CN114174320B - Chimeric human papillomavirus 18-type L1 protein - Google Patents

Abstract

The invention discloses chimeric human papillomavirus type 18L 1 protein and polynucleotide encoding the same, and also discloses HPV type 18 virus-like particles and a preparation method thereof. The chimeric human papillomavirus type 18L 1 protein comprises an N-terminal fragment derived from HPV type 18L 1 protein, the N-terminal fragment maintaining the immunogenicity of HPV type 18L 1 protein; and a C-terminal fragment derived from a second type papillomavirus L1 protein, said second type papillomavirus L1 protein having characteristics of better expression and solubility than other types of L1 proteins; wherein the chimeric HPV 18 type L1 protein has the immunogenicity of an HPV 18 type L1 protein.

Description

Chimeric human papillomavirus 18-type L1 protein

Technical Field

The present invention relates to Human Papillomavirus (HPV) L1 protein and polynucleotides encoding the same, as well as HPV virus-like particles and methods of making the same.

Background

Papillomaviruses (papilloma virus, PV) belong to the papillomaviridae family (Papillomaviridae) and can cause papillomas in humans, cattle, dogs, rabbits, etc. The member human papillomavirus (Human Papillomavirus, HPV) is a non-enveloped DNA virus. The genome of the virus is double-stranded closed-loop DNA, has a size of about 7.2-8kb and has 8 open reading frames, and can be functionally divided into three regions: (1) Early region (E), about 4.5kb, encodes a total of 6 nonstructural proteins associated with viral replication, transcription, and transformation, E1, E2, E4-E7; (2) Late region (L), about 2.5kb, encoding major capsid protein L1 and minor capsid protein L2; (3) A long regulatory region (LCR) located between the end of the L region and the beginning of the E region, about 800-900bp long, does not encode any protein, but has DNA replication and expression regulatory elements.

The L1 and L2 proteins are synthesized in the middle and late stages of the HPV infection cycle. The L1 protein is the major capsid protein and has a molecular weight of 55-60 kDa. The L2 protein is a minor capsid protein. The 72L 1 protein pentamers constitute the outer shell (45-55 nm in diameter) of icosahedral HPV virions, which encapsulate closed-loop double stranded DNA. The L2 protein is located inside the L1 protein (Structure of Small Virus-like Particles Assembled from the L1 Protein of Human Papillomavirus 16 Chen,X.S.,R.L.Garcea,Mol.Cell.5(3)：557-567,2000).

The ORF of the L1 protein is the most conserved gene in the PV genome and can be used to identify new PV types. If the complete genome is cloned and the DNA sequence of the L1 ORF differs by more than 10% from the nearest known PV type, it is assumed that a new PV type is isolated. Differences at 2% and 10% homology are defined as different subtypes, differences less than 2% are defined as different varieties of the same subtype (e.—m.de VILLIERS ET al./Virology 324 (2004) 17-27).

In the late stages of HPV infection, newly synthesized L1 protein in the cytoplasm is delivered into terminally differentiated keratin nuclei, together with L2 protein, packaging the replicated HPV genomic DNA forms infectious virus (Nelson,L.M,et al.2002.Nuclear import strategies of high risk HPV16 L1 major capsid protein.J.Biol.Chem.277：23958-23964)., suggesting that nuclear import of L1 protein plays a very important role in HPV infection and production. The ability of the virus to enter the nucleus is determined by the Nuclear Localization Signal (NLS) of the C-terminal of the HPV L1 protein, one feature of which is the enrichment of basic amino acids (Garcia-Bustos,J.,et al.1991.Nuclear protein localization.Biochimica et Biophysica Acta 1071：83-101).

15 High Risk (HR) HPV types can lead to cervical, anal, penile, vaginal, vulvar and oropharyngeal cancers. Among them, HPV-16 and HPV-18 types are the most common cause of cancer to date, accounting for about 70% of cervical cancers, the remainder being caused by other HR-HPV types (31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73 and 82). HPV-16 accounts for about 95% of HPV-positive oropharyngeal cancers (OPCs). Persistent low risk genotypes HPV-6 and HPV-11 lead to most anogenital warts and respiratory papillomas, but are rarely associated with cancer (Human Papillomavirus in Cervical Cancer and Oropharyngeal Cancer：One Cause,Two Diseases Tara A.Bermanand John T.Schiller,PhD2Cancer 2017;123：2219-29).

The L1 protein is recombinantly expressed using vaccinia virus, baculovirus, or yeast systems, and the L1 protein self-assembles to form virus-like particles (VLPs), containing approximately 72L 1 proteins, similar to the viral housing. VLPs have no indication. VLPs can induce neutralizing antibodies in vaccinated animals, protecting experimental animals from subsequent challenge with infectious virus. Thus, VLPs appear to be excellent candidates for papillomavirus vaccines (Structure of Small Virus-like Particles Assembled from the L1 Protein of Human Papillomavirus 16 Chen,X.S.,R.L.Garcea,Mol.Cell.5(3)：557-567,2000).

From the company of GelansuIs a bivalent recombinant HPV vaccine. Wherein the recombinant baculovirus expression vector system is used for expressing the obtained HPV 16 type recombinant L1 protein and HPV 18 type recombinant L1 protein in noctuid (Trichoplusia ni) insect cells. The L1 protein self-assembles into virus-like particles for the prevention of cervical cancer, grade 2 or grade 3 cervical intraepithelial neoplasia and carcinoma in situ, and grade 1 cervical intraepithelial neoplasia (carcinogenesis) in women aged 9-25 by type 16 and 18 HPVs (https:// www.fda.gov/downloads/BiologicsBloodVaccines/Vaccines/ApprovedProducts/UCM 186981. Pdf).

Is a human papillomavirus tetravalent (types 6, 11, 16 and 18) recombinant vaccine produced by merck company, used for girls and women aged 9-26 for preventing cervical cancer genital warts (condyloma acuminatum) and precancerous or dysproliferative lesions caused by HPV types 6, 11, 16, 18; and for the prevention of anal cancer, genital warts (condyloma acuminatum) and precancerous or dysplastic lesions caused by HPV types 6, 11, 16, 18 in men and men 9-26 years old (https:// www.fda.gov/vaccines-blood-biologics/vaccines/gardasil).

Is a human papillomavirus nine-valent recombinant vaccine produced by merck company, comprising virus-like particles of HPV types 6, 11, 16, 18, 31, 33, 45, 52, and 58L 1 proteins produced by fermentation of saccharomyces cerevisiae, self-assembled into VLPs. For women and women aged 9-45 to prevent cervical, vulvar, vaginal and anal cancers caused by HPV types 16, 18, 31, 33, 45, 52 and 58, genital warts (condyloma acuminatum) caused by HPV types 6 and 11 and precancerous or dysproliferative lesions caused by HPV types 6, 11, 16, 18, 31, 33, 45, 52 and 58; and 9-45 years old boys and men are used for preventing anal cancers caused by types 16, 18, 31, 33, 45, 52 and 58, genital warts caused by HPV6 and 11 (condyloma acuminatum) and precancerous or dysplastic lesions caused by HPV types 6, 11, 16, 18, 31, 33, 45, 52 and 58 (https:// www.fda.gov/vaccines-blood-biologics/vaccines/gardasil-9).

In the specification of (a), it is stated that HPV types 16 and 18 are responsible for about 70% of cervical cancer, and the remaining 20% of cases are responsible for types 31, 33, 45, 52 and 58, byCan prevent 90% of cervical cancer occurrence (https:// www.fda.gov/BiologicsBloodVaccines/Vaccines/ApprovedPro ducts/ucm426445. Htm).

The key factor in HPV vaccine development is that virus-like particles can be mass produced. The currently more common systems for producing virus-like particles are largely divided into eukaryotic and prokaryotic expression systems.

Common eukaryotic expression systems are poxvirus expression systems, insect baculovirus expression systems, yeast expression systems. The HPV L1 protein expressed in eukaryotic expression systems has less disruption of natural conformation and can spontaneously assemble to form virus-like particles, but with lower yields. The prokaryotic expression system is mainly an escherichia coli expression system, has high yield, exists in an inclusion body form, is unfavorable for purification, and has a complex production process.

Thus, there remains a need in the art to obtain high yields of HPV virus-like particles.

Disclosure of Invention

In one aspect, the invention provides a chimeric Human Papillomavirus (HPV) type 18L 1 protein comprising, from its N-terminal to C-terminal direction, a. An N-terminal fragment derived from HPV type 18L 1 protein, the N-terminal fragment maintaining the immunogenicity of HPV type 18L 1 protein; c-terminal fragments derived from a second type of papillomavirus L1 protein, said second type of papillomavirus L1 protein having better expression and solubility characteristics than other types of L1 proteins; wherein the chimeric HPV 18 type L1 protein has the immunogenicity of an HPV 18 type L1 protein.

In another aspect, the invention provides an HPV type 18 virus-like particle comprising a chimeric HPV type 18L 1 protein.

In another aspect, the invention provides an immunogenic composition for preventing HPV-related diseases or infections comprising HPV type 18 virus-like particles and an adjuvant.

In another aspect, the invention provides an isolated polynucleotide encoding a chimeric HPV type 18L 1 protein.

In another aspect, the invention provides a vector comprising a polynucleotide encoding a chimeric HPV type 18L 1 protein.

In another aspect, the invention provides a baculovirus comprising a polynucleotide encoding a chimeric HPV type 18L 1 protein.

In another aspect, the invention provides a host cell comprising a polynucleotide, vector, or baculovirus as described above.

In another aspect, the invention provides a method of preparing HPV type 18 virus-like particles comprising culturing a host cell as described above to express the chimeric HPV type 18L 1 protein and assembling into virus-like particles; and purifying the HPV type 18 virus-like particle.

Drawings

FIG. 1 HPV 18 L1:33C, expression of L1 protein. M: a Marker; l: cell lysate; E-S: supernatant collected after centrifugation of the lysate.

FIG. 2 transmission electron microscopy of HPV 18L1:33C virus-like particles.

FIG. 3C expression of truncated HPV16L1 (1-474). M: a Marker; l: cell lysate; E-S: supernatant collected after centrifugation of the lysate.

Detailed Description

In one aspect, the invention provides a chimeric Human Papillomavirus (HPV) type 18L 1 protein comprising, from its N-terminal to C-terminal orientation: a. an N-terminal fragment derived from HPV type 18L 1 protein, said N-terminal fragment maintaining the immunogenicity of HPV type 18L 1 protein; c-terminal fragments derived from a second type of papillomavirus L1 protein, said second type of papillomavirus L1 protein having better expression and solubility characteristics than other types of L1 proteins; wherein the chimeric HPV 18 type L1 protein has the immunogenicity of an HPV 18 type L1 protein.

In one embodiment, the N-terminal fragment is a fragment obtained by truncating the C-terminal end of the natural sequence of HPV type 18L 1 protein to any amino acid position within its α5 region, and a fragment having at least 98% identity thereto; and the C-terminal fragment is a fragment obtained by truncating the N-terminal of the natural sequence of the L1 protein of the second type papillomavirus to any amino acid site in the alpha 5 region thereof, and a functional variant generated by further mutating, deleting and/or adding the fragment.

In another embodiment, the N-terminal fragment has at least 98.5%, 99%, 99.5% or 100% identity to a fragment obtained by truncating the C-terminal end of the natural sequence of HPV type 18L 1 protein to any amino acid position within its α5 region.

In another embodiment, the C-terminal fragment contains one or more nuclear localization sequences.

In one embodiment, the second type papillomavirus L1 protein is selected from HPV type 1, type 2, type 3, type 4, type 6, type 7, type 10, type 11, type 13, type 16, type 18, type 22, type 26, type 28, type 31, type 32, type 33, type 35, type 39, type 42, type 44, type 45, type 51, type 52, type 53, type 56, type 58, type 59, type 60, type 63, type 66, type 68, type 73, or type 82L 1 proteins;

Preferably, the second type papillomavirus L1 protein is selected from HPV type 16, type 28, type 33, type 59, or type 68L 1 proteins;

More preferably, the type II papillomavirus L1 protein is selected from HPV 33 type or HPV 59 type L1 proteins.

In one embodiment, the second papillomavirus type L1 protein is HPV 33 type L1 protein and the C-terminal fragment is SEQ ID No:2; or a fragment thereof of length m amino acids, preferably encompassing SEQ ID No:2 from amino acid 1 to amino acid m; wherein m is an integer of 8 to 26.

In one embodiment, the C-terminal fragment of HPV type 33L 1 protein has a nuclear localization sequence. In another embodiment, the C-terminal fragment of HPV type 33L 1 protein has two nuclear localization sequences. In some embodiments, the chimeric HPV type 18L 1 protein comprises one or more C-terminal fragments of HPV type 33L 1 protein. The C-terminal fragments of the plurality of HPV 33 type L1 proteins may be the same or different. In one embodiment, SEQ ID No:2 and amino acid sequences (KRKK) of amino acid numbers 7-8 and amino acid sequence numbers 20-23 are the nuclear localization sequences of the C-terminal fragment of HPV type 33L 1 protein.

In another embodiment, the second papillomavirus type L1 protein is HPV59 type L1 protein and the C-terminal fragment is SEQ ID No:13; or a fragment thereof of length n amino acids, preferably encompassing SEQ ID No:13 from amino acid 1 to n; wherein n is an integer from 16 to 38.

In one embodiment, the C-terminal fragment of HPV 59 type L1 protein has a nuclear localization sequence. In another embodiment, the C-terminal fragment of HPV 59 type L1 protein has two nuclear localization sequences. In some embodiments, the chimeric HPV type 18L 1 protein comprises one or more C-terminal fragments of HPV type 59L 1 protein. The C-terminal fragments of the plurality of HPV 59 type L1 proteins may be the same or different. In one embodiment, SEQ ID No:13 and amino acid sequences (KRVKRRK) of amino acid numbers 14-16 and amino acid sequence numbers 28-34 are the nuclear localization sequences of the C-terminal fragment of HPV type 59L 1 protein.

In one embodiment, the chimeric HPV type 18L1 protein comprises both a C-terminal fragment of HPV type 33L 1 protein and a C-terminal fragment of HPV type 59L 1 protein.

In one embodiment, the N-terminal fragment is identical to the sequence of SEQ ID No:1, and the resulting fragment has 98%, 98.5%, 99%, 99.5% or 100% identity to any amino acid position within its alpha 5 region.

In one embodiment, the C-terminus of the N-terminal fragment is directly linked to the N-terminus of the C-terminal fragment or linked by a linker.

The linker does not affect the immunogenicity of the N-terminal fragment and does not affect the expression level or solubility of the protein. In one embodiment, the N-terminal fragment and the C-terminal fragment are connected by a linker consisting of 1,2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids. In one embodiment, the linker is an artificial sequence. In another embodiment, the linker is a naturally occurring sequence in HPV L1 protein. In one embodiment, the linker may be part of the sequence of HPV type 18L 1 protein. In another embodiment, the linker may be part of the sequence of HPV type 33L 1 protein. In another embodiment, the linker may be part of the sequence of HPV type 59L 1 protein.

In one embodiment, when the C-terminus of the N-terminal fragment is linked to the N-terminus of the C-terminal fragment, the following contiguous amino acid sequences are present within plus or minus 4 amino acid positions of the point of attachment: RKFL; preferably, the following contiguous amino acid sequences are present within plus or minus 6 amino acid positions of the point of attachment: LGRKFL.

In one embodiment, the chimeric HPV type 18L1 protein is identical to SEQ ID No:3 has 98%, 98.5%, 99%, 99.5% or 100% identity.

In another aspect, the invention provides an HPV type 18 virus-like particle comprising a chimeric HPV type 18L 1 protein as described above. In one embodiment, the HPV type 18 virus-like particle is an icosahedron consisting of pentamers of 72 of said chimeric HPV type 18L 1 proteins. In one embodiment, HPV type 18 virus-like particles have correctly formed disulfide bonds and thus have a good native conformation. In one embodiment, HPV type 18 virus-like particles self-assemble in an in vivo expression system.

In one aspect, the invention provides an immunogenic composition for preventing HPV-related diseases or infections comprising HPV type 18 virus-like particles according to the foregoing and an adjuvant. The prophylaxis may be considered as treatment, both being used interchangeably.

In one aspect, the above immunogenic composition is administered to a subject. In one embodiment, the subject is a human.

In one aspect, the invention provides an isolated polynucleotide encoding a chimeric HPV type 18L 1 protein as described hereinbefore. In one embodiment, the polynucleotide is a polynucleotide codon optimized for a different expression system. In one embodiment, the polynucleotide is a polynucleotide codon optimized for an insect baculovirus expression system.

In one aspect, the invention provides an isolated polynucleotide having the sequence of SEQ ID No: 4.

In one aspect, the invention provides a vector comprising a polynucleotide as described above. In one embodiment, the vector is a baculovirus vector. In one embodiment, the vector may be a transfer vector for a baculovirus expression system. In another embodiment, the vector may be an expression vector for a baculovirus expression system. In another embodiment, the vector may be a recombinant vector for a baculovirus expression system.

In one aspect, the invention provides a baculovirus comprising a polynucleotide as described above.

In one aspect, the invention provides a host cell comprising a polynucleotide, vector, or baculovirus as described above. In one embodiment, the host cell is an insect cell, preferably selected from Sf9 cells, sf21 cells, hi5 cells and S2 cells.

In one aspect, the present invention provides a method of preparing HPV type 18 virus-like particles according to the foregoing, comprising: culturing a host cell as described above to express the chimeric HPV 18 type L1 protein and assembling into a virus-like particle; and purifying the HPV type 18 virus-like particle.

In one embodiment, the host cell is an insect cell. In one embodiment, the host cell is a Hi5 cell. In one embodiment, the chimeric HPV 18 type L1 protein self-assembles into HPV 18 type virus-like particles in the host cell. In one embodiment, the chimeric HPV type 18L 1 protein self-assembles in a host cell into HPV type 18 virus-like particles having an icosahedron of pentamers of 72 of said chimeric HPV type 18L 1 proteins. In one embodiment, HPV type 18 virus-like particles have correctly formed disulfide bonds and thus have a good native conformation.

In one embodiment, the purification employs cation exchange chromatography. In one embodiment, the purification employs strong cation exchange chromatography. In another embodiment, the purification employs weak cation exchange chromatography. In one embodiment, the purification employs a combination of multiple cation exchange chromatography. In one embodiment, the purification employs HS strong cation exchange chromatography. In another embodiment, the purification uses MMA ion exchange chromatography. In another embodiment, the purification uses two steps of HS-MMA chromatography.

The papillomavirus L1 protein expressed by the eukaryotic expression system can be spontaneously assembled into virus-like particles, but has the defects of low expression quantity and difficult mass production.

The sequence of the L1 protein of each type of HPV can be found from https: the values of/(www.uniprot.org) are readily available. Each type of HPV L1 may be derived from a different strain and thus have multiple versions of its amino acid sequence, any one version of the natural sequence may be used in the present invention, and the HPV L1 protein sequence of a given type may be different from that used in the examples in the conception and design of the present invention, but such differences do not affect the judgment and conclusion of the inventors.

It is widely recognized by those skilled in the art that the C-terminal end of the L1 protein does not contain a major neutralizing epitope and thus attempts to increase expression by truncating the C-terminal end of HPV L1 protein, for example by truncating the C-terminal end of HPV 16L 1 protein by 1-34 amino acids, preferably 26 amino acids, in U.S. Pat. No. 6361778B1 to the company Grandin, declare a many-fold, preferably at least a 10-fold, and in particular about 10-to 100-fold increase in VLP production. In light of this, the inventors tried to truncate the C-terminal of HPV type 16L 1 by 31 amino acids, designated HPV 16L 1 (1-474). However, the protein expression amount is high but the protein solubility is poor, and the extraction and purification are difficult (see comparative examples).

The poor protein solubility resulting from such truncations may be due to a deletion of the C-terminal nuclear localization sequence, and the invention is not limited to this assumption. The inventor discovers that the expression quantity and the solubility of the HPV 16 type L1 protein, the HPV 28 type L1 protein, the HPV 33 type L1 protein, the HPV59 type L1 protein and the HPV 68 type L1 protein are better than those of other types of L1 proteins in the research and production process, and inspires that the inventor replaces the C end of the HPV type which is not easy to extract or has low expression quantity with the C end of the L1 protein with the better expression quantity and the better solubility. Namely, the inventors constructed a chimeric protein: the N-terminal fragment derived from the L1 protein of the first type papillomavirus (e.g. HPV L1 protein) and the C-terminal fragment derived from the L1 protein of the second type papillomavirus (e.g. HPV L1 protein) are included from the N-terminal to the C-terminal direction, the former provides the immunogenicity of the first type papillomavirus (e.g. HPV), and the latter provides the characteristics of better expression and solubility. The two can be directly connected or connected through a joint.

To maintain the immunogenicity of the HPV L1 protein of type i and to ensure that it is capable of forming VLPs, the inventors determined the length of the N-terminal fragment of the appropriate HPV L1 protein. The following reports relate to epitope studies of common HPV subtypes:

Sunanda Baidya et al report that epitope 48EEYDLQFIFQLCKITLTA65,45RHGEEYDLQFIFQLCKITLTA65,63LPDPNKF69,79PETQRLVWAC88,36PVPGQYDA43,77YNPETQRLVWAC88,188DTGYGAMD195,36PVPGQYDATK45,45KQDIPKVSAYQYRVFRV61,130RDNVSVDYKQTQLCI144 and49YSRHVEEY DLQFIF62 of the L1 protein can be used as a tool for designing HPV type 16 and 18 vaccines (see Epitope design of L1protein for vaccine production against Human Papilloma Virus types 16and 18,Bioinformation 13(3)：86-93March 2017,, incorporated herein by reference in its entirety).

KATHARINA SLUPETZKY et al report that the regions near aa 282-286 and 351-355 of HPV-16 contribute to neutralizing epitopes, and that the latter is an immunodominant site (see Chimeric papillomavirus-like particles expressing a foreign epitope on capsid surface loops,Journal of General Virology(2001),82,2799-2804,, incorporated herein by reference in its entirety).

Brooke Bishop et al prepared the following 3 variants of HPV11, 16, 18 and 35L1 proteins: the former two were reported to be unable to assemble into VLPs but the latter were not reported to have a deletion of 9 amino acids at the N-terminus, a deletion of alpha 4 (corresponding to amino acid residues 404-436 of HPV 16) and a deletion of 31 amino acids at the C-terminus

(Crystal Structures of Four Types of Human Papillomavirus L1 Capsid Proteins UNDERSTANDING THE SPECIFICITY OF NEUTRALIZING MONOCLONAL ANTIBODIES,The Journal of Biological Chemistry,282,31803-31811. Incorporated by reference in its entirety). The individual Loop regions of each type of HPV L1 protein alpha helix, beta sheet can be readily determined by sequence analysis software commonly used in the art. Wherein the alpha helical region comprises an alpha 1 region, an alpha 2 region, an alpha 3 region, an alpha 4 region, and an alpha 5 region.

The inventors performed sequence alignment on HPV L1 proteins of 14 types (types 6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58 and 59), and then performed secondary structure prediction according to the above-cited document (Crystal Structures of Four Types of Human Papillomavirus L1 Capsid Proteins UNDERSTANDING THE SPECIFICITY OF NEUTRALIZING MONOCLONAL ANTIBODIES,The Joumal of Biological Chemistry,282,31803-31811), and the results are shown below, with the portion between the downward arrows corresponding to the region involved in the document deleted for the preparation of variants.

In addition to the methods of alignment used by the inventors, protein secondary structure prediction software useful for prediction includes, but is not limited to:

1.JPred：http：//www.compbio.dundee.ac.uk/jpred/index.html

2.ProtPredicct：http：//predictprotein.org

3.PsiPred：http：//bioinf.cs.ucl.ac.uk/psipred

4.SCRATCH-1D：http：//download.igb.uci.edu

5.Nnpredict：http：//www.cmpharm.ucsf.edu/～nomi/nnpredict

6.

Predictprotein：http：//www.embl-heidelberg.de/predictprotein/SOPMA

http：//www.ibcp.fr/predict.html

7.

SSPRED：http：//www.embl-heidelberg.de/sspred/ssprd_info.html。

In one embodiment of the invention, the inventors determine the length of the N-terminal fragment derived from HPV L1 protein of the first type in the following manner: the native sequence of the L1 protein is truncated in its region of alpha 5 and its vicinity, leaving the newly generated sequence from its N-terminus to the C-terminus of the alpha 5 region. Such truncated sequences may ensure that they are immunogenic in the native form and are capable of forming VLPs.

The N-terminal fragment derived from HPV L1 protein of the first type may be further engineered to ensure that it is immunogenic of the type and capable of forming VLPs.

The inventors determined the length of the C-terminal fragment derived from HPV L1 protein of the second type in the following manner. The native sequence of the L1 protein is truncated in its alpha 5 region and its vicinity, leaving the newly generated N-terminal to C-terminal sequence from its alpha 5 region. Such truncated sequences do not possess a major neutralizing epitope and do not interfere with the immunogenicity of the chimeric protein formed.

The C-terminal fragment derived from the HPV L1 protein of the second type may be further mutated, deleted and/or added, preferably retaining at least one of its nuclear localization sequences. The Yang et al predicts nuclear localization sequences (Yang et al.Predicting the nuclear localization signals of 107 types of HPV L1 proteins by bioinformatic analysis.Geno.Prot.Bioinfo.Vol.4 No.1 2006 of the 107 HPV subtypes, which are incorporated herein by reference in their entirety, and the nuclear localization sequences of each of the individual HPV L1 proteins can be readily determined by sequence analysis software commonly used in the art.

The ligation of the N-terminal fragment and the C-terminal fragment occurs at the newly generated C-terminal end of the former and the newly generated N-terminal end of the latter. Either directly or through a joint. Considering the connection point as the origin, it is negative on the N-terminal side of the origin and positive on the C-terminal side.

Amino acid sequences 453 to 469 of the HPV 6L 1 protein and a corresponding stretch of the various types of HPV L1 proteins are shown below. It can be seen that these sequences are highly similar. This sequence coincides with the α5 region. The numbers in brackets indicate the last amino acid position of the listed sequences, where for HPV type 45, an additional 26 amino acids are present at the N-terminus of the L1 protein of some HPV type 45 strains, whereas the additional 26 amino acids are not present at the N-terminus of the L1 protein of other HPV type 45 strains, and are therefore indicated as (478) +26.

HPV6 ELDQYPLGRKFLLQSGY(469)

HPV11 ELDQFPLGRKFLLQSGY(470)

HPV16 DLDQFPLGRKFLLQAGL(474)

HPV18 DLDQYPLGRKFLVQAGL(475)

HPV31 DLDQFPLGRKFLLQAGY(475)

HPV35 DLDQFPLGRKFLLQAGL(472)

HPV39 ELDQFPLGRKFLLQARV(474)

HPV45 DLDQYPLGRKFLVQAGL(478)+26

HPVS1 DLDQFALGRKFLLQVGV(474)

HPV52 DLDQFPLGRKFLLQAGL(478)

HPV56 DLDQFPLGRKFLMQLGTRS(474)

HPV58 DLDQFPLGRKFLLQSGL(473)

HPV33 DLDQFPLGRKFLLQAGL (473) KAKPKLKRAAPTSTRTSSAKRKKVKK wherein KR at positions 480-481 and KRKK at positions 493-496 are nuclear localization sequences.

HPV59

DLDQFPLGRKFLLQLGA (475) RPKPTIGPRKRAAPAPTSTPSPKRVKRRKSSRK wherein RKR of 484-486 and KRVKRRK of 498-504 are nuclear localization sequences.

In one embodiment of the present invention, the inventors conveniently accomplished C-terminal substitution of L1 proteins between different types by virtue of sequence similarity of the α5 region and its vicinity between HPV types.

In the most preferred embodiment of the invention, the inventors have noted that each type of HPV L1 protein has a tetrapeptide RKFL, more advantageously a hexapeptide LGRKFL, in a similar position. The inventors have cleverly utilized this highly conserved sequence to design the junction of the chimeric protein at any amino acid position in this stretch of oligopeptides. From one aspect, the sequence from the N-terminus of the chimeric protein to RKFL or LGRKFL is identical to the sequence of the N-terminal fragment derived from the HPV L1 protein of the first type, and from another aspect from RKFL or LGRKFL to the C-terminus of the chimeric protein is identical to the sequence of the C-terminal fragment derived from the L1 protein of the second type.

The chimeric proteins so produced remain highly similar to the native HPV L1 protein and are expected to perform well during production and even later in the medical or prophylactic process.

Those skilled in the art will appreciate that because HPV of the same type have different strains, their natural sequences are different and chimeric proteins constructed using different strains are also within the scope of the invention.

It will be appreciated by those skilled in the art that because of the high degree of similarity of HPV L1 of different types, if during construction of the chimeric protein the N-terminal fragment derived from HPV L1 protein of the first type is extended more amino acid residues towards the C-terminus or the C-terminal fragment derived from HPV L1 protein of the second type is extended more amino acid residues towards the N-terminus, it is also possible to form chimeric proteins consistent with the structure of the present invention due to the identity or similarity of the amino acids at the corresponding sites. Chimeric proteins so formed also fall within the invention.

Those skilled in the art will appreciate that, on the basis of the chimeric proteins of the above-described embodiments, variants of the chimeric proteins may be formed by mutation, deletion and/or addition of amino acid residues. These variants are likely to be immunogenic for HPV L1 proteins of the first type, form VLPs, and have good yield and solubility. Chimeric proteins so formed also fall within the invention.

Advantageous effects of the invention

Expression systems currently in common use for the production of virus-like particles are divided into eukaryotic and prokaryotic expression systems. The papillomavirus L protein expressed by the eukaryotic expression system can be spontaneously assembled into virus-like particles, but has the defects of low expression quantity and difficult mass production. The natural conformation of papillomavirus L protein expressed by a prokaryotic expression system is often destroyed, virus-like particles can be obtained by in vitro treatment in a later period, the yield is low, and industrialization is difficult to realize.

The invention modifies the C end of the L protein of papillomavirus (such as human papillomavirus), for example, replaces the C end fragment in HPV 16 type L1 protein, HPV 28 type L1 protein, HPV 33 type L1 protein, HPV 59 type L1 protein or HPV 68 type L1 protein, and can improve the expression quantity and the solubility of the L protein of the papillomavirus in an expression system (such as host cells, such as insect cells). This can be used for large-scale production of vaccines, such as HPV vaccines.

The inventors have found by themselves that the L1 proteins of HPV 16 type, L1 proteins of HPV 28 type, L1 proteins of HPV 33 type, L1 proteins of HPV 59 type and L1 proteins of HPV 68 type are better expressed and soluble than the L1 proteins of other types, and that the increased protein expression and solubility depend on the C-terminal sequence of the HPV L1 proteins. In the type 107 HPV L1 protein, most have a nuclear localization sequence at the C-terminus, and the C-terminal sequences have some similarity.

For papillomavirus L proteins which cannot be expressed at present, have very low expression level or are insoluble after expression, the C-terminal fragment of the papillomavirus L proteins is replaced by the C-terminal fragment in HPV 16 type L1 protein, HPV 28 type L1 protein, HPV 33 type L1 protein, HPV 59 type L1 protein or HPV 68 type L1 protein, so that the soluble expression and the subsequent purification of the papillomavirus L proteins with extremely low expression level or insoluble expression level are possible. This can be used for the mass production of more multivalent vaccines (e.g. HPV vaccines) making it possible to more comprehensively prevent infection by a variety of papillomaviruses, in particular HPV.

The HPV 18 type L1 protein has low expression level in insect cells and poor solubility, and is unfavorable for subsequent purification and vaccine production. Furthermore, in yeast cells, virus-like particles assembled from HPV type 18L 1 proteins lack a good conformation because disulfide bonds cannot be properly formed.

Compared with the unmodified HPV 18 type L1 protein, the chimeric HPV 18 type L1 protein has greatly improved expression quantity and solubility in insect cells. Can be used for large-scale production of HPV 18 vaccine. In addition, the chimeric HPV 18 type L1 protein can correctly form disulfide bonds in insect cells to assemble HPV 18 type virus-like particles with good conformation. This may increase the immunogenicity of HPV type 18 virus-like particles, resulting in a better immune response.

Definition of the definition

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. For the convenience of understanding the present invention, the following terms are given in their ordinary meanings.

As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. The terms "include/comprise," "have," "such as," and the like are intended to be inclusive and not limiting unless expressly stated otherwise.

The term "immunogenicity" refers to the ability of a substance, such as a protein or polypeptide, to stimulate an immune response, i.e., to stimulate the production of antibodies, particularly to produce a humoral or cell-mediated response.

The term "antibody" refers to an immunoglobulin molecule that is capable of binding an antigen. Antibodies may be polyclonal mixtures or monoclonal. Antibodies may be intact immunoglobulins derived from natural sources or from recombinant sources or may be immunoreactive portions of intact immunoglobulins. Antibodies can exist in a variety of forms including, for example, fv, fab ', F (ab') 2, and in single chains.

The term "antigenic" refers to the ability of a substance, such as a protein or polypeptide, to produce antibodies that specifically bind thereto.

The term "epitope" includes any protein determinant capable of specific binding to an antibody or T cell receptor. Epitope determinants are generally composed of chemically active surface groupings of molecules (e.g., amino acids or sugar side chains, or combinations thereof) and generally have specific three-dimensional structural characteristics as well as specific charge characteristics.

The terms "subtype" or "type" are used interchangeably herein to refer to a genetic variant of the viral antigen such that one subtype is recognized by the immune system differently than a different subtype. For example, HPV 16 is immunologically distinguishable from HPV 33.

The term "HPV L1 protein" as used herein, the terms "HPV'" and "human papillomavirus" refer to non-enveloped double-stranded DNA viruses of the papillomaviridae family. Their genomes are circular and are about 8 kilobase pairs in size. Most HPVs encode eight major proteins, six in the "early" region (E1-E2), and two in the "late" region (L1 (major capsid protein) and L2 (minor capsid protein)). More than 120 HPV types have been identified and are indicated by numbers (e.g., HPV-16, HPV-18, etc.).

The term "HPV'" or "HPV virus" refers to papillomaviruses of the papillomaviridae family, non-enveloped DNA viruses, whose genome is double-stranded closed-loop DNA, approximately 8kb in size, and can be generally divided into three regions: ① Early region (E) containing 6 open reading frames encoding nonstructural proteins involved in replication, transcription and transformation of the E1, E2, E4-E7 virus, and E3 and E8 open reading frames; ② The late region (L) contains the reading frame encoding the major capsid protein L1 and the minor capsid protein L2; ③ The long regulatory region (LCR) does not encode any protein, but has a replication origin and multiple transcription factor binding sites.

The terms "HPV L1 protein" and "HPV L2 protein" refer to proteins encoded by the late region (L) of the HPV gene, synthesized late in the HPV infection cycle. The L1 protein is the major capsid protein and has a molecular weight of 55-60 kDa. The L2 protein is the minor capsid protein. 72L 1 pentamers constitute the outer shell of icosahedral HPV virions, encapsulating closed-loop double-stranded DNA microcolonies. The L2 protein is located inside the L1 protein.

The term "virus-like particle" is a hollow particle containing one or more structural proteins of a virus, without viral nucleic acid.

The "HPV pseudovirus" is formed by wrapping free DNA by VLPs consisting of HPV L1 and L2 expressed in cells or introducing foreign plasmids, using the characteristics of non-specifically wrapping nucleic acids of HPV VLPs. Is an ideal HPV in-vitro neutralization experimental model.

The "pseudo-virus neutralization method" is a method for evaluating neutralizing activity of antibodies, cells are infected after the serum of an immunized animal is incubated with a certain amount of pseudo-viruses, cells decrease along with the increase of neutralizing antibodies in the serum, and linear negative correlation can exist in a certain range, so that the neutralizing activity of the antibodies in the serum can be evaluated by detecting the change of the number of expressed cells.

The term "fragment thereof" or "variant thereof" refers to a part of a nucleotide or amino acid sequence according to the invention that is deleted, inserted and/or substituted. Preferably, fragments or variants of the polypeptides provided herein are capable of eliciting a humoral and/or cellular immune response in an animal or human.

The term "chimeric" means that polypeptides or nucleotide sequences derived from different parent molecules are linked together via an amide bond or a 3',5' -phosphodiester bond, respectively. Preferably, they are not separated by additional linker sequences, but are directly adjacent to each other.

The term "truncated" means by removing one or more amino acids from the N-and/or C-terminus of a polypeptide or deleting amino acids within one or more polypeptides.

The term "nuclear localization sequence" is an amino acid sequence that directs a protein into the nucleus of a cell. In some HPV L1 proteins, two tightly basic residue clusters (i.e., nuclear localization sequences) (e.g., one is KRKR, KRKK, KRKRK, KRKKRK, KRVKRRK, etc., the other is KR, RKR, KRK, etc.) have a 10-14 amino acid spacer in between. The above basic residue clusters belong to the nuclear localization sequences. In other HPV L1 proteins, the nuclear localization sequences are tightly clustered basic residues formed by arginine and/or lysine. Nuclear localization sequences include but are not limited to the basic residue clusters of examples. See Jun Yang et al ,Predicting the Nuclear Localization Signals of 107 Types of HPV L1 Proteins by Bioinformatic Analysis,Genomics,Proteomics&Bioinformatics Volume 4,Issue 1,2006,Pages 34-41,, incorporated herein by reference in its entirety.

The term "functional variant" is a version of a polypeptide or protein that retains a desired activity or characteristic after truncation, mutation, deletion, and/or addition.

"Sequence identity" between two polypeptide or nucleic acid sequences means the number of identical residues between the sequences as a percentage of the total number of residues, and is calculated based on the size of the smaller of the molecules compared. In calculating the percent identity, sequences being compared are aligned in a manner that produces a maximum match between the sequences, with gaps in the alignment (if any) being resolved by a particular algorithm. Preferred computer program methods for determining identity between two sequences include, but are not limited to, the GCG program package, including GAP, BLASTP, BLASTN and FASTA (Altschul et al, 1990, J.mol. Biol. 215:403-410). The above procedure is publicly available from the international biotechnology information center (NCBI) and other sources. A well-known SMITH WATERMAN algorithm may also be used to determine identity.

Non-critical amino acids can be conservatively substituted without affecting the normal function of the protein. Conservative substitutions are intended to mean substitutions of amino acids with chemically or functionally similar amino acids. Conservative substitution tables providing similar amino acids are well known in the art. For example, in some embodiments, the sets of amino acids provided in tables 1-3 are considered conservative substitutions for one another.

Table 1 in certain embodiments, selected groups of amino acids that are considered to be conservative substitutions for one another

Acidic residues	D and E
		Basic residues	K. r and H
Hydrophilic uncharged residues	S, T, N and Q
		Aliphatic uncharged residues	G. A, V, L and I
Non-polar uncharged residues	C. M and P
		Aromatic residues	F. Y and W

Table 2 in certain embodiments, other selected groups of amino acids are considered to be conservative substitutions for one another

Group 1	A. S and T
		Group 2	D and E
Group 3	N and Q
		Group 4	R and K
Group 5	I. L and M
		Group 6	F. Y and W

Table 3 in certain embodiments, other selected groups of amino acids are considered to be conservative substitutions for one another

The term "amino acid" means twenty common naturally occurring amino acids. Naturally occurring amino acids include alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp; D), cysteine (Cys; C); glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y) and valine (Val; V).

The term "adjuvant" refers to a compound or mixture that enhances an immune response. In particular, the vaccine may comprise an adjuvant. Adjuvants for use in the present invention may include, but are not limited to, one or more of the following: mineral-containing adjuvant compositions, oil-emulsion adjuvants, saponin adjuvant formulations, bacterial or microbial derivatives.

The term "vector" means a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures as well as vectors that are integrated into the genome of a host cell into which the vector has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which such vectors are operably linked.

The term "host cell" means a cell into which exogenous nucleic acid has been introduced, as well as the progeny of such a cell. Host cells include "transformants" (or "transformed cells"), "transfectants" (or "transfected cells"), or "infectious agents" (or "infected cells"), each of which includes primary transformed, transfected, or infected cells and progeny derived therefrom. Such progeny may not be exactly identical in nucleic acid content to the parent cell and may contain mutations.

The amount administered is preferably a "prophylactically effective amount" (prophylaxis may be considered herein as treatment, both being used interchangeably) sufficient to show benefit to the individual.

Examples

Example 1: construction of chimeric Gene in which the C-terminal HPV18L1 is replaced with the C-terminal HPV33L1

1.1 Construction of pFB-HPV18L1 as template

The HPV18L1 gene is synthesized by the gene of Thermo Fisher company [ original English Weijie (Shanghai) trade Co., ltd ], and the two ends of the synthesized sequence are respectively provided with KpnI and XbaI restriction sites, and the sequence is shown in SEQ ID NO:5. the synthesized gene fragment was ligated to pcDNA3 vector (vendor Thermo Fisher) via KpnI and XbaI cleavage sites to obtain plasmid pcDNA3-HPV18-L1 containing nucleotide sequences encoding HPV18L11-507 amino acids.

The resulting pcDNA3-HPV18-L1 plasmid was subjected to KpnI and XbaI double digestion to obtain fragments of the gene of HPV18L1 (1-507). Then, this fragment was ligated with the pFastBac ^TM vector (vendor Thermo Fisher) digested with KpnI and XbaI to obtain a bacmid vector containing the HPV18L1 (1-507) gene fragment, designated pFB-HPV18L1.

1.2 Construction of pFB-HPV33L1 used as template

The HPV33L1 gene is synthesized by the gene of Thermo Fisher company [ original English Weijie (Shanghai) trade Co., ltd.), and the two ends of the synthesized sequence are respectively provided with KpnI and XbaI restriction sites, and the sequence of the gene fragment is shown in SEQ ID NO:6. the synthesized gene fragment was ligated to pcDNA3 vector (vendor Thermo Fisher) via KpnI and XbaI cleavage sites to obtain plasmid pcDNA3-HPV33-L1 containing nucleotide sequences encoding amino acids 33L11-499 of HPV.

The resulting pcDNA3-HPV33-L1 plasmid was subjected to KpnI and XbaI double digestion to obtain fragments of the gene of HPV33L1 (1-499). This fragment was then ligated with a KpnI and XbaI double digested pFastBacTM vector (vendor Thermo Fisher) to give a bacmid vector containing the HPV33L1 (1-499) gene fragment, designated pFB-HPV33L1.

1.3PFB-HPV18L1:33C construction

Chimeric gene with HPV18L 1C-terminal substitution to HPV33L 1C-terminal: the successfully constructed recombinant plasmid pFB-HPV18L1 is taken as a gene template, a gene fragment with the length of 1429bp is amplified by using primers F1 and R1, and the primer sequence F1 is shown as SEQ ID No:7, R1 is shown as SEQ ID No: shown at 8.

The gene fragment comprises a gene fragment encoding 1-470 amino acids of HPV18L1, 10 bases overlapped with a gene fragment of 474-499 amino acids of HPV33L1 and a KpnI cleavage site (GGTAC ≡C) segment, and the amplified sequence is shown as SEQ ID No:9 shows:

PCR amplification parameters: pre-denaturation at 94℃for 5min; denaturation at 98℃for 10s, annealing at 69℃for 15s, annealing at 72℃for 1kb/1min, and 30 cycles; extending at 72 ℃ for 5min; ending at 16 ℃.

The recombinant plasmid pFB-HPV33L1 is used as a gene template, primers F2 and R2 are used for amplifying a gene fragment with the length of 101bp, and the primer sequence F2 is shown as SEQ ID No:10, R2 is shown as SEQ ID No: 11.

The gene fragment contains 26 (474-499) amino acid gene fragments of HPV33L 1C end, 10bp basic groups overlapped with 1-470 amino acid C end gene fragments of HPV18L1 and XbaI (T CTAGA) enzyme cleavage sites, and the amplified sequence is shown as SEQ ID No: shown at 12.

PCR amplification parameters: pre-denaturation at 94℃for 5min; denaturation at 98℃for 10s, annealing at 69℃for 15s, annealing at 72℃for 1kb/1min, and 30 cycles; extending at 72 ℃ for 5min; ending at 16 ℃.

PCR splice sequence:

the splicing primers are F1 and R2 respectively, and fragments amplified by the primers (fragments amplified by F1 and R1 and fragments amplified by F2 and R2) are used as templates.

PCR splice parameters: pre-denaturation at 94℃for 5min; denaturation at 98℃for 10s, annealing at 52℃for 15s, 1kb/1min at 72℃for 5 cycles; denaturation at 98℃for 10s, annealing at 68℃for 15s, annealing at 72℃for 1kb/1min, 25 cycles; extending at 72 ℃ for 5min; ending at 16 ℃.

Finally, SEQ ID NO:4, a nucleotide sequence consisting of 1-470 amino acids of HPV18L1 and 26 (474-499) amino acids at the C-terminus of HPV33L1, and KpnI and XbaI cleavage sites (hereinafter referred to as splice sequences) at both ends.

The pFastBac ^TM vector and the spliced sequence fragment were double digested with KpnI+XbaI, and the spliced sequence was cloned into the pFastBac ^TM vector to obtain the recombinant plasmid pFB-HPV18L1:33C. Namely, the chimeric gene with HPV18L 1C-terminal replaced by HPV33L 1C-terminal.

Example 2: HPV 18L1:33C recombinant baculovirus package

PFB-HPV 18L1 constructed in example 1: after the recombinant plasmid of 33C is identified and sequenced correctly, it is transformed into DH10Bac bacterial competent cellsKit, purchased from Thermo Fisher), cultured and amplified at 37 ℃ and subjected to plate streak culture, white bacterial plaque is selected and amplified, bacterial liquid is collected after overnight culture, and recombinant bacmid DNA is extracted by an alkaline lysis method.

Packaging of recombinant baculovirus seed was performed by transfection into insect cell SF9 with cationic transfection reagent (purchased from Sino Biological). The specific operation is as follows:

a. Taking SF9 cells in logarithmic growth phase, inoculating dish according to the density of 0.6X10 ⁶ cell/dish, standing the dish inoculated with SF9 cells for 2h at room temperature, and attaching.

B. The extracted plasmid 20. Mu.L of Bacmid DNA was added to 200. Mu.L of Grace's Medium (serum free, additive free, purchased from Gibico) and mixed upside down 5 times.

Mu.L of 0.2x TF1 (transfection reagent, purchased from Sino Biological) was added dropwise to 200. Mu.L of Grace's Meduim and gently mixed.

D. Mixing b and c. Incubating for 15-45min at room temperature.

E. When the DNA was incubated with cellfectin (purchased from Sino Biological), the cell supernatant was discarded and Grace Medium 0.8 mL/disch without serum addition was added.

F. and d, dripping the incubated DNA and transfection reagent complex into the dish.

Incubation at 27℃for 2hr.

H. cell culture broth was discarded and 2.5mL/dish complete growth medium (SCD6SF+10% FBS) (SCD 6 SF was purchased from Sino Biological, FBS was purchased from Gibico) was added.

The cells were incubated at 27℃for 7 days to observe the presence of viral infection.

After transfection, the virus supernatant is collected after the cells have developed obvious lesions and typically cultured for 7-11 days. Sterile collection of virus supernatant by pipettor, namely HPV18L1:33C P1 is used as a seed for virus. HPV18L1 was used: the 33C P1 virus seed is used for infecting SF9 cells according to the proportion of 1:50 (V/V), the infection density of the SF9 cells is 2 multiplied by 10 ⁶ cells/mL, the SF9 cells are cultured and amplified for 3 days at the temperature of 27 ℃,1000 g+/-200 g is centrifuged at room temperature for 10min, and the collected virus supernatant is the P2 virus, and can be used for infection production.

Example 3: HPV18L1:33C expression production

HPV 18L1 was used as obtained in example 2: the baculovirus of the 33C recombinant gene is used for infecting High Five cells, the infection ratio is 1:200 (V/V), 1000 g+/-100 g room temperature is used for centrifuging and collecting cell sediment, PBS or MOPS buffer solution (pH is 6.0-7.0, the salt concentration is 100 mM-1M) is used for ultrasonically lysing the cell sediment, the cell sediment is crushed for 3min at low temperature, centrifugal force of more than 10000g is used for centrifuging for 10min, and supernatant fluid after centrifugation is collected and detected by SDS-PAGE electrophoresis. Lane 1: marker (7 purified proteins, molecular weight size 14.4 to 116kDa, manufacturer Thermo Scientific); lane 2: cell lysate; lane 3: supernatant collected after centrifugation of the lysate.

The results are shown in FIG. 1, and HPV 18L1 prepared by the method: the 33C L1 protein has the yield of more than 100mg/L and the protein size of about 56KD, and can be used for large-scale production.

Example 4: HPV 18L1: purification preparation of 33C Virus-like particles

HPV 18L1: the 33C virus-like particle purification method was a two-step chromatography, namely HS-MMA method, and the supernatant collected in example 3 was purified to finally obtain virus-like particles of high purity.

First step chromatography:

Medium: produced by Thermo Fisher company 50HS strong cation exchange media.

Volume of medium: the volume of the medium is 150mL, and the linear flow rate is 30mL/min.

Chromatographic conditions: equilibration buffer (pH 6.2, salt concentration 50mM phosphate, 0.5M sodium chloride); washing buffer (50 mM phosphate, 0.75M sodium chloride, pH6.2; saline concentration)

The column was equilibrated with 5CV buffer and then loaded. After loading, the mixed proteins were eluted with 5CV of equilibration buffer and wash buffer, respectively.

Elution conditions: pH6.2, elution with 1.25M sodium chloride in 50mM phosphate buffer containing 50mM arginine hydrochloride.

And a second step of chromatography:

medium: MMA ion exchange medium produced by Shanghai Bogu company was used.

Volume of medium: the volume of the medium is 150mL, and the linear flow rate is 30mL/min.

Chromatographic conditions: equilibration buffer 50mM PB,1.25M NaCl,pH6.2. The column was equilibrated with 4CV equilibration buffer and then loaded. After loading, the target protein was collected by washing the hetero-protein with 5CV equilibration buffer and then eluting the target protein with elution buffer.

Elution conditions: 100mM NaAC,150mM NaCl,0.01%Tween 80,pH4.5.

Example 5: HPV 18L1: morphological detection of 33C virus-like particles

A 10 μl sample was taken for transmission electron microscopy. Fixing the sample on a carbon spray copper net for adsorption for 2min, sucking residual liquid by using filter paper, dyeing by using phosphotungstic acid (concentration is 2% and pH is 6.5) twice, sucking residual dyeing liquid by using filter paper each time for 30 seconds, and observing under a transmission electron microscope after airing. The transmission electron microscope (brand: hitachi, model: H-7650) was 80KV and magnification was 80,000. The electron microscope observation results are shown in FIG. 2, and the C-terminal modified HPV 18L1 is shown in FIG. 2: 33C can form virus-like particles with uniform size, and the average diameter is about 60 nm.

Example 6: HPV 18L1: evaluation of immunogenicity of 33C Virus-like particle animals

6.1 Modeling of pseudovirus neutralizing cells

Because HPV is difficult to culture in vitro and has strong host specificity, it is difficult to reproduce organisms other than human bodies, and a proper animal model is lacking. It is therefore necessary to build a suitably efficient in vitro neutralization experimental model for the evaluation of vaccine immunoprotection.

HPV pseudoviruses are ideal experimental models for HPV in vitro neutralization: by utilizing the characteristic of HPV VLPs with nonspecific nucleic acid encapsulation, VLPs composed of intracellular expressed HPV L1 and L2 encapsulate free DNA or are introduced into exogenous plasmids to form HPV pseudoviruses.

The immunogenicity of the animal serum samples after sample immunization was analyzed by pseudovirus neutralization. The HPV18 virus-like particle sample, upon immunization of an animal, produces neutralizing antibodies against HPV18, neutralizing pseudoviruses of HPV18 type. After the immunized animal serum is incubated with a certain amount of pseudoviruses and then the cells are infected, the cells capable of expressing GFP fluorescence can be reduced along with the increase of neutralizing antibodies in the serum, and linear negative correlation can exist in a certain range, so that the neutralizing activity of the antibodies in the serum can be evaluated by detecting the change of the number of the cells capable of expressing GFP.

The pseudovirus construction method comprises the following steps: the pCMV3-3-HPV18L1+L2 (L1 sequence from Uniprot Q80B70, L2 sequence from Uniprot P06793) plasmid (available from Sino Biological) and a fluorescent plasmid (PSEU-GFP Spark, available from Sino Biological) of HPV type 18 were co-transfected into 293FT adherent cells (available from Thermo Fisher). The method reference (Pastrana D V,Buck C B,Pang Y S,Thompson C D,Castle P E,FitzGerald P C,Kjaer S K,Lowy D R,Schiller J T.Reactivity of human sera in a sensitive,high-throughput pseudovirus-based papillomavirus neutralization assay for HPV16and HPV18.[J]Virology 2004,321：205-216.). collects the pseudovirus supernatant and sub-packages, and stores in a refrigerator at-80 ℃ for standby.

6.2HPV 18L1: evaluation of immunoprotection in 33C Virus-like particle animals

Mouse immunization procedure:

HPV 18L1:33C virus-like particles are adsorbed on aluminum phosphate adjuvant, 200 mu L of the mixture is taken after mixing and used for immunizing mice, the immune dose of each mouse is 0.15 mu g, 10 mice are immunized, the diluted samples are respectively used for immunizing the mice on the 0 th day, the 7 th day and the 21 st day of the experiment, a blank serum control group is established, eyeballs of the mice are taken for blood collection on the 28 th day of the experiment, and the serum is separated for pseudovirus neutralization titer detection.

Mouse EC50 detection:

After 30 minutes of inactivation at 56 ℃, the mice serum was centrifuged at 6000g for 5 minutes and the supernatant was taken for detection. 4-8 hours prior to detection, 293FT cells were plated at a density of 15000 cells/well in 96-well plates and incubated at 37℃in a carbon dioxide incubator at 5% CO ₂. The serum of mice after immunization and the serum of blank control are respectively mixed with HPV18 pseudovirus prepared in 6.1 according to the volume ratio of 1:1 after serial dilution by a neutralization culture medium. After incubation for 1 hour in a refrigerator at 2-8 ℃ 100 μl/well was added to 293FT cells plated 4-8 hours in advance, 2 duplicate wells per sample, with blank serum control wells, false virus positive control wells and negative control wells established. The cells after the sample addition were further cultured in a carbon dioxide incubator at 37℃with 5% CO ₂ for 62-96 hours, and then subjected to fluorescent scanning photographing and counting in an ELISA (model: S6 Universal-V Analyzer, manufacturer: CTL). The neutralization inhibition rate of each mouse serum sample is calculated, and the maximum dilution factor of the serum, namely the half-effective dilution factor EC ₅₀, when the neutralization inhibition rate of the serum is 50% is calculated according to a Reed-Muench method.

The neutralization titer of HPV18 serum pseudovirus is shown in Table 4.

TABLE 4 results of mice serum neutralization titer assay EC ₅₀ (GMT.+ -. SEM)

Remarks:

1. Number of animals, n=10;

Gmt (geometry MEAN TITER): geometric mean titer;

sem (Standard Error of Mean): standard error.

The detection result shows that HPV 18L1 prepared by the invention: the 33C virus-like particle has better immunogenicity, can generate high-titer neutralizing antibodies in animals, and can be used for preparing vaccines for preventing HPV infection.

Comparative example 1: expression of C-terminally truncated HPV16L1 (1-474)

The inventors tried to truncate the C-terminal of HPV16L1 by 31 amino acids, designated HPV16L1 (1-474) (SEQ ID NO: 14). However, in the study, it was found that truncated HPV16L1 (1-474) protein was expressed in high quantity but protein solubility was poor, and extraction purification was difficult, and specific expression and extraction results are shown in FIG. 3.

While the invention has been described in detail in the foregoing description and with reference to the embodiments, it is for an understanding of the nature and character of the invention to be considered as obvious and obvious to those skilled in the art, without departing from the spirit or scope of the appended claims.

Appendix 1: sequence listing

Claims (16)

1. A chimeric HPV type 18L1 protein having the sequence of SEQ ID No. 3.

2. An HPV type 18 virus-like particle comprising the chimeric HPV type 18L 1 protein of claim 1.

3. The HPV type 18 virus-like particle of claim 2, which is an icosahedron consisting of pentamers of 72 of the chimeric HPV type 18L 1 proteins.

4. An immunogenic composition for preventing HPV-related diseases or infections comprising HPV type 18 virus-like particles according to claim 2 or 3 and an adjuvant.

5. An isolated polynucleotide encoding the chimeric HPV type 18L 1 protein of claim 1.

6. An isolated polynucleotide having the sequence set forth in SEQ ID No. 4.

7. A vector comprising the polynucleotide of claim 5 or 6.

8. The vector of claim 7, wherein the vector is a baculovirus vector.

9. A baculovirus comprising the polynucleotide of claim 5 or 6.

10. A host cell comprising the polynucleotide of claim 5 or 6, the vector of claim 7 or 8, or the baculovirus of claim 9.

11. The host cell of claim 10, which is an insect cell.

12. The host cell of claim 11, wherein the insect cell is selected from Sf9 cells, sf21 cells, hi5 cells, and S2 cells.

13. A method of preparing HPV type 18 virus-like particles according to claim 2 or 3, comprising:

Culturing the host cell of any one of claims 10 to 12 to express the chimeric HPV type 18L 1 protein and assemble into virus-like particles; and

Purifying the HPV type 18 virus-like particle.

14. The method of claim 13, wherein the host cell is a Hi5 cell.

15. The method of claim 13, wherein the purifying employs cation exchange chromatography.

16. The method of claim 15, wherein the cation exchange chromatography is HS-MMA two-step chromatography.