HK40066218A - Chimeric papillomavirus li protein - Google Patents

Description

Chimeric human papillomavirus L1 protein

Technical Field

This invention relates to the human papillomavirus (HPV) L1 protein and the polynucleotide encoding the protein, as well as HPV virus-like particles and methods for their preparation.

Background Technology

Papillomavirus (PV) belongs to the Papillomaviridae family and can cause papillomas in humans, cattle, dogs, rabbits, etc. Its member, human papillomavirus (HPV), is a non-enveloped DNA virus. The genome of this virus is a double-stranded closed circular DNA, about 7.2-8kb in size, with 8 open reading frames, which can be divided into three regions according to function: (1) early region (E), about 4.5kb, encoding 6 non-structural proteins E1, E2, E4-E7, which are related to viral replication, transcription and transformation; (2) late region (L), about 2.5kb, encoding the major capsid protein L1 and the minor capsid protein L2; (3) long regulatory region (LCR), which is located between the end of the L region and the beginning of the E region, about 800-900bp in length, does not encode any protein, but has DNA replication and expression regulatory elements.

L1 and L2 proteins are synthesized in the mid-to-late stages of the HPV infection cycle. L1 protein is the major capsid protein and has a molecular weight of 55-60 kDa. L2 protein is the minor capsid protein. 72 L1 protein pentamers constitute the outer shell of the icosahedral HPV virus particle (45-55 nm in diameter), which encapsulates closed circular double-stranded DNA. L2 protein is located inside 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 L1 protein ORF is the most conserved gene in the PV genome and can be used to identify new PV types. A new PV type is identified if the complete genome is cloned and the DNA sequence of the L1 ORF differs from the closest known PV type by more than 10%. Differences between 2% and 10% homology are defined as different subtypes, and differences less than 2% are defined as different variants of the same subtype (E.-M. de Villiers et al./Virology 324(2004)17-27).

In the later stages of HPV infection, newly synthesized L1 protein in the cytoplasm is transported to the nucleus of terminally differentiated keratinocytes, where it, along with L2 protein, packages the replicated HPV genomic DNA to form 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). This indicates that nuclear importation of L1 protein plays a crucial role in HPV infection and production. The ability of the virus to enter the nucleus is determined by the nuclear localization signal (NLS) at the C-terminus of the HPV L1 protein, a characteristic of which is its high content of basic amino acids (Garcia-Bustos, J., et al. 1991. Nuclear protein localization. Biochimica et Biophysica Acta 1071: 83-101).

Fifteen high-risk (HR) HPV types can cause cervical, anal, penile, vaginal, vulvar, and oropharyngeal cancers. HPV-16 and HPV-18 are the most common causes of these cancers, accounting for approximately 70% of cervical cancers. The remainder are caused by other HR-HPV types (31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73, and 82). HPV-16 accounts for approximately 95% of HPV-positive oropharyngeal cancers (OPCs). Persistently low-risk HPV-6 and HPV-11 genotypes cause 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. Berman and John T. Schiller, PhD2 Cancer 2017;123:2219-29).

The L1 protein is recombinantly expressed using vaccinia virus, baculovirus, or yeast systems. The L1 protein can self-assemble to form virus-like particles (VLPs), which contain approximately 72 L1 proteins and resemble the viral capsid. VLPs have no indications. VLPs can induce neutralizing antibodies in vaccinated animals, protecting experimental animals from subsequent attack by infectious viruses. Therefore, 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).

GlaxoSmithKline's vaccine is a bivalent recombinant HPV vaccine. It contains recombinant L1 proteins for HPV types 16 and 18, obtained by expressing a recombinant baculovirus expression vector system in the cells of the cutworm (Trichoplusia ni). The L1 proteins self-assemble into virus-like particles and are used to prevent cervical cancer, grade 2 or 3 cervical intraepithelial neoplasia and adenocarcinoma in situ, and grade 1 cervical intraepithelial neoplasia (carcinogenic) caused by HPV types 16 and 18 in women aged 9-25 (https://www.fda.gov/downloads/BiologicsBloodVaccines/Vaccines/ApprovedProducts/UCM186981.pdf).

It is a quadrivalent (types 6, 11, 16, and 18) recombinant human papillomavirus vaccine manufactured by Merck. It is used for the prevention of cervical cancer, genital warts (condyloma acuminata), and precancerous or proliferative lesions caused by HPV types 6, 11, 16, and 18 in girls and women aged 9-26; and for the prevention of anal cancer, genital warts (condyloma acuminata), and precancerous or developmental lesions caused by HPV types 6, 11, 16, and 18 in boys and men aged 9-26 (https://www.fda.gov/vaccines-blood-biologics/vaccines/gardasil).

It is a nine-valent recombinant human papillomavirus vaccine produced by Merck, which contains virus-like particles of the L1 protein of HPV types 6, 11, 16, 18, 31, 33, 45, 52 and 58. The L1 protein is produced by fermentation of Saccharomyces cerevisiae and self-assembles into VLP. For use in girls and women aged 9–45 years to prevent cervical, vulvar, vaginal, and anal cancers caused by HPV types 16, 18, 31, 33, 45, 52, and 58; genital warts (condyloma acuminata) caused by HPV types 6 and 11; and precancerous or proliferative lesions caused by HPV types 6, 11, 16, 18, 31, 33, 45, 52, and 58; and in boys and men aged 9–45 years to prevent anal cancer caused by HPV types 16, 18, 31, 33, 45, 52, and 58; genital warts (condyloma acuminata) caused by HPV types 6 and 11; and precancerous or developmental 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).

The product information states that HPV types 16 and 18 are responsible for approximately 70% of cervical cancer cases, while the remaining 20% are attributed to types 31, 33, 45, 52, and 58, thus preventing 90% of cervical cancer cases (https://www.fda.gov/BiologicsBloodVaccines/Vaccines/ApprovedProducts/ucm426445.htm).

A key factor in HPV vaccine development is the ability to mass-produce virus-like particles (VMPs). Currently, the most common systems for producing VMPs are mainly divided into eukaryotic expression systems and prokaryotic expression systems.

Commonly used eukaryotic expression systems include poxvirus, baculovirus, and yeast expression systems. HPV L1 protein expressed in eukaryotic systems exhibits less native conformational disruption and can spontaneously assemble into virus-like particles, but yields are low. Prokaryotic expression systems, primarily E. coli systems, offer high yields, but the protein mostly exists in inclusion body form, which is detrimental to purification and results in complex production processes.

Therefore, there remains a need in this field to obtain high yields of HPV virus-like particles.

Summary of the Invention

In one aspect, the present invention provides a chimeric human papillomavirus (HPV) L1 protein comprising, from its N-terminus to its C-terminus, a. an N-terminal fragment derived from a first type of HPV L1 protein, said N-terminal fragment maintaining the immunogenicity of the corresponding HPV type L1 protein; and b. a C-terminal fragment derived from a second type of HPV L1 protein, said second type of HPV L1 protein having better expression levels and solubility compared to other types of L1 proteins; wherein the chimeric HPV L1 protein has the immunogenicity of the first type of HPV L1 protein.

In another aspect, the present invention provides a papillomavirus-like particle comprising a chimeric papillomavirus L1 protein.

In another aspect, the present invention provides an immune composition for preventing papillomavirus-related diseases or infections, comprising papillomavirus-like particles and an adjuvant as described above.

In another aspect, the present invention provides an isolated polynucleotide encoding a chimeric human papillomavirus L1 protein.

In another aspect, the present invention provides a vector comprising a polynucleotide encoding a chimeric human papillomavirus L1 protein.

In another aspect, the present invention provides a baculovirus comprising a polynucleotide encoding a chimeric human papillomavirus L1 protein.

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

In another aspect, the present invention provides a method for preparing papillomavirus-like particles, comprising culturing host cells as described above to express the chimeric papillomavirus L1 protein and assembling them into virus-like particles; and purifying the papillomavirus-like particles.

Attached Figure Description

Figure 1A. Expression of L1 protein in HPV 6 L1:33C. M: Marker; L: Cell lysis buffer; E-S: Supernatant collected after centrifugation of lysis buffer.

Figure 1B. Expression of L1 protein in HPV 11 L1:33C. M: Marker; L: Cell lysis buffer; E-S: Supernatant collected after centrifugation of lysis buffer.

Figure 1C HPV 16 L1: Expression of L1 protein in 33C. M: Marker; L: Cell lysis buffer; E-S: Supernatant collected after centrifugation of lysis buffer.

Figure 1D. Expression of L1 protein in HPV 18 L1:33C. M: Marker; L: Cell lysis buffer; E-S: Supernatant collected after centrifugation of lysis buffer.

Figure 1E HPV 31 L1: Expression of L1 protein in 33C. M: Marker; L: Cell lysis buffer; E-S: Supernatant collected after centrifugation of lysis buffer.

Figure 1F. Expression of L1 protein in HPV 35 L1:33C. M: Marker; L: Cell lysis buffer; E-S: Supernatant collected after centrifugation of lysis buffer.

Figure 1. Expression of L1 protein in G HPV 39 L1:59C. M: Marker; L: Cell lysis buffer; E-S: Supernatant collected after centrifugation of lysis buffer.

Figure 1H. Expression of L1 protein in HPV 45 L1:33C. M: Marker; L: Cell lysis buffer; E-S: Supernatant collected after centrifugation of lysis buffer.

Figure 1I. Expression of L1 protein in HPV 51 L1:33C. M: Marker; L: Cell lysis buffer; E-S: Supernatant collected after centrifugation of lysis buffer.

Figure 1. Expression of L1 protein in J HPV 52 L1:33C. M: Marker; L: Cell lysis buffer; E-S: Supernatant collected after centrifugation of lysis buffer.

Figure 1. Expression of L1 protein in K HPV 56 L1:33C. M: Marker; L: Cell lysis buffer; E-S: Supernatant collected after centrifugation of lysis buffer.

Figure 1. Expression of L1 protein in L HPV 58 L1:33C. M: Marker; L: Cell lysis buffer; E-S: Supernatant collected after centrifugation of lysis buffer.

Figure 2A shows HPV 6 L1:33C virus-like particles observed by transmission electron microscopy.

Figure 2B shows HPV 11 L1:33C virus-like particles observed by transmission electron microscopy.

Figure 2C shows HPV 16 L1:33C virus-like particles observed by transmission electron microscopy.

Figure 2D shows HPV 18 L1:33C virus-like particles observed by transmission electron microscopy.

Figure 2E shows HPV 31 L1:33C virus-like particles observed by transmission electron microscopy.

Figure 2F shows HPV 35 L1:33C virus-like particles observed by transmission electron microscopy.

Figure 2G shows HPV 39 L1:59C virus-like particles observed by transmission electron microscopy.

Figure 2H shows HPV 45 L1:33C virus-like particles observed by transmission electron microscopy.

Figure 2I shows HPV 51 L1:33C virus-like particles observed by transmission electron microscopy.

Figure 2J shows HPV 52 L1:33C virus-like particles observed by transmission electron microscopy.

Figure 2K shows HPV 56 L1:33C virus-like particles observed by transmission electron microscopy.

Figure 2L shows HPV 58 L1:33C virus-like particles observed by transmission electron microscopy.

Figure 3. Expression of C-terminal truncated HPV16L1(1-474). M: Marker; L: Cell lysis buffer; E-S: Supernatant collected after centrifugation of lysis buffer.

Detailed Implementation

In one aspect, the present invention provides a chimeric human papillomavirus (HPV) L1 protein comprising, from its N-terminus to its C-terminus: a. an N-terminal fragment derived from a first type of HPV L1 protein, said N-terminal fragment maintaining the immunogenicity of the corresponding HPV type L1 protein; and b. a C-terminal fragment derived from a second type of HPV L1 protein, said second type of HPV L1 protein having better expression levels and solubility compared to other types of L1 proteins; wherein the chimeric HPV L1 protein has the immunogenicity of the first type of HPV L1 protein.

In one embodiment, the N-terminal fragment is a fragment obtained by truncating the C-terminus of the natural sequence of the first type of papillomavirus L1 protein to any amino acid site within its α5 region, and a fragment having at least 98% identity with it; the C-terminal fragment is a fragment obtained by truncating the N-terminus of the natural sequence of the second type of papillomavirus L1 protein to any amino acid site within its α5 region, and a functional variant of the fragment resulting from further mutation, deletion, and/or addition.

In another embodiment, the N-terminal fragment has at least 98.5%, 99%, 99.5%, or 100% identity with a fragment obtained by truncating the C-terminus of the natural sequence of the first type of papillomavirus L1 protein to any amino acid site in its α5 region.

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

In one embodiment, the papillomavirus L1 protein is the HPV L1 protein.

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

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

More preferably, the second type of human papillomavirus L1 protein is selected from HPV type 33 or HPV type 59 L1 protein.

In one embodiment, the second type of human papillomavirus L1 protein is HPV 33 L1 protein, and the C-terminal fragment is SEQ ID No: 2; or a fragment of length m1 amino acids, preferably covering the first to the first m1 amino acids of SEQ ID No: 2; wherein m1 is an integer from 8 to 26; or the C-terminal fragment is SEQ ID No: 132; or a fragment of length m2 amino acids, preferably covering the first to the first m2 amino acids of SEQ ID No: 132; wherein m2 is an integer from 13 to 31.

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

In another embodiment, the second type of human papillomavirus L1 protein is HPV59 L1 protein, and the C-terminal fragment is SEQ ID No: 13; or a fragment of length n amino acids, preferably covering the first to nth amino acids of SEQ ID No: 13; where n is an integer from 16 to 38.

In one embodiment, the C-terminal fragment of the HPV 59 L1 protein has a nuclear localization sequence. In another embodiment, the C-terminal fragment of the HPV 59 L1 protein has two nuclear localization sequences. In some embodiments, the chimeric papillomavirus L1 protein comprises one or more C-terminal fragments of the HPV 59 L1 protein. The multiple C-terminal fragments of the HPV 59 L1 proteins may be the same or different.

In one embodiment, the first type of human papillomavirus L1 protein is selected from HPV types 6, 11, 16, 18, 31, 35, 39, 45, 51, 52, 56 or 58 L1 proteins.

In one embodiment, the C-terminus of the N-terminal segment is directly connected to the N-terminus of the C-terminal segment or connected via a connector.

The adapter does not affect the immunogenicity of the N-terminal fragment, nor does it affect the protein expression level or solubility. In one embodiment, the N-terminal fragment and the C-terminal fragment are linked by an adapter consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. In one embodiment, the adapter is an artificial sequence. In another embodiment, the adapter is a naturally occurring sequence in the HPV L1 protein. In another embodiment, the adapter may be a partial sequence of the HPV 33 L1 protein. In another embodiment, the adapter may be a partial sequence of the HPV 59 L1 protein.

In one embodiment, when the C-terminus of the N-terminal fragment is connected to the N-terminus of the C-terminal fragment, the following continuous amino acid sequence exists within a range of 4 amino acid sites plus or minus the connection point: RKFL; preferably, the following continuous amino acid sequence exists within a range of 6 amino acid sites plus or minus the connection point: LGRKFL.

In one aspect, the present invention provides a papillomavirus-like particle comprising the chimeric papillomavirus L1 protein as described above. In one embodiment, the papillomavirus-like particle is an HPV virus-like particle, and in another embodiment, the HPV virus-like particle is an icosahedron composed of 72 pentamers of the chimeric HPV L1 protein. In one embodiment, the HPV virus-like particle has correctly formed disulfide bonds, thus exhibiting a well-defined native conformation. In one embodiment, the HPV virus-like particle self-assembles in an in vivo expression system.

In one aspect, the present invention provides an immunogenic composition for preventing papillomavirus-related diseases or infections, comprising papillomavirus-like particles and an adjuvant as described above. The prevention can be considered as treatment, and the two are used interchangeably.

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

In one aspect, the present invention provides an isolated polynucleotide encoding a chimeric papillomavirus L1 protein as described above. In one embodiment, the polynucleotide is a codon-optimized polynucleotide for different expression systems. In one embodiment, the polynucleotide is a codon-optimized polynucleotide for an insect baculovirus expression system.

In one aspect, the present invention provides a vector comprising the polynucleotides 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 yet another embodiment, the vector may be a recombinant vector for a baculovirus expression system.

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

In one aspect, the present invention provides a host cell comprising the 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 for preparing papillomavirus-like particles as described above, comprising: culturing host cells as described above to express the chimeric papillomavirus L1 protein and assembling them into virus-like particles; and purifying the papillomavirus-like particles.

In one embodiment, the host cell is an insect cell. In another embodiment, the host cell is a Hi5 cell. In one embodiment, the chimeric papilloma L1 protein is a chimeric HPV L1 protein that self-assembles into HPV virus-like particles within the host cell. In one embodiment, the chimeric HPV L1 protein self-assembles into HPV virus-like particles within the host cell, having an icosahedron composed of 72 pentamers of the chimeric HPV L1 protein. In one embodiment, the HPV virus-like particles have correctly formed disulfide bonds, thus possessing a well-defined native conformation.

In one embodiment, purification is performed using cation exchange chromatography. In another embodiment, purification is performed using strong cation exchange chromatography. In yet another embodiment, purification is performed using weak cation exchange chromatography. In one embodiment, purification is performed using a combination of multiple cation exchange chromatography steps. In one embodiment, purification is performed using HS strong cation exchange chromatography. In another embodiment, purification is performed using MMA ion exchange chromatography. In yet another embodiment, purification is performed using HS-MMA two-step chromatography.

The L1 protein of papillomavirus expressed by the eukaryotic expression system can spontaneously assemble into virus-like particles, but it has the disadvantage of low expression level and difficulty in large-scale production.

The sequences of L1 proteins for each HPV type can be easily obtained from https://www.uniprot.org. Each HPV L1 type can originate from different strains, resulting in multiple versions of its amino acid sequence. Any of these natural sequences can be used in this invention. During the conceptualization and design of this invention, the HPV L1 protein sequence for a given type may differ from the sequence used in the examples; however, such differences do not affect the inventors' judgments and conclusions.

Those skilled in the art generally believe that the C-terminus of the L1 protein does not contain a major neutralizing antigenic epitope. Therefore, attempts have been made to increase the expression level of HPV L1 protein by truncating the C-terminus. For example, in GlaxoSmithKline's US patent US6361778B1, the C-terminus of HPV16 L1 protein is truncated by 1-34 amino acids, preferably 26 amino acids, claiming that VLP yield is increased many times, preferably at least 10 times, and particularly about 10 to 100 times. Inspired by this, the inventors attempted to truncate the C-terminus of HPV type 16 L1 by 31 amino acids, naming it HPV16 L1 (1-474). However, while its protein expression level is high, its protein solubility is poor, making extraction and purification difficult (see comparative example).

The poor protein solubility caused by this truncation may be due to the deletion of the nuclear localization sequence at the C-terminus, but this invention is not limited to this speculation. During research and production, the inventors discovered that HPV 16 L1 proteins, HPV 28 L1 proteins, HPV 33 L1 proteins, HPV 59 L1 proteins, and HPV 68 L1 proteins have better expression levels and solubility compared to other L1 protein types. Inspired by this, the inventors replaced the C-terminus of HPV types that are difficult to extract or have low expression levels with the C-terminus of L1 proteins from types with better expression levels and solubility. That is, the inventors constructed a chimeric protein containing, from its N-terminus to its C-terminus, an N-terminal fragment derived from a type 1 papillomavirus L1 protein (e.g., HPV L1 protein) and a C-terminal fragment derived from a type 2 papillomavirus L1 protein (e.g., HPV L1 protein). The former provides the immunogenicity of the type 1 papillomavirus (e.g., HPV), while the latter provides better expression levels and solubility. The two can be connected directly or through a connector.

To maintain the immunogenicity of HPV type 1 L1 protein and ensure its ability to form VLPs, the inventors determined the appropriate length of the N-terminal fragment of the HPV L1 protein. The following reports pertain to epitope studies of common HPV subtypes:

Sunanda Baidya et al. reported that the epitopes of the L1 protein are 48EEYDLQFIFQLCKITLTA65, 45RHGEEYDLQFIFQLCKITLTA65, 63LPDPNKF69, 79PETQRLVWAC88, 36PVPGQYDA43, 77YNPETQRLVWAC88, 188DTGYGAMD195, 36PVPGQYDATK45, 45KQDIPKVSAYQYRVFRV61, and 130RDNVSVDYKQTQ. LCI144 and 49YSRHVEEY DLQFIF62 can be used as tools for designing vaccines for HPV types 16 and 18 (see Epitope design of L1 protein for vaccine production against Human Papilloma Virus types 16 and 18, Bioinformation 13(3): 86-93 March 2017, incorporated herein by reference in its entirety).

Katharina Slupetzky et al. reported that regions around HPV-16 aa 282-286 and 351-355 contribute to neutralizing epitopes, and that the latter are immune-dominant sites (see Chimeric papillomavirus-like particles expressing a foreign epitope on capsid surface loops, Journal of General Virology (2001), 82, 2799-2804, all incorporated herein by reference).

Brooke Bishop et al. prepared three variants of HPV11, 16, 18, and 35 L1 proteins: one with a 9-amino acid deletion at the N-terminus, one with a deletion of α4 (corresponding to amino acid residues 404-436 of HPV16), and one with a 31-amino acid deletion at the C-terminus. They reported that the first two variants could not assemble into a VLP, but did not report this phenomenon in the latter.

(Crystal Structures of Four Types of Human Papillomavirus L1 Capsid Proteins Understanding the Speciality of Neutralizing Monoclonal Antibodies, The Journal of Biological Chemistry, 282, 31803-31811. All are incorporated herein by reference). The α-helix and β-sheet loop regions of each HPV L1 protein type can be easily determined using commonly used sequence analysis software in this field. The α-helix region includes α1, α2, α3, α4, and α5 regions.

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 based on the above-cited literature (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). The results are shown below, where the parts between the downward arrows correspond to the regions missing in the literature for the preparation of variants.

In addition to the sequence alignment method used by the inventors, protein secondary structure prediction software that can be used for prediction includes, but is not limited to:

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

2.ProtPredict: 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/ SOPMAhttp://www.ibcp.fr/predict.html

7.SSPRED: http://www.embl-heidelberg.de/sspred/ssprdinfo.html .

In one embodiment of the invention, the inventors determined the length of the N-terminal fragment of the HPV L1 protein derived from the first type by truncating the natural sequence of the L1 protein in its α5 region and surrounding area, retaining the sequence from its N-terminus to the newly generated C-terminus in the α5 region. This truncated sequence ensures that it retains the immunogenicity of the HPV type and can form a VLP.

The N-terminal fragment of the HPV L1 protein derived from type 1 can be further modified to ensure that it has the immunogenicity of this type and can form VLPs.

The inventors determined the length of the C-terminal fragment of the HPV L1 protein derived from HPV type 2 by truncating the native L1 protein sequence in and around its α5 region, while retaining the newly generated N-terminal to C-terminal sequence from its α5 region. This truncated sequence does not possess a major neutralizing antigenic epitope and does not interfere with the immunogenicity of the resulting chimeric protein.

The C-terminal fragments of HPV L1 proteins derived from HPV type 2 can be further mutated, deleted, and/or added, preferably retaining at least one nuclear localization sequence. Yang et al. predicted the nuclear localization sequences of 107 HPV subtypes (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, all of which are incorporated herein by reference). The nuclear localization sequences of HPV L1 proteins of each type can be conveniently determined using sequence analysis software commonly used in the field.

The connection between the N-terminal and C-terminal segments mentioned above occurs at the newly generated C-terminus of the former and the newly generated N-terminus of the latter. This connection can be direct or via a connector. If the connection point is considered the origin, then the N-terminal side of the origin is negative, while the C-terminal side is positive.

The following shows the amino acid sequence of HPV6 L1 protein from positions 453 to 469, and a corresponding sequence of multiple HPV L1 protein types. It can be seen that these sequences are highly similar. This sequence overlaps with the α5 region. The numbers in parentheses indicate the position of the last amino acid in the listed sequence. For HPV type 45, some HPV 45 strains have an additional 26 amino acids at the N-terminus of their L1 protein, while others do not, so it is represented 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

HPV51 DLDQFALGRKFLLQVGV(474)

HPV52 DLDQFPLGRKFLLQAGL(478)

HPV56 DLDQFPLGRKFLMQLGTRS(474)

HPV58 DLDQFPLGRKFLLQSGL(473)

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

HPV59DLDQFPLGRKFLLQLGA(475)RPKPTIGPRKRAAPAPTSTPSPKRVKRRKSSRK, where... RKR in 484-486 and KRVKRRK in 498-504 are nuclear localization sequences.

In one embodiment of the present invention, the inventors conveniently completed the C-terminal substitution of the L1 protein between different HPV types by taking advantage of the sequence similarity of the α5 region and its surrounding regions among multiple HPV types.

In the most preferred embodiment of the invention, the inventors noted that all types of HPV L1 proteins have a tetrapeptide RKFL at similar positions, and more advantageously, a hexapeptide LGRKFL. The inventors cleverly utilized this highly conserved sequence to design the linker of the chimeric protein at any amino acid site of this oligopeptide. From one perspective, the sequence from the N-terminus of the chimeric protein to RKFL or LGRKFL is identical to the N-terminal segment of the HPV L1 protein derived from type 1; from another perspective, the sequence from RKFL or LGRKFL to the C-terminus of the chimeric protein is identical to the C-terminal segment of the L1 protein derived from type 2.

The resulting chimeric protein maintains a high degree of similarity to the natural HPV L1 protein, and can be expected to perform well in production and subsequent medical or preventative processes.

Those skilled in the art will understand that because there are different strains of the same type of HPV, their natural sequences are different, and chimeric proteins constructed using different strains also fall under the scope of this invention.

Those skilled in the art will understand that, due to the high similarity between different HPV L1 types, if, during the construction of the chimeric protein, the N-terminal fragment of the HPV L1 protein derived from the first type is extended to the C-terminus with more amino acid residues, or the C-terminal fragment of the HPV L1 protein derived from the second type is extended to the N-terminus with more amino acid residues, it is possible to form a chimeric protein with the same structure as the present invention due to the similarity or identical amino acids at the corresponding sites. Such chimeric proteins also fall under the scope of the present invention.

Those skilled in the art will understand that, based on the chimeric protein of the embodiments described above, variants of the chimeric protein can be formed by mutations, deletions, and/or additions of amino acid residues. These variants may possess the immunogenicity of type 1 HPV L1 protein, be able to form VLPs, and have good yield and solubility. Chimeric proteins thus formed also fall under the scope of this invention.

Beneficial effects of the invention

Currently, expression systems commonly used for producing virus-like particles are divided into eukaryotic expression systems and prokaryotic expression systems. Eukaryotic expression systems can spontaneously assemble the L protein of papillomavirus into virus-like particles, but they suffer from low expression levels, making large-scale production difficult. Prokaryotic expression systems often disrupt the native conformation of the L protein of papillomavirus, requiring subsequent in vitro processing to obtain virus-like particles, and their yields are also low, making industrialization challenging.

This invention modifies the C-terminus of the L protein of papillomavirus (e.g., human papillomavirus), for example, by replacing it with a C-terminal fragment from the L1 protein of HPV16, HPV28, HPV33, HPV59, or HPV68. This can increase the expression level and solubility of the papillomavirus L protein in expression systems (e.g., host cells, such as insect cells). This can be used for the large-scale production of vaccines, such as HPV vaccines.

The inventors independently discovered that HPV 16 L1 proteins, HPV 28 L1 proteins, HPV 33 L1 proteins, HPV 59 L1 proteins, and HPV 68 L1 proteins exhibit better expression levels and solubility compared to other L1 protein types. Furthermore, they found that the increased protein expression levels and solubility depend on the C-terminal sequence of the HPV L1 proteins. In HPV 107 L1 proteins, most possess nuclear localization sequences at the C-terminus, and these C-terminal sequences show a certain degree of similarity.

For papillomavirus L proteins that are currently not expressed, have very low expression levels, or are insoluble after expression, replacing their C-terminal fragments with C-terminal fragments from HPV 16 L1, HPV 28 L1, HPV 33 L1, HPV 59 L1, or HPV 68 L1 proteins makes it possible to solublely express and subsequently purify the previously extremely low-expression or insoluble L proteins. This can be used for the large-scale production of multivalent vaccines (such as HPV vaccines), enabling more comprehensive prevention of infection by multiple papillomaviruses, especially HPV.

To achieve large-scale vaccine production, there is a need to increase the expression level and solubility of HPV L1 protein in insect cells. Furthermore, in yeast cells, the virus-like particles assembled from HPV L1 protein lack a proper conformation because they cannot correctly form disulfide bonds.

For HPV L1 protein, which has low expression levels and poor solubility in insect cells, modifying its C-terminal fragment to the C-terminal fragment of HPV type 33 or 59 L1 protein significantly improves expression levels and solubility, making it suitable for large-scale production of HPV vaccines.

For HPV L1 proteins, such as HPV 16, HPV 28, and HPV 68 L1 proteins, which exhibit better expression levels and solubility in insect cells compared to other L1 protein types, there is a need to further improve expression levels and solubility to achieve large-scale vaccine production. In this invention, for example, by modifying the C-terminal fragment of the HPV 16 L1 protein to the C-terminal fragment of the HPV 33 L1 protein, the expression level and solubility of the modified chimeric HPV 16 protein are improved, which is beneficial for the large-scale production of HPV vaccines.

In summary, the chimeric HPV L1 protein exhibits significantly increased expression levels and solubility in insect cells compared to the unmodified HPV L1 protein. This makes it suitable for large-scale production of HPV vaccines. Furthermore, the chimeric HPV L1 protein can correctly form disulfide bonds and assemble into well-conformed HPV virus-like particles in insect cells. This can enhance the immunogenicity of HPV virus-like particles and generate a better immune response.

definition

Unless otherwise stated, all technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains. For ease of understanding, the following terms are used with reference to their ordinary meanings.

When used herein and in the appended claims, the singular forms “a,” “another,” and “the” include the plural referents unless the context clearly indicates otherwise. Unless otherwise expressly stated, the terms “comprising,” “including,” “having,” etc., are intended to convey inclusion rather than limitation.

The term "immunogenicity" refers to the ability of a substance, such as a protein or polypeptide, to stimulate an immune response, that is, to stimulate the production of antibodies, especially humoral or cell-mediated responses.

The term "antibody" refers to an immunoglobulin molecule that binds to an antigen. Antibodies can be polyclonal mixtures or monoclonal. Antibodies can be complete immunoglobulins derived from natural or recombinant sources, or they can be immunoreactive portions of complete immunoglobulins. Antibodies can exist in various forms, including, for example, Fv, Fab', F(ab')2, and as single chains.

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

The term "epitaph" refers to any protein determinant that can specifically bind to an antibody or T-cell receptor. Epitope determinants are typically composed of chemically active surface groups of a molecule (such as amino acid or sugar side chains, or combinations thereof) and usually have specific three-dimensional structural features as well as specific charge features.

The terms "subtype" or "type" are used interchangeably herein to refer to a genetic variant of the viral antigen that allows one subtype to be recognized by the immune system in a way that distinguishes it from a different subtype. For example, HPV 16 is immunologically distinct from HPV 33.

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

The term "HPV" or "HPV virus" refers to papillomavirus of the Papillomaviridae family. It is a non-enveloped DNA virus with a double-stranded closed circular DNA genome of approximately 8 kb in size. It can generally be divided into three regions: ① Early region (E), which contains six open reading frames encoding non-structural proteins related to viral replication, transcription, and transformation, namely E1, E2, E4-E7, as well as open reading frames E3 and E8; ② Late region (L), which contains reading frames encoding the major capsid protein L1 and the minor capsid protein L2; ③ Long regulatory region (LCR), which does not encode any proteins but has the origin of replication 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 a minor capsid protein. 72 L1 pentamers constitute the outer shell of the icosahedral HPV viral particle, encapsulating a closed circular double-stranded DNA microchromosome. The L2 protein is located inside the L1 protein.

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

"HPV pseudoviruses" utilize the non-specific encapsulation of nucleic acids by HPV VLPs. They are formed by encapsulating free DNA with VLPs composed of HPV L1 and L2 expressed intracellularly, or by introducing exogenous plasmids. They are an ideal experimental model for HPV neutralization in vitro.

The "pseudovirus neutralization method" is a method for evaluating the neutralizing activity of antibodies. After immunizing an animal, the serum is incubated with a certain amount of pseudovirus and then infects cells. The number of cells decreases as the number of neutralizing antibodies in the serum increases. Within a certain range, there can be a linear negative correlation. Therefore, the neutralizing activity of antibodies in the serum can be evaluated by detecting changes in the number of expressing cells.

The terms "fragment thereof" or "variant thereof" refer to the deletion, insertion, and/or substitution of a portion of the nucleotide or amino acid sequence according to the invention. Preferably, the fragments or variants of the polypeptides provided by the present invention can elicit humoral and/or cellular immune responses in animals or humans.

The term "chimerism" refers to polypeptide or nucleotide sequences derived from different parent molecules linked together via amide bonds or 3',5'-phosphodiester bonds. Preferably, they are directly adjacent to each other without being separated by additional linker sequences.

The term "truncation" refers to removing one or more amino acids from the N and/or C-terminus of a polypeptide or deleting one or more amino acids from within the polypeptide.

The term "nuclear localization sequence" refers to an amino acid sequence that guides a protein into the cell nucleus. In some HPV L1 proteins, there is a 10-14 amino acid spacer between two tight clusters of basic residues (i.e., nuclear localization sequences) (e.g., one is KRKR, KRKK, KRKRK, KRKKRK, KRVKRRK, etc., and the other is KR, RKR, KRK, etc.). These basic residue clusters are nuclear localization sequences. In other HPV L1 proteins, the nuclear localization sequence is a tight cluster of basic residues formed by arginine and/or lysine. Nuclear localization sequences include, but are not limited to, examples of basic residue clusters as described above. 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, the entire contents of which are incorporated herein by reference.

The term "functional variant" refers to a version of a polypeptide or protein that retains the desired activity or characteristics even after being truncated, mutated, deleted, and/or added.

The “sequence identity” between two polypeptide or nucleic acid sequences represents the percentage of identical residues between the sequences, calculated based on the smaller of the compared molecules. In calculating the identity percentage, the sequences being compared are aligned in a manner that produces the maximum possible match between them, and gaps in the alignment (if present) are resolved using a specific algorithm. Preferred computer program methods for determining identity between two sequences include, but are not limited to, the GCG package, including GAP, BLASTP, BLASTN, and FASTA (Altschul et al., 1990, J. Mol. Biol. 215: 403-410). These programs are publicly available from the National Center for Biotechnology Information (NCBI) and other sources. The well-known Smith-Waterman algorithm can also be used to determine identity.

Non-critical amino acids can be conservatively substituted without affecting the normal function of the protein. A conservative substitution means replacing an amino acid with one that is chemically or functionally similar. Providing tables of conservative substitutions for similar amino acids is well known in the art. For example, in some embodiments, the amino acid groups provided in Tables 1-3 are considered to be mutually conservative substitutions.

Table 1 lists the selected groups of amino acids considered to be mutually conserved substitutions in some embodiments.

acid 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 Nonpolar, uncharged residues C, M, and P Aromatic residues F, Y and W

Table 2 lists other selected groups of amino acids that are considered to have mutually conserved substitutions in some embodiments.

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 lists other selected groups of amino acids that are considered to have mutually conserved substitutions in some embodiments.

Group A A and G Group B D and E Group C N and Q Group D R, K and H Group E I, L, M, V Group F F, Y and W Group G S and T Group H C and M

The term "amino acid" refers to twenty common, naturally occurring amino acids. These 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 (I1e; 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, vaccines may contain adjuvants. Adjuvants used in this invention may include, but are not limited to, one or more of the following: mineral adjuvant compositions, oil-emulsion adjuvants, saponin adjuvant formulations, bacterial or microbial derivatives.

The term "vector" refers to a nucleic acid molecule capable of proliferating another nucleic acid linked to it. This term includes vectors as self-replicating nucleic acid structures and vectors that integrate into the host cell genome into which they have been introduced. Some vectors can guide the expression of nucleic acids linked to them in an operative manner.

The term "host cell" refers to a cell in which foreign nucleic acids have been introduced, as well as the offspring of such cells. Host cells include "transformers" (or "transformed cells"), "transfectants" (or "transfected cells"), or "infected cells," each comprising the primary transformed, transfected, or infected cell and its derived offspring. Such offspring may not be identical to the parent cells in terms of nucleic acid content and may contain mutations.

The preferred dosage is the "preventive effective dose" (prevention can be considered as treatment in this article, and the two are used interchangeably), which is sufficient to show benefit to the individual.

Example

Example 1: Construction of chimeric genes

Example 1.1: Construction of a chimeric gene with HPV6L1 C-terminus replaced by HPV33L1 C-terminus

1.1.1 Construction of pFB-HPV6L1 as a template

The HPV6L1 gene was synthesized by Thermo Fisher Scientific (formerly Invitrogen (Shanghai) Trading Co., Ltd.), and the synthesized sequence had KpnI and XbaI restriction sites at both ends, as shown in SEQ ID NO: 5. The synthesized gene fragment was ligated into the pcDNA3 vector (marketed by Thermo Fisher) through the KpnI and XbaI restriction sites to obtain the plasmid pcDNA3-HPV6-L1 containing a nucleotide sequence encoding 1-500 amino acids of HPV6L1.

The obtained pcDNA3-HPV6-L1 plasmid was double-digested with KpnI and XbaI to obtain a fragment of the HPV6L1 (1-500) gene. This fragment was then ligated with the pFastBac ^™ 1 vector (marketed by Thermo Fisher) double-digested with KpnI and XbaI to obtain a rod-like vector containing the HPV6L1 (1-500) gene fragment, named pFB-HPV6L1.

1.1.2 Construction of pFB-HPV33L1 used as a template

The HPV33L1 gene was synthesized by Thermo Fisher Scientific (formerly Ingenic Semiconductor (Shanghai) Trading Co., Ltd.), and the synthesized sequence has KpnI and XbaI restriction sites at both ends, respectively. The gene fragment sequence is shown in SEQ ID NO: 6. The synthesized gene fragment was ligated into the pcDNA3 vector (marketed by Thermo Fisher) through the KpnI and XbaI restriction sites to obtain the plasmid pcDNA3-HPV33-L1 containing the nucleotide sequence encoding amino acids 1-499 of HPV33L1.

The obtained pcDNA3-HPV33-L1 plasmid was double-digested with KpnI and XbaI to obtain a fragment of the HPV33L1 (1-499) gene. This fragment was then ligated into the pFastBacTM1 vector (marketed by Thermo Fisher) double-digested with KpnI and XbaI to obtain a rod-like vector containing the HPV33L1 (1-499) gene fragment, named pFB-HPV33L1.

1.1.3 Construction of pFB-HPV6L1:33C

The chimeric gene with HPV6L1 C-terminus replaced by HPV33L1 C-terminus: Using the successfully constructed recombinant plasmid pFB-HPV6L1 as a gene template, a 1426bp gene fragment was amplified using primers F1 and R1. Primer sequence F1 is shown in SEQ ID No: 7, and R1 is shown in SEQ ID No: 8.

The gene fragment contains a gene segment encoding amino acids 1-469 of HPV6L1, a 10-base overlap with a gene segment encoding amino acids 474-499 of HPV33L1, and a KpnI restriction site (GGTAC^C). The amplified sequence is shown in SEQ ID No: 9.

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

Using recombinant plasmid pFB-HPV33L1 as a gene template, a 101 bp gene fragment was amplified using primers F2 and R2. Primer sequence F2 is shown in SEQ ID No: 10, and primer R2 is shown in SEQ ID No: 11.

The gene fragment contains a 26-amino acid (474-499) C-terminal segment of HPV33L1, a 10 bp base overlap with the 1-469 amino acid C-terminal segment of HPV6L1, and an XbaI (T^CTAGA') restriction site. The amplified sequence is shown in SEQ ID No: 12.

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

PCR assembly sequence:

The splicing primers were F1 and R2, and the fragments amplified by the above primers (fragments amplified by F1 and R1, and fragments amplified by F2 and R2) were used as templates.

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

The final result is SEQ ID NO: 4, which encodes a nucleotide sequence consisting of amino acids 1-469 of HPV6L1 and 26 (474-499) amino acids at the C-terminus of HPV33L1, with KpnI and XbaI restriction sites at both ends (hereinafter referred to as the spliced sequence).

The pFastBac ^™ 1 vector and the spliced sequence fragment were digested with KpnI and XbaI, and the spliced sequence was cloned into the pFastBac ^™ 1 vector to obtain the recombinant plasmid pFB-HPV6L1:33C. This is a chimeric gene in which the C-terminus of HPV6L1 is replaced with the C-terminus of HPV33L1.

Example 1.2 Construction of a chimeric gene with HPV11L1 C-terminus replaced by HPV33L1 C-terminus

The experimental methods and procedures are the same as in Example 1.1, and the relevant sequences are shown in Appendix 2.

Example 1.3 Construction of a chimeric gene with HPV16L1 C-terminus replaced by HPV33L1 C-terminus

The experimental methods and procedures are the same as in Example 1.1, and the relevant sequences are shown in Appendix 3.

Example 1.4 Construction of a chimeric gene with HPV18L1 C-terminus replaced by HPV33L1 C-terminus

The experimental methods and procedures are the same as in Example 1.1, and the relevant sequences are shown in Appendix 4.

Example 1.5 Construction of a chimeric gene with HPV31L1 C-terminus replaced by HPV33L1 C-terminus

The experimental methods and procedures are the same as in Example 1.1, and the relevant sequences are shown in Appendix 5.

Example 1.6 Construction of a chimeric gene with HPV35L1 C-terminus replaced by HPV33L1 C-terminus

The experimental methods and procedures are the same as in Example 1.1, and the relevant sequences are shown in Appendix 6.

Example 1.7 Construction of a chimeric gene with HPV39L1 C-terminus replaced by HPV59L1 C-terminus

1.7.1 Construction of pFB-HPV39L1 as a template

The HPV39L1 gene was synthesized by Thermo Fisher Scientific (formerly Ingenic Semiconductor (Shanghai) Trading Co., Ltd.), and the synthesized sequence had KpnI and XbaI restriction sites at both ends, respectively. The sequence is shown in SEQ ID NO: 83. The synthesized gene fragment was ligated to the pcDNA3 vector (marketed by Thermo Fisher) through the KpnI and XbaI restriction sites to obtain the plasmid pcDNA3-HPV39-L1 containing the nucleotide sequence encoding 1-505 amino acids of HPV39L1.

The obtained pcDNA3-HPV39-L1 plasmid was double-digested with KpnI and XbaI to obtain a fragment of the HPV39L1 (1-505) gene. This fragment was then ligated with the pFastBac ^™ 1 vector (marketed by Thermo Fisher) double-digested with KpnI and XbaI to obtain a rod-like vector containing the HPV39L1 (1-505) gene fragment, named pFB-HPV39L1.

1.7.2 Construction of pFB-HPV59L1 as a template

The HPV59L1 gene was synthesized by Thermo Fisher Scientific (formerly Ingenic Semiconductor (Shanghai) Trading Co., Ltd.), and the synthesized sequence had KpnI and XbaI restriction sites at both ends, respectively. The gene fragment sequence is shown in SEQ ID NO: 84. The synthesized gene fragment was ligated to the pcDNA3 vector (marketed by Thermo Fisher) through the KpnI and XbaI restriction sites to obtain the plasmid pcDNA3-HPV59-L1 containing the nucleotide sequence encoding amino acids 1-508 of HPV59L1.

The obtained pcDNA3-HPV59-L1 plasmid was double-digested with KpnI and XbaI to obtain a fragment of the HPV59L1 (1-508) gene. This fragment was then ligated into the pFastBacTM1 vector (marketed by Thermo Fisher) double-digested with KpnI and XbaI to obtain a rod-like vector containing the HPV59 L1 (1-508) gene fragment, named pFB-HPV59L1.

1.7.3 Construction of pFB-HPV39L1:59C

The HPV39L1 C-terminus was replaced with the HPV59L1 C-terminus chimeric gene: Using the successfully constructed recombinant plasmid pFB-HPV39L1 as a gene template, a 1428bp gene fragment was amplified using primers F1 and R1. The primer sequence F1 is shown in SEQ ID No: 85, and R1 is shown in SEQ ID No: 86.

The gene fragment contains a gene segment encoding amino acids 1-469 of HPV39L1, a 12-base overlap with a gene segment encoding amino acids 471-508 of HPV59L1, and a KpnI restriction site (GGTAC^C). The amplified sequence is shown in SEQ ID No: 87.

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

Using recombinant plasmid pFB-HPV59L1 as a gene template, a 139bp gene fragment was amplified using primers F2 and R2. Primer sequence F2 is shown in SEQ ID No: 88, and primer R2 is shown in SEQ ID No: 89.

The gene fragment contains a 38-amino acid (471-508) C-terminal segment of HPV59L1, a 12 bp base overlap with the 1-469 amino acid C-terminal segment of HPV39L1, and an XbaI (T^CTAGA) restriction site. The amplified sequence is shown in SEQ ID No: 90.

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

PCR assembly sequence:

The splicing primers were F1 and R2, and the fragments amplified by the above primers (fragments amplified by F1 and R1, and fragments amplified by F2 and R2) were used as templates.

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

The final result is SEQ ID NO: 82, which encodes a nucleotide sequence consisting of amino acids 1-469 of HPV39L1 and 38 (471-508) amino acids at the C-terminus of HPV59L1, with KpnI and XbaI restriction sites at both ends (hereinafter referred to as spliced sequence).

The pFastBac ^™ 1 vector and the spliced sequence fragment were digested with KpnI and XbaI, and the spliced sequence was cloned into the pFastBac ^™ 1 vector to obtain the recombinant plasmid pFB-HPV39L1:59C. This is a chimeric gene in which the C-terminus of HPV39L1 is replaced with the C-terminus of HPV59L1.

Example 1.8 Construction of a chimeric gene with HPV45L1 C-terminus replaced by HPV33L1 C-terminus

The experimental methods and procedures are the same as in Example 1.1, and the relevant sequences are shown in Appendix 8.

Example 1.9 Construction of a chimeric gene with HPV51L1 C-terminus replaced by HPV33L1 C-terminus

The experimental methods and procedures are the same as in Example 1.1, and the relevant sequences are shown in Appendix 9.

Example 1.10 Construction of a chimeric gene with HPV52L1 C-terminus replaced by HPV33L1 C-terminus

The experimental methods and procedures are the same as in Example 1.1, and the relevant sequences are shown in Appendix 10.

Example 1.11 Construction of a chimeric gene with HPV56L1 C-terminus replaced by HPV33L1 C-terminus

The experimental methods and procedures are the same as in Example 1.1, and the relevant sequences are shown in Appendix 11.

Example 1.12 Construction of a chimeric gene with HPV58L1 C-terminus replaced by HPV33L1 C-terminus

The experimental methods and procedures are the same as in Example 1.1, and the relevant sequences are shown in Appendix 12.

Example 2 Packaging of Recombinant Baculovirus

Example 2.1: Packaging of HPV 6L1:33C recombinant baculovirus

After the recombinant plasmid pFB-HPV 6L1:33C constructed in Example 1 was identified and sequenced correctly, it was transformed into DH10Bac bacterial competent cells (kit purchased from Thermo Fisher), cultured and amplified at 37°C, and then streak-cultured on plates. White colonies were selected and amplified. After overnight culture, the bacterial culture was collected, and recombinant rod-like DNA was extracted using the alkaline lysis method.

The recombinant baculovirus seed was packaged by transfecting SF9 insect cells with a cationic transfection reagent (purchased from Sino Biological). The specific procedures are as follows:

SF9 cells in logarithmic growth phase were seeded into dishes at a density of 0.6 × ^10⁶ cells/dish. The dishes seeded with SF9 cells were placed at room temperature for 2 hours to allow them to adhere.

b. Add 20 μL of extracted plasmid DNA to 200 μL of Grace’s Medium (serum-free, additive-free, purchased from Gibico), mix and invert 5 times.

c. Add 25 μL of 0.2x TF1 (transfection reagent, purchased from Sino Biological) dropwise to 200 μL of Grace’s Meduim and mix gently.

d. Mix b and c. Incubate at room temperature for 15-45 minutes.

When the DNA was incubated with cellfectin (purchased from Sino Biological), the cell supernatant was discarded and 0.8 mL/dish of serum-free Grace Medium was added.

f. Add the DNA and transfection reagent complex incubated in step d to the dish.

Incubate at 27℃ for 2 hours.

h. Discard the cell culture medium and add 2.5 mL/dish of complete growth medium (SCD6 SF + 10% FBS) (SCD6 SF was purchased from Sino Biological, and FBS was purchased from Gibico).

i. Incubate at 27℃ for 7 days and observe for viral infection.

After transfection, collect the viral supernatant once the cells show obvious cytopathic effects, generally after 7-11 days of culture. Aseptically collect the viral supernatant using a pipette; this is the HPV6L1:33C P1 generation virus. Infect SF9 cells with the HPV6L1:33C P1 generation virus at a ratio of 1:50 (V/V), with an infection density of 2 × ^10⁶ cells/mL. Amplify the cells by culturing at 27°C for 3 days, followed by centrifugation at 1000g ± 200g for 10 min at room temperature. The collected viral supernatant is the P2 generation virus, which can be used for infection production.

Example 2.2: HPV 11L1:33C recombinant baculovirus packaging

The experimental methods and procedures are the same as in Example 2.1.

Example 2.3: Packaging of HPV 16L1:33C recombinant baculovirus

The experimental methods and procedures are the same as in Example 2.1.

Example 2.4: Packaging of HPV 18L1:33C recombinant baculovirus

The experimental methods and procedures are the same as in Example 2.1.

Example 2.5: Packaging of HPV 31L1:33C recombinant baculovirus

The experimental methods and procedures are the same as in Example 2.1.

Example 2.6: Packaging of HPV 35L1:33C recombinant baculovirus

The experimental methods and procedures are the same as in Example 2.1.

Example 2.7: Packaging of HPV 39L1:59C recombinant baculovirus

The experimental methods and procedures are the same as in Example 2.1.

Example 2.8: Packaging of HPV 45L1:33C recombinant baculovirus

The experimental methods and procedures are the same as in Example 2.1.

Example 2.9: HPV 51L1:33C recombinant baculovirus packaging

The experimental methods and procedures are the same as in Example 2.1.

Example 2.10: HPV 52L1:33C recombinant baculovirus packaging

The experimental methods and procedures are the same as in Example 2.1.

Example 2.11: HPV 56L1:33C recombinant baculovirus packaging

The experimental methods and procedures are the same as in Example 2.1.

Example 2.12: HPV 58L1:33C recombinant baculovirus packaging

The experimental methods and procedures are the same as in Example 2.1.

Example 3 Expression of chimeric proteins

Example 3.1: HPV 6L1:33C Expression and Production

High Five cells were infected with baculovirus containing the HPV 6L1:33C recombinant gene obtained in Example 2 at an infection ratio of 1:200 (V/V). Cell pellets were collected by centrifugation at 1000g ± 100g at room temperature. The cell pellets were lysed using PBS or MOPS buffer (pH 6.0-7.0, salt concentration 100mM-1M) by sonication, followed by low-temperature sonication for 3 min and centrifugation at a force greater than 10000g for 10 min. The supernatant was collected and analyzed by SDS-PAGE electrophoresis. Lane 1: Marker (Markers consist of 7 purified proteins with molecular weights ranging from 14.4 to 116 kDa, manufactured by Thermo Scientific); Lane 2: Cell lysis buffer; Lane 3: Supernatant collected after centrifugation of the lysis buffer.

As shown in Figure 1A, the HPV 6L1:33C L1 protein prepared by this method has a yield of more than 100 mg/L and a protein size of about 56 KD, which can be used for large-scale production.

Example 3.2: HPV 11L1:33C Expression and Production

The experimental methods and procedures are the same as in Example 3.1.

The results are shown in Figure 1B. The HPV 11L1:33C L1 protein prepared by this method has a yield of more than 100 mg/L and a protein size of about 56 KD, which can be used for large-scale production.

Example 3.3: HPV 16L1:33C Expression and Production

The experimental methods and procedures are the same as in Example 3.1.

As shown in Figure 1C, the HPV 16L1:33C L1 protein prepared by this method has a yield of more than 100 mg/L and a protein size of about 56 KD, which can be used for large-scale production.

Example 3.4: HPV 18L1:33C Expression and Production

The experimental methods and procedures are the same as in Example 3.1.

As shown in Figure 1D, the HPV 18L1:33C L1 protein prepared by this method has a yield of more than 100 mg/L and a protein size of about 56 KD, which can be used for large-scale production.

Example 3.5: HPV 31L1:33C Expression and Production

The experimental methods and procedures are the same as in Example 3.1.

As shown in Figure 1E, the HPV 31L1:33C L1 protein prepared by this method has a yield of more than 100 mg/L and a protein size of about 56 KD, which can be used for large-scale production.

Example 3.6: HPV 35L1:33C Expression and Production

The experimental methods and procedures are the same as in Example 3.1.

The results are shown in Figure 1F. The HPV 35L1:33C L1 protein prepared by this method has a yield of more than 100 mg/L and a protein size of about 56 KD, which can be used for large-scale production.

Example 3.7: HPV 39L1:59C Expression and Production

The experimental methods and procedures are the same as in Example 3.1.

As shown in Figure 1G, the HPV 39L1:59C L1 protein prepared by this method has a yield of more than 100 mg/L and a protein size of about 56 KD, which can be used for large-scale production.

Example 3.8: HPV 45L1:33C Expression Production

The experimental methods and procedures are the same as in Example 3.1.

The results are shown in Figure 1H. The HPV 45L1:33C L1 protein prepared by this method has a yield of more than 100 mg/L and a protein size of about 56 KD, which can be used for large-scale production.

Example 3.9: HPV 51L1:33C Expression Production

The experimental methods and procedures are the same as in Example 3.1.

The results are shown in Figure 1I. The HPV 51L1:33C L1 protein prepared by this method has a yield of more than 100 mg/L and a protein size of about 56 KD, which can be used for large-scale production.

Example 3.10: HPV 52L1:33C Expression and Production

The experimental methods and procedures are the same as in Example 3.1.

The results are shown in Figure 1J. The HPV 52L1:33C L1 protein prepared by this method has a yield of more than 100 mg/L and a protein size of about 56 KD, which can be used for large-scale production.

Example 3.11: HPV 56L1:33C Expression Production

The experimental methods and procedures are the same as in Example 3.1.

As shown in Figure 1K, the HPV 56L1:33C L1 protein prepared by this method has a yield of more than 100 mg/L and a protein size of about 56 KD, which can be used for large-scale production.

Example 3.12: HPV 58L1:33C Expression Production

The experimental methods and procedures are the same as in Example 3.1.

The results are shown in Figure 1L. The HPV 58L1:33C L1 protein prepared by this method has a yield of more than 100 mg/L and a protein size of about 56 KD, which can be used for large-scale production.

Example 4: Purification and Preparation of Virus-like Particles

Example 4.1: Purification and preparation of HPV 6L1:33C virus-like particles

The purification method for HPV 6L1:33C virus-like particles is a two-step chromatography method, namely the HS-MMA method. The supernatant collected in Example 3 is purified to finally obtain high-purity virus-like particles.

Step 1 Chromatography:

Medium: 50 HS strong cation exchange medium manufactured by Thermo Fisher Scientific.

Medium volume: 150 mL, linear flow rate: 30 mL/min.

Chromatographic conditions: Equilibration buffer (pH 6.2, salt concentration: 50 mM phosphate, 0.5 M sodium chloride); Washing buffer (salt concentration: 50 mM phosphate, 0.75 M sodium chloride, pH 6.2);

The chromatography column was first equilibrated with 5 CV buffer before sample loading. After sample loading, contaminating proteins were eluted with 5 CV equilibration buffer and washing buffer, respectively.

Elution conditions: pH 6.2, elution salt concentration of 1.25M sodium chloride, eluted with 50mM phosphate buffer containing 50mM arginine hydrochloride.

Second step: Chromatography

Medium: MMA ion exchange medium produced by Shanghai Bogelon Company.

Medium volume: 150 mL, linear flow rate: 30 mL/min.

Chromatographic conditions: 50 mM PB equilibration buffer, 1.25 M NaCl, pH 6.2. The column was first equilibrated with 4 CV equilibration buffer, then the sample was loaded. After loading, the column was washed with 5 CV equilibration buffer to remove contaminating proteins, and then the target protein was eluted with elution buffer to collect the protein.

Elution conditions: 100 mM NaAC, 150 mM NaCl, 0.01% Tween 80, pH 4.5.

Example 4.2: Purification and preparation of HPV 11L1:33C virus-like particles

The experimental methods and procedures are the same as in Example 4.1.

Example 4.3: Purification and preparation of HPV 16L1:33C virus-like particles

The experimental methods and procedures are the same as in Example 4.1.

Example 4.4: Purification and preparation of HPV 18L1:33C virus-like particles

The experimental methods and procedures are the same as in Example 4.1.

Example 4.5: Purification and preparation of HPV 31L1:33C virus-like particles

The experimental methods and procedures are the same as in Example 4.1.

Example 4.6: Purification and preparation of HPV 35L1:33C virus-like particles

The experimental methods and procedures are the same as in Example 4.1.

Example 4.7: Purification and preparation of HPV 39L1:59C virus-like particles

The experimental methods and procedures are the same as in Example 4.1.

Example 4.8: Purification and preparation of HPV 45L1:33C virus-like particles

The experimental methods and procedures are the same as in Example 4.1.

Example 4.9: Purification and preparation of HPV 51L1:33C virus-like particles

The experimental methods and procedures are the same as in Example 4.1.

Example 4.10: Purification and preparation of HPV 52L1:33C virus-like particles

The experimental methods and procedures are the same as in Example 4.1.

Example 4.11: Purification and preparation of HPV 56L1:33C virus-like particles

The experimental methods and procedures are the same as in Example 4.1.

Example 4.12: Purification and preparation of HPV 58L1:33C virus-like particles

The experimental methods and procedures are the same as in Example 4.1.

Example 5: Morphological detection of virus-like particles

Example 5.1: Morphological detection of HPV 6L1:33C virus-like particles

Take 10 μL of sample for transmission electron microscopy (TEM) observation. Fix the sample onto a carbon-sprayed copper grid for 2 min, remove any residual liquid with filter paper, and then stain twice with phosphotungstic acid (Beijing Zhongjing Scientific Instruments Technology Co., Ltd., concentration 2%, pH 6.5), 30 seconds each time. Remove any residual staining solution with filter paper, allow to dry, and then observe under a TEM. The TEM (brand: Hitachi, model: H-7650) is 80 kV with a magnification of 80,000x.

The electron microscopy results are shown in Figure 2A. As can be seen from Figure 2A, the C-terminal modified HPV 6L1:33C can form virus-like particles of uniform size with an average diameter of about 60 nm.

Example 5.2: Morphological detection of HPV 11L1:33C virus-like particles

The experimental methods and procedures are the same as in Example 5.1.

The electron microscopy results are shown in Figure 2B. As can be seen from Figure 2B, the C-terminal modified HPV 11L1:33C can form virus-like particles of uniform size with an average diameter of about 60 nm.

Example 5.3: Morphological detection of HPV 16L1:33C virus-like particles

The experimental methods and procedures are the same as in Example 5.1.

The electron microscopy results are shown in Figure 2C. As can be seen from Figure 2C, the C-terminal modified HPV 16L1:33C can form virus-like particles of uniform size with an average diameter of about 60 nm.

Example 5.4: Morphological detection of HPV 18L1:33C virus-like particles

The experimental methods and procedures are the same as in Example 5.1.

The electron microscopy results are shown in Figure 2D. As can be seen from Figure 2D, the C-terminal modified HPV 18L1:33C can form virus-like particles of uniform size with an average diameter of about 60 nm.

Example 5.5: Morphological detection of HPV 31L1:33C virus-like particles

The experimental methods and procedures are the same as in Example 5.1.

The electron microscopy results are shown in Figure 2E. As can be seen from Figure 2E, the C-terminal modified HPV 31L1:33C can form virus-like particles of uniform size with an average diameter of about 60 nm.

Example 5.6: Morphological detection of HPV 35L1:33C virus-like particles

The experimental methods and procedures are the same as in Example 5.1.

The electron microscopy results are shown in Figure 2F. As can be seen from Figure 2F, the C-terminal modified HPV 35L1:33C can form virus-like particles of uniform size with an average diameter of about 60 nm.

Example 5.7: Morphological detection of HPV 39L1:59C virus-like particles

The experimental methods and procedures are the same as in Example 5.1.

The electron microscopy results are shown in Figure 2G. As can be seen from Figure 2G, the C-terminal modified HPV 39L1:59C can form virus-like particles of uniform size with an average diameter of about 60 nm.

Example 5.8: Morphological detection of HPV 45L1:33C virus-like particles

The experimental methods and procedures are the same as in Example 5.1.

The electron microscopy results are shown in Figure 2H. As can be seen from Figure 2H, the C-terminal modified HPV 45L1:33C can form virus-like particles of uniform size with an average diameter of about 60 nm.

Example 5.9: Morphological detection of HPV 51L1:33C virus-like particles

The experimental methods and procedures are the same as in Example 5.1.

The electron microscopy results are shown in Figure 2I. As can be seen from Figure 2I, the C-terminal modified HPV 51L1:33C can form virus-like particles of uniform size with an average diameter of about 60 nm.

Example 5.10: Morphological detection of HPV 52L1:33C virus-like particles

The experimental methods and procedures are the same as in Example 5.1.

The electron microscopy results are shown in Figure 2J. As can be seen from Figure 2J, the C-terminal modified HPV 52L1:33C can form virus-like particles of uniform size with an average diameter of about 60 nm.

Example 5.11: Morphological detection of HPV 56L1:33C virus-like particles

The experimental methods and procedures are the same as in Example 5.1.

The electron microscopy results are shown in Figure 2K. As can be seen from Figure 2K, the C-terminal modified HPV 56L1:33C can form virus-like particles of uniform size with an average diameter of about 60 nm.

Example 5.12: Morphological detection of HPV 58L1:33C virus-like particles

The experimental methods and procedures are the same as in Example 5.1.

The electron microscopy results are shown in Figure 2L. As can be seen from Figure 2L, the C-terminal modified HPV 58 L1:33C can form virus-like particles of uniform size with an average diameter of about 60 nm.

Example 6: Immunogenicity evaluation of virus-like particles in animals

Example 6.1: Immunogenicity evaluation of HPV 6L1:33C virus-like particles in animals

6.1.1 Establishment of a model for neutralizing cells using pseudoviruses

Because HPV is difficult to culture in vitro and has strong host specificity, it is difficult for it to reproduce in organisms other than humans, and suitable animal models are lacking. Therefore, it is necessary to establish suitable and effective in vitro neutralization experimental models for evaluating the immunoprotective effect of vaccines.

HPV pseudoviruses are an ideal experimental model for HPV neutralization in vitro: taking advantage of the non-specific nucleic acid encapsulation property of HPV VLPs, HPV L1 and L2 expressed in cells encapsulate free DNA or introduce foreign plasmids to form HPV pseudoviruses.

Immunogenicity analysis was performed on serum samples from immunized animals using a pseudovirus neutralization method. Immunization of animals with HPV6 virus-like particles produced neutralizing antibodies against HPV6, which could neutralize HPV6 pseudoviruses. After incubating the immunized animal serum with a certain amount of pseudovirus and then infecting cells, the number of cells expressing GFP fluorescence decreased with increasing levels of neutralizing antibodies in the serum, exhibiting a linear negative correlation within a certain range. Therefore, the neutralizing activity of antibodies in the serum could be evaluated by detecting changes in the number of cells expressing GFP.

Pseudovirus construction method: HPV6 type pCMV3-3-HPV6L1+L2 plasmid (L1 sequence from Uniprot P69898, L2 sequence from Uniprot Q84297) (purchased from Sino Biological) and fluorescent plasmid (PSEU-GFP Spark, purchased from Sino Biological) were co-transfected into 293FT adherent cells (purchased from Thermo Fisher). The specific methods are referenced in the literature (Pastrana D V, Buck C B, Pang Y S, Thompson C D, Castle PE, FitzGerald P C, Kjaer SK, Lowy D R, Schiller J T. Reactivity of human sera in a sensitive, high-throughput pseudovirus-based papillomavirus neutralization assay for HPV16 and HPV18. [J] Virology 2004, 321: 205-216.). The pseudovirus supernatant was collected, aliquoted, and stored at -80°C for later use.

6.1.2 Evaluation of the immunoprotective effect of HPV 6L1:33C virus-like particles in animals

Mouse immunization program:

HPV 6L1:33C virus-like particles were adsorbed onto aluminum phosphate adjuvant. After mixing, 200 μL was used to immunize mice. Each mouse received an immunization dose of 0.15 μg, and 10 mice were immunized. The diluted sample was used to immunize mice on days 0, 7, and 21 of the experiment. A blank serum control group was set up at the same time. On day 28 of the experiment, blood was collected from the eyeballs of mice, and the serum was separated for pseudovirus neutralization titer detection.

Mouse EC50 detection:

Mouse serum was inactivated at 56℃ for 30 minutes, centrifuged at 6000g, and the supernatant was collected after 5 minutes for detection. 4-8 hours before detection, 293FT cells were seeded at a density of 15000 cells/well in 96-well plates and cultured at 37℃ in a ₅ % CO2 incubator. Immunized mouse serum and blank control serum were serially diluted with neutralizing medium and mixed with the HPV6 pseudovirus prepared in section 6.1 at a 1:1 volume ratio. After incubation at 2-8℃ for 1 hour, 100 μL/well was added to the 293FT cells seeded 4-8 hours prior, with two replicates per sample. Blank serum control wells, pseudovirus positive control wells, and negative control wells were also included. Cells were further cultured at 37℃ in a ₅ % CO2 incubator for 62-96 hours, followed by fluorescence scanning and cell counting using an ELISA speckle analyzer (model: S6 Universal-V Analyzer, manufacturer: CTL). By calculating the neutralization inhibition rate of each mouse serum sample, the maximum serum dilution factor, i.e., the half effective dilution factor _EC50 , was calculated according to the Reed-Muench method when the serum neutralization inhibition rate was 50%.

The results of HPV6 serum pseudovirus neutralization titer detection are detailed in Table 4.

Table 4. Results of mouse serum neutralization titers (EC ₅₀ (GMT±SEM))

Remark:

1. Number of animals, N = 10;

2. GMT (Geometric Mean Titer): Geometric mean titer;

3. SEM (Standard Error of Mean): Standard error.

The above test results show that the HPV 6L1:33C virus-like particles prepared by this invention have good immunogenicity and can produce high titers of neutralizing antibodies in animals, and can be used to prepare a vaccine to prevent HPV infection.

Example 6.2: Immunogenicity evaluation of HPV 11L1:33C virus-like particles in animals

The experimental methods and procedures were the same as in Example 6.1. The L1 sequence was derived from Uniprot P04012, and the L2 sequence was derived from Uniprot P04013.

The results of HPV11 serum pseudovirus neutralization titer detection are detailed in Table 5.

Table 5. Results of mouse serum neutralization titers (EC ₅₀ (GMT±SEM))

The above test results show that the HPV 11L1:33C virus-like particles prepared by this invention have good immunogenicity and can produce high titers of neutralizing antibodies in animals, and can be used to prepare a vaccine to prevent HPV infection.

Example 6.3: Immunogenicity evaluation of HPV 16L1:33C virus-like particles in animals

The experimental methods and procedures were the same as in Example 6.1. The L1 sequence was derived from Uniprot P03101, and the L2 sequence was derived from Uniprot P03107.

The results of HPV16 serum pseudovirus neutralization titer detection are detailed in Table 6.

Table 6. Results of mouse serum neutralization titers (EC ₅₀ (GMT±SEM))

The above test results show that the HPV 16L1:33C virus-like particles prepared by this invention have good immunogenicity and can produce high titers of neutralizing antibodies in animals, and can be used to prepare a vaccine to prevent HPV infection.

Example 6.4: Immunogenicity evaluation of HPV 18L1:33C virus-like particles in animals

The experimental methods and procedures were the same as in Example 6.1. The L1 sequence was derived from Uniprot Q80B70, and the L2 sequence was derived from Uniprot P06793.

The results of HPV18 serum pseudovirus neutralization titer detection are detailed in Table 7.

Table 7. Results of mouse serum neutralization titers (EC ₅₀ (GMT±SEM))

The above test results show that the HPV 18L1:33C virus-like particles prepared by this invention have good immunogenicity and can produce high titers of neutralizing antibodies in animals, and can be used to prepare a vaccine to prevent HPV infection.

Example 6.5: Immunogenicity evaluation of HPV 31L1:33C virus-like particles in animals

The experimental methods and procedures were the same as in Example 6.1. The L1 sequence was derived from Uniprot P17388, and the L2 sequence was derived from Uniprot P17389.

The results of HPV31 serum pseudovirus neutralization titer detection are detailed in Table 8.

Table 8. Results of mouse serum neutralization titers (EC ₅₀ , GMT±SEM)

The above test results show that the HPV 31L1:33C virus-like particles prepared by this invention have good immunogenicity and can produce high titers of neutralizing antibodies in animals, and can be used to prepare a vaccine to prevent HPV infection.

Example 6.6: Immunogenicity evaluation of HPV 35L1:33C virus-like particles in animals

The experimental methods and procedures were the same as in Example 6.1. The L1 sequence was derived from Uniprot P27232, and the L2 sequence was derived from Uniprot P27234.

The results of HPV35 serum pseudovirus neutralization titer detection are detailed in Table 10.

Table 9. Results of mouse serum neutralization titers (EC ₅₀ , GMT±SEM)

The above test results show that the HPV 35L1:33C virus-like particles prepared by this invention have good immunogenicity and can produce high titers of neutralizing antibodies in animals, and can be used to prepare a vaccine to prevent HPV infection.

Example 6.7: Immunogenicity evaluation of HPV 39L1:59C virus-like particles in animals

The experimental methods and procedures were the same as in Example 6.1. The L1 sequence was derived from Uniprot P24838, and the L2 sequence was derived from Uniprot P24839.

The results of HPV39 serum pseudovirus neutralization titer detection are detailed in Table 11.

Table 10. Results of mouse serum neutralization titers (EC ₅₀ , GMT±SEM)

The above test results show that the HPV 39L1:59C virus-like particles prepared by this invention have good immunogenicity and can produce high titers of neutralizing antibodies in animals, and can be used to prepare a vaccine to prevent HPV infection.

Example 6.8: Immunogenicity evaluation of HPV 45L1:33C virus-like particles in animals

The experimental methods and procedures were the same as in Example 6.1. The L1 sequence was derived from Uniprot P36741, and the L2 sequence was derived from Uniprot P36761.

The results of HPV45 serum pseudovirus neutralization titer detection are detailed in Table 12.

Table 11 Results of mouse serum neutralization titers (EC ₅₀ (GMT±SEM))

The above test results show that the HPV 45L1:33C virus-like particles prepared by this invention have good immunogenicity and can produce high titers of neutralizing antibodies in animals, and can be used to prepare a vaccine to prevent HPV infection.

Example 6.9: Immunogenicity evaluation of HPV 51L1:33C virus-like particles in animals

The experimental methods and procedures were the same as in Example 6.1. The L1 sequence was derived from Uniprot P26536, and the L2 sequence was derived from Uniprot P26539.

The results of HPV51 serum pseudovirus neutralization titer detection are detailed in Table 13.

Table 12 Results of mouse serum neutralization titers (EC ₅₀ (GMT±SEM))

The above test results show that the HPV 51L1:33C virus-like particles prepared by this invention have good immunogenicity and can produce high titers of neutralizing antibodies in animals, and can be used to prepare a vaccine to prevent HPV infection.

Example 6.10: Immunogenicity evaluation of HPV 52L1:33C virus-like particles in animals

The experimental methods and procedures were the same as in Example 6.1. The L1 sequence was derived from Uniprot Q05138, and the L2 sequence was derived from Uniprot F8S4U2.

The results of HPV52 serum pseudovirus neutralization titer detection are detailed in Table 14.

Table 13 Results of mouse serum neutralization titer detection (EC ₅₀ (GMT±SEM))

The above test results show that the HPV 52L1:33C virus-like particles prepared by this invention have good immunogenicity and can produce high titers of neutralizing antibodies in animals, and can be used to prepare a vaccine to prevent HPV infection.

Example 6.11: Immunogenicity evaluation of HPV 56L1:33C virus-like particles in animals

The experimental methods and procedures were the same as in Example 6.1. The L1 sequence was derived from Uniprot P36743, and the L2 sequence was derived from Uniprot P36765.

The results of HPV56 serum pseudovirus neutralization titer detection are detailed in Table 15.

Table 14 Results of mouse serum neutralization titer detection (EC ₅₀ (GMT±SEM))

The above test results show that the HPV 56L1:33C virus-like particles prepared by this invention have good immunogenicity and can produce high titers of neutralizing antibodies in animals, and can be used to prepare a vaccine to prevent HPV infection.

Example 6.12: Immunogenicity evaluation of HPV 58L1:33C virus-like particles in animals

The experimental methods and procedures were the same as in Example 6.1. The L1 sequence was derived from Uniprot P26535, and the L2 sequence was derived from Uniprot B6ZB12.

The results of HPV58 serum pseudovirus neutralization titer detection are detailed in Table 16.

Table 15 Results of mouse serum neutralization titer detection (EC ₅₀ (GMT±SEM))

The above test results show that the HPV 58L1:33C virus-like particles prepared by this invention have good immunogenicity and can produce high titers of neutralizing antibodies in animals, and can be used to prepare a vaccine to prevent HPV infection.

Comparative Example 1: Expression of C-terminal truncated HPV16L1 (1-474)

The inventors attempted to truncate the C-terminus of HPV16L1 by 31 amino acids, naming it HPV16L1(1-474) (SEQ ID NO: 27). However, the study found that the truncated HPV16L1(1-474) protein had high expression levels but poor protein solubility, making it difficult to extract and purify. The specific expression and extraction results are shown in Figure 3.

Although the invention has been described in detail by way of illustration and examples, it is intended for ease of understanding. It will be apparent to those skilled in the art that various modifications and improvements can be made to the technical solutions of the invention without departing from the spirit or scope of the appended claims.

Appendix 1: Sequence Listing - Chimeric Human Papillomavirus Type 6 L1 Protein

Appendix 2: Sequence Listing - Chimeric Human Papillomavirus Type 11 L1 Protein

Appendix 3: Sequence Listing - Chimeric Human Papillomavirus Type 16 L1 Protein

Appendix 4: Sequence Listing - Chimeric Human Papillomavirus Type 18 L1 Protein

Appendix 5: Sequence Listing - Chimeric Human Papillomavirus Type 31 L1 Protein

Appendix 6: Sequence Listing - Chimeric Human Papillomavirus Type 35 L1 Protein

Appendix 7: Sequence Listing - Chimeric Human Papillomavirus Type 39 L1 Protein

Appendix 8: Sequence Listing - Chimeric Human Papillomavirus Type 45 L1 Protein

Appendix 9: Sequence Listing - Chimeric Human Papillomavirus Type 51 L1 Protein

Appendix 10: Sequence Listing - Chimeric Human Papillomavirus Type 52 L1 Protein

Appendix 11: Sequence Listing - Chimeric Human Papillomavirus Type 56 L1 Protein

Appendix 12: Sequence Listing - Chimeric Human Papillomavirus Type 58 L1 Protein

Claims (1)

1. An immunogenic composition for preventing human papillomavirus infection, comprising human papillomavirus-like particles and aluminum phosphate adjuvant, The human papillomavirus-like particles described herein comprise any one of the following chimeric human papillomavirus L1 proteins: HPV 6L1:33C protein, whose amino acid sequence is SEQ ID NO:3; HPV 11L1:33C protein, whose amino acid sequence is SEQ ID NO:16; HPV 16L1:33C protein, whose amino acid sequence is SEQ ID NO:29; HPV 18L1:33C protein, the amino acid sequence of which is SEQ ID NO:42; HPV 31L1:33C protein, the amino acid sequence of which is SEQ ID NO:55; HPV 35L1:33C protein, whose amino acid sequence is SEQ ID NO:68; HPV 39L1:59C protein, whose amino acid sequence is SEQ ID NO:81; HPV 45L1:33C protein, the amino acid sequence of which is SEQ ID NO:94; HPV 51L1:33C protein, whose amino acid sequence is SEQ ID NO:107; HPV 52L1:33C protein, whose amino acid sequence is SEQ ID NO:120; HPV 56L1:33C protein, whose amino acid sequence is SEQ ID NO:133; HPV 58L1:33C protein, whose amino acid sequence is SEQ ID NO:146.