50 centrifuge head, Beckman ultra-speed centrifuge). The centrifugation fractions were collected, each fraction being about 500 μL, and assayed for the content of HPV 16 L1 protein using Western blotting. The fractions having highest concentration of L1 protein were combined as pseudotype virus solution, which was then aliquoted and stored at −80° C.
293FT cells were plated onto 24-well cell culture plate (1.5×105 cells/well). Twenty-four hours later, neutralization experiment was done as follows. Different serum samples were serially diluted 100-fold with DMEM medium. An aliquot of 50 μL of each dilution was mixed with 50 μL of the pseudotype virus solution diluted in DMEM medium, and each mixture was incubated at 4° C. for 1 hour. Then each incubated mixture was added into the 24-well cell culture plate pre-plated with 293FT cells and incubated at 37° C. for 48 hours. After incubation, the cells were observed for expression of green fluorescent protein under an OLUMPUSCKX41F32FL inverted fluorescence microscope, and fluorescence images were collected, as shown in FIG. 19. Fluorescence was seen in FIG. 19A, which indicates that the HPV 16 pseudotype virus had infected the 293FT cells. Little fluorescence remained in FIG. 19B, which indicates that mouse serum had neutralized the HPV 16 pseudotype virus, thereby reducing the viral infection of the 293FT cells. It is therefore clear that mice immunized with recombinant HPV 16L1 VLPs vaccine could generate neutralizing antibody which would inhibit entry of the pseudotype virus into cells.
In the present invention, nucleotide sequence SEQ ID NO: 8 or SEQ ID NO: 9 can be substituted for SEQ ID NO: 7 in the above examples to prepare HPV 16 L1 protein of the present invention using the methods as described in the above examples. In addition, the above-said genes can also be cloned, in appropriate manner, into other existing Pichia yeast expression vector, such as pPIC6, pGAPZ or pAO815. Then the recombinant expression vectors obtained can be used to transform other Pichia yeast strains, such as Pichia pastoris GS115, KM71 or SMD1168 strains, so as to construct genetically engineered strains. Using conventional methods of culturing and fermentation, and separation and purification (which are not detailed herein), HPV 16 L1 protein (HPV 16 L1 VLPs) of the present invention can be obtained from the genetically engineered strains. The thus prepared HPV 16 L1 protein will have similar immunogenicity to that of the HPV 16 L1 protein prepared in the above examples.
All references cited in the present disclosure are hereby incorporated herein by reference as if each was individually incorporated herein by reference. In addition, it is understood that those skilled in the art will, in light of the teaching described hereinabove, make various changes and modifications to the present invention without departing from the spirit of the present invention, and these equivalents are deemed to fall within the scope of the present invention as defined in the appended claims.

## Claims

1. An isolated gene encoding the major capsid protein L1 of human papilloma virus, wherein said isolated gene is selected from the group consisting of: SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6.

2. An expression vector, wherein said expression vector comprises the sequence of an isolated gene encoding the major capsid protein L1 of human papilloma virus, wherein said isolated gene is selected from the group consisting of: SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6.

3. A genetically engineered isolated host cell, wherein said cell comprises the expression vector according to claim 2.

4. The isolated host cell according to claim 3, wherein said cell is Pichia yeast cell.

5. A method for preparing an immunogenic macromolecule having a diameter of 50 to 80 nm and being essentially self-assembled from major capsid proteins L1 of human papilloma virus, said major capsid proteins L1 of human papilloma virus being expressed by Pichia yeast, wherein said method comprises:
(1) culturing the isolated host cell according to claim 4, to allow said major capsid protein L1 of human papilloma virus to be expressed and to be self-assembled into said immunogenic macromolecule in said host cell;
(2) isolating said immunogenic macromolecule from the isolated host cell.

6. The method according to claim 5, wherein said step (2) includes:
(a) disrupting the isolated host cells obtained from step (1) to obtain a supernatant containing said immunogenic macromolecule; and
(b) purifying the supernatant obtained from step (a) using a first chromatographic column and a second chromatographic column to obtain said immunogenic macromolecule.

7. The method according to claim 6, wherein in said step (b), purification using the first chromatographic column is performed as follows:
(i) the supernatant obtain from step (a) is loaded onto a column having been cleaned and equilibrated to allow said immunogenic macromolecule to bind to the column;
(ii) the column is eluted with a linear gradient of 100% buffer A to 100% buffer B, and the chromatographic peaks at 70-100 ms/cm are collected,
wherein said buffer A contains 50+20 mM MOPS, 0.75+0.3 M NaCl and 0.05+0.02% polysorbate-80 at pH 6.5+1, and wherein said buffer B contains 50+20 mM MOPS, 1.5M NaCl and 0.05+0.02% polysorbate-80 at pH 6.5+1; and/or purification using the second chromatographic column is performed as follows:
(iii) the product purified from the first chromatographic column is loaded onto the second chromatographic column having been cleaned and equilibrated;
(iv) the second column is eluted with a linear gradient of 100% buffer C to 100% buffer D, and the chromatographic peaks 50-70 ms/cm are collected,
wherein said buffer C contains 50+20 mM MOPS, 0.5+0.2 M NaCl, 0.04+0.02 M phosphate buffer (PB) and 0.05+0.02% polysorbate-80 at pH 6.5+1, and said buffer D contains 0.5+0.2 M NaCl, 200 mM PB, and 0.05+0.02% polysorbate-80 at pH 6.5+1.

8. A genetically engineered isolated host cell, wherein said cell has a gene integrated into its genome, wherein said gene is selected from the group consisting of: SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6.

9. The isolated host cell according to claim 8, wherein said cell is a Pichia yeast cell.

10. A method for preparing an immunogenic macromolecule having a diameter of 50 to 80 nm and being essentially self-assembled from major capsid proteins L1 of human papilloma virus, said major capsid proteins L1 of human papilloma virus being expressed by Pichia yeast, wherein said method comprises:
(1) culturing the host cell according to claim 9, to allow said major capsid protein L1 of human papilloma virus to be expressed and to be self-assembled into said immunogenic macromolecule in said host cell;
(2) isolating said immunogenic macromolecule from the isolated host cell.

11. The method according to claim 10, wherein said step (2) includes:
(a) disrupting the isolated host cells obtained from step (1) to obtain a supernatant containing said immunogenic macromolecule; and
(b) purifying the supernatant obtained from step (a) using a first chromatographic column and a second chromatographic column to obtain said immunogenic macromolecule.

12. The method according to claim 11, wherein in said step (b), purification using the first chromatographic column is performed as follows:
(i) the supernatant obtain from step (a) is loaded onto a column having been cleaned and equilibrated to allow said immunogenic macromolecule to bind to the column;
(ii) the column is eluted with a linear gradient of 100% buffer A to 100% buffer B, and the chromatographic peaks at 70-100 ms/cm are collected,
wherein said buffer A contains 50+20 mM MOPS, 0.75+0.3 M NaCl and 0.05+0.02% polysorbate-80 at pH 6.5+1, and wherein said buffer B contains 50+20 mM MOPS, 1.5M NaCl and 0.05+0.02% polysorbate-80 at pH 6.5+1; and/or purification using the second chromatographic column is performed as follows:
(iii) the product purified from the first chromatographic column is loaded onto the second chromatographic column having been cleaned and equilibrated;
(iv) the second column is eluted with a linear gradient of 100% buffer C to 100% buffer D, and the chromatographic peaks 50-70 ms/cm are collected,
wherein said buffer C contains 50+20 mM MOPS, 0.5+0.2 M NaCl, 0.04+0.02 M phosphate buffer (PB) and 0.05+0.02% polysorbate-80 at pH 6.5+1, and said buffer D contains 0.5+0.2 M NaCl, 200 mM PB, and 0.05+0.02% polysorbate-80 at pH 6.5+1.