HK1166347A - Recombinant virus-like particles encoded by multi-gene vector - Google Patents

Description

Recombinant virus-like particles encoded by multigene vectors

Technical Field

The present invention relates to recombinant virus-like particles comprising virus from different virus strains, and vectors encoding them.

Background

The use of small natural biomolecules in different aspects of biomedicine, nanotechnology and material science is of increasing interest. Virus mimetics, virus capsids or virus-like particles are very attractive due to their regular structure, their uniform particle size, their stability, ease of production and the possibility of manipulation. Virus-like particles have a dynamic structure, their interior is accessible, and the coating is additionally modifiable. Depending on the application, virus-like particles may or may not have an envelope, and may be selected as virus mimetics. These embodiments can be used as new biological entities or targets, as vaccines, as antigens for antibody production, as research tools, as diagnostic tools, for drug delivery and bioconjugates. These virus mimetics are formed by the self-assembly of the envelope or capsid proteins of a variety of viruses. The size varies between 22-150 nm depending on the particular virus morphology. Virus mimetics are non-infectious in that they do not incorporate genetic material for assembly. Depending on the application, exogenous genetic material may be included in the virus mimetics described herein.

A promising application of these virus mimetics is the production of vaccines against various diseases, as repeated, high-density display of their epitopes often elicits strong immune responses. For uptake by dendritic cells, a small particle size is an advantage. Chimeric virus mimetics offer great possibilities in terms of selective, multi-epitope, multi-protein, multi-serotype, multi-strain or multi-species presentation.

There are many expression systems for the production of viral mimetics, including baculovirus/insect cell systems, various mammalian cell lines, all stably or transiently transfected or transduced with viral expression vectors, and including Saccharomyces cerevisiae (R)Saccharomyces cerevisiae) And Pichia pastoris (Pichia pastoris) Various yeast species of (2), and Escherichia coli (E.coli) (II)Escherichia coli) And other bacteria.

Vaccination depends on the development of sufficient immunity to protect against infectious diseases. Most used attenuated virus vaccines rely on the limited replication of the virus in the host following immunization. However, such vaccination may cause severe reactions in some patients. The development of virus-like particles (VLPs) as subunit vaccines is thus advantageous, since the particles typically lack DNA or RNA genomes, but have the true conformation of the native virus.

Vaccination is one of the most effective and low cost countermeasures against threats such as seasonal or pandemic influenza outbreaks. The susceptibility to transmission as aerosols and the resulting severe illness for particularly sensitive people are the major causes of influenza, the most devastating viral disease. The current approved seasonal vaccine is only partially protective and egg-based production is very time consuming and costly. This strategy is vulnerable to the unexpected emergence of epidemic strains with poor or no match at all for the vaccine. Due to the risk of ever-emerging strains of avian influenza or influenza of other origin, new vaccine regimens are necessary.

On the other hand, the study in the field of several important viruses such as HCV, HIV, Ebola, etc. is very difficult due to biosafety issues. Until now, only a few models have been used for studies of virus entry and virus delivery. Due to the lack of suitable non-infectious virus models, diagnostic tools are based on the genomes of these viruses.

Current commercial human influenza vaccines contain hemagglutinin as their sole or major viral antigen. Their production starts with viruses grown on embryonated chicken eggs or, more recently, in mammalian cells in tissue culture. Production in eggs requires selection of high-producing, rearranged virus strains, is limited in capacity, is time-consuming (6-8 months) and expensive. In addition to that, it may cause problems in vaccinated people who are allergic to egg proteins. It is only possible to produce and use non-lethal strains of birds. One of the most important drawbacks of egg-based production is the limited capacity. For a pandemic, it is necessary to stop the production of seasonal influenza vaccines to help with the production of pandemic influenza vaccines, which may cause more fatal events in the long term.

Vaccines against viral diseases typically rely on inactivation of attenuated virus strains or infectious viruses. Suitable environments are necessary for highly pathogenic or hemorrhagic viruses, which limits production possibilities due to biosafety levels (e.g., BL3/BL 4). For some viruses, such as human papilloma virus, attenuation will not be sufficient because the virus cannot be propagated in vitro. The ability to generate vaccines based on Human Papilloma Virus (HPV) -like particles (Gardasil, Cervarix) has changed the prospects for preventing cervical cancer in women.

Due to the risk of ever-emerging avian influenza or other source of strains, new vaccine regimens leading to enhanced protection are necessary.

Summary of The Invention

The present invention relates to recombinant virus-like particles comprising two or more different epitopes or different epitope-containing proteins selected from (a) different virus strains of the same virus and/or (b) different serotypes of the same virus and/or (c) different virus strains specific for different hosts. These recombinant virus-like particles are useful as vaccines, and the present invention also relates to such vaccines.

Furthermore, the present invention relates to vectors comprising two or more polynucleotides encoding different epitopes or encoding different epitope-containing proteins selected from (a) different virus strains of the same virus and/or (b) different serotypes of the same virus and/or (c) different virus strains specific for different hosts, as well as to host cells comprising these vectors.

Brief description of the drawings

FIG. 1 of the drawingsSchematic representation of vector constructs expressing polyepitope virus-like particles.

(A) (SEQ ID NO: 1) multivalent influenza A virus mimetics containing different epitopes (M1, M2) from the H1N1 virus strain as well as the H3N2 (HA, NA) virus strain.

(B) (SEQ ID NO: 2) chimeric human papillomavirus-like particles containing epitopes from HPV serotypes 16 and 18 (L1)

(C) (SEQ ID NO: 3) expression vector constructs with embedded epitopes (HA, NA, M1, M2) from influenza B/Florida isolates

(D) (SEQ ID NO: 4) vector constructs for the expression of the epitope of HPV16 and HPV18 serotype (L1), where both genes are under the control of different promoters.

(E) (SEQ ID NO: 5) same vector construct as (B) with deletion of promoter p10

The vector contains two promoters P1 and P2: (,) Selected from polh, p10 and p_XIV Very late baculovirus promoter, vp39 baculovirus late promoter, vp39polh baculovirus late/very late hybrid promoter, pca/polh,pcna，etl，p35，egt，da26a baculovirus early promoter; CMV, SV40, UBc, EF-1, RSVLTR, MT, P_DS47，Ac5，P_GALAnd P_ADH . The terminator sequences T1 and T2 (T) are selected from SV40, HSVtk or BGH (bovine growth hormone). In addition, the vector contains transposon sites TnL and TnR (L, R) for MultiBacbacmid production, loxP sites (LP) for site-specific homologous recombination (plasmid fusion), replication origin (O), ampicillin (A) and gentamicin (G) resistance genes and defined restriction sites.

FIG. 2 of the drawingsAnalysis of expressed chimeric influenza virus-like particles.

(A) Secreted (A, lane 2) and intracellular VLPs (prepared from SEQ ID NO:1, A, lane 1) were verified by immunoblotting using specific antibodies against the proteins HA (H3), NA (H3) and M2 (H1). Lane 3= ladder, protein size in kDa. Epitopes are co-localized, meaning that they are assembled into one particle.

(B) Visualization of chimeric influenza virus-like particles (prepared from SEQ ID NO: 3) by electron microscopy using negative staining with uranium acetate. Spikes presenting epitope HA are visible. The size of the particles is in the range of 50-80 nm.

FIG. 3 of the drawingsFrom SEQ ID NO:1 chromatographic purification and analysis of the secreted multi-epitope influenza virus-like particles prepared.

(A) Gel filtration of the purified chromatogram. The first peak (1) contains virus-like particles (VLPs). The other peaks (2-6) represent contaminating proteins.

(B) Coomassie stained SDS-PAGE. Multiple epitopes of the virus-like particle were verified by analyzing different fractions from the first peak (1), representing the beginning (lane 1), middle (lane 2) and end (lane 3) portions of the peak containing VLPs, respectively. The ladder [ kDa ] is presented on the left side of the first lane. Detection of the epitope is indicated by an arrow.

(C) Western blot analysis using anti-HA antibody according to Coomassie stained gels.

FIG. 4 shows a schematic representation of a drawingFunctionality of native-like influenza virus-like particles (VLPs).

Two-fold serial dilution series (1: 2 to 1: 2048) of purified VLPs (prepared from SEQ ID NO: 3) were analyzed by standard hemagglutination analysis. 50 μ L of the purified particle solution was coated onto 96-well plates incubated with red blood cells (erythrocytes). Influenza VLPs (1, upper) are capable of agglutinating red blood cells in a dose-dependent manner. The highest dilution was 1: 1024. In contrast, pbs (c) used as a control resulted in only the precipitation of red blood cells, appearing as a "dot" in the center of the reaction well.

FIG. 5 shows a schematic representation of the structureIn vivo evaluation of multi-epitope influenza virus-like particles.

Mice were prepared with 50 ng (mouse 1-5) or 100 ng (mouse 6-10) of the peptide from SEQ ID NO:3, and as a control, was immunized with PBS (mouse 11-12). Antibody titers after the initial injection (3 weeks post-injection) are shown in white boxes, titers after the booster injection are shown in black boxes (6 weeks post-injection). Titers were expressed as dilutions of mouse serum (y-axis). VLPs effectively stimulate antibody immune responses. The best results were obtained when immunised with 100. mu.l (mice 6-10), indicating a dose-dependent immune response. A clear increase in anti-VLP antibodies was observed after boosting. As expected, control animals (mice 11-12) showed no immune response.

FIG. 6 shows a schematic representation of a drawingConfirmation of specific immune response to multi-epitope influenza VLPs by hemagglutination inhibition assay.

ELISA assays were performed with sera collected 6 weeks after injection to analyze the presence of specific anti-HA antibodies. From SEQ ID NO:3 the prepared polyepitopic virus-like particles are coated on 96-well plates, mixed with serum, and incubated for 30 minutes. Sera were tested in a series of two-fold dilutions (1: 2 to 1: 1024). After incubation, red blood cells were added and incubated for a further 30 minutes. Specific anti-influenza HA antibodies from different mice bind to the polyepitopic virus-like particles, causing inhibition of hemagglutination to reach dilutions 1:128 (1) and 1:256 (2), emerging as red blood cell pellets ("dots") in the center of the reaction well. No hemagglutination inhibition was observed using serum samples of control mice (C).

FIG. 7 of the drawingsScreening of optimal expression conditions using a 50 mL bioreactor.

The range 1-2X 10 was determined by dot blot analysis⁶Amount of primary cells in the range of one cell/mL (TOI, 1 or 2), virus inoculum (MOI, 0.01-2) and harvest time (days post infection, d2-d 6).

(A) Determination of the optimal expression parameters of the expression construct with only one epitope L1 used as a control. Detection by specific anti-HPV 18 antibody (Abcam).

(B) Determination of optimal expression parameters for a multiple epitope expression construct (SEQ ID NO: 2) carrying two epitopes from different serotypes. Detection by specific anti-HPV 16-antibody (Camvir, Santa Cruz) and anti-HPV 18- (Abcam) antibodies against the two epitopes HPV 16L 1 and HPV 18L 1.

FIG. 8 of the drawingsFrom SEQ ID NO:2 chromatographic purification and analysis of the prepared polyepitope human papilloma virus-like particles.

(A) Chromatography by anion exchange chromatography using a DEAE-Sepharose column. The flow-through (1), wash (2) and elution peaks (3-5) are indicated by numbers (1-5). The increased ionic strength of the elution buffer is shown by line [% ]. 3= eluted with 300 mM NaCl, 4= eluted with 420 mM NaCl, 5= eluted with 680 mM NaCl.

(B) Coomassie stained SDS-PAGE. The presence of multiple epitopes of virus-like particles is verified by analyzing different parts of the chromatogram. Lane 1= ladder [ kDa ], lane 2= VLP before purification, lane 3= wash step, lane 4= eluted with 300 mM NaCl, lane 5= eluted with 420 mM NaCl, lane 6= eluted with 500 mM NaCl, lane 7= eluted with 680 mM NaCl. Epitopes are indicated by arrows (L1).

(C) Western blot analysis of Coomassie stained gels using antibodies specific for the two epitopes indicated by the arrows (L1).

Detailed description of the invention

The present invention aims to produce novel virus-like particles for use as vaccines, diagnostic tools and R & D tools based on recombinant DNA and cell culture techniques for production. The particles of the present invention meet the need for a vaccine suitable for combating potential influenza outbreaks. The recombinant virus-like particles of the invention are assembled from polypeptide chains that incorporate several, particularly two or more, e.g., two, three, four or five, or multiples of three, e.g., six, nine, twelve different epitopes or different epitope-containing proteins selected from (a) different virus strains of the same virus and/or (b) different serotypes of the same virus and/or (c) different virus strains specific for different hosts. These epitopes are then displayed on the surface of the particles. Epitope selection from different strains, serotypes, and/or viruses specific for different hosts results in versatile virus-like particles that mimic their natural alteration of the viruses that occur in nature, such as that observed during the outbreak of swine influenza 4 months in 2009. The state of the art virus-like particles consist of a single protein or comprise up to three different epitopes from the same virus strain. The particles of the invention are encoded by a single DNA vector based on a virus or plasmid, which is used for production in host cells such as insect cells, bacterial cells and mammalian cells. In a preferred embodiment, the DNA vector is a baculovirus vector and the host cell is an insect cell.

The epitopes of the invention are immunogenic peptides consisting of between 4 and 1000 amino acids, preferably between 6 and 100 amino acids, preferably neutralizing epitopes. Neutralizing epitopes are epitopes that, when bound by an antibody as a result of an immunogenic response, cause neutralization of a virus bearing such neutralizing epitopes. An epitope as understood herein may be repetitive and may be part of a larger protein, in particular part of an antigen, part of a viral surface protein or part of a viral membrane protein. Such epitopes incorporated into viral surface proteins or viral membrane proteins are preferred. If the intended use of the virus-like particle according to the invention is as an R & D tool, a diagnostic tool or a virus mimic, it is important that the epitope is part of the complete viral protein providing a complete viral-type surface.

The different virus strains of the invention are, for example, different strains of influenza virus, e.g., influenza a virus strains H1N1, H5N1, H9N1, H1N2, H2N2, H3N2 or H9N2, or influenza B virus or influenza C virus.

Different serotypes of the invention are, for example, different serotypes of Human Papilloma Virus (HPV), such as serotypes 6, 11, 16, 18, 31, 33, 35, 39, 45, 48, 52, 58, 62, 66, 68, 70, 73, and 82, as well as HPV types 5, 8, 14, 17, 20, and 47 from proto-cancerous types, or HPV types 6, 11, 13, 26, 28, 32, and 60 from papilloma-associated.

Virus strains specific for different hosts are meant to be specifically adapted to the respective host, e.g. human influenza virus strains, swine influenza virus strains and avian influenza virus strains. In this context, specific to a host means that a virus is readily transmitted from one host to another of the same type, but not to a different type of host. For example, avian virus strains are readily transmitted from birds to other birds, but not to other animals or humans.

In a preferred embodiment, particles comprising epitopes from different strains, serotypes and/or viruses specific to different hosts are combined with B cell and/or T cell epitopes to induce a broader immune response.

In another preferred embodiment, the virus-like particle consists of proteins forming an intact virus-like surface, optionally further comprising capsid and nucleopore proteins. The virus-like particles of the invention may further comprise fluorescent proteins, proteins useful for purification purposes of the particles or for attachment of tags, and the structure of the proteins required for transport processes and stability.

The polypeptides and virus-like particles described herein are produced in shorter time and in unlimited amounts compared to the actual vaccine manufacturing process due to the application of specific genetic and process engineering tools. The ability to assemble desired viral genes by modern molecular biological methods, e.g., MultiBac technology (WO 2005/085456; I. Berger et al, Nature Biotechnology 22, 1583, 2004), Polybac technology (WO 2007/054250) or gene synthesis, e.g., allows for rapid assembly of encoding DNA vectors. The use of these techniques does not require any physical transfer of the original, potentially dangerous virus during the development, preparation or administration of the virus-like particles and vaccines of the present invention. For the construction of the particles of the invention, it is sufficient to use nucleotide sequences from infected individuals. This is in great contrast to classical egg-based methods for producing vaccines, which require genetically modified viruses as seed strain viruses. The particles of the present invention are manufactured using modern disposable tissue culture techniques, which allows for high throughput. In a preferred embodiment of baculovirus vectors and insect cells as host cells, the manufacturing process can be established quickly, with short production times, i.e. in the range of weeks, rather than months as compared to egg-based methods. In addition, the construction of disposable tissue culture devices is less time consuming and less costly than the construction of egg-based devices. As a result, a large number of vaccines for the entire population can be produced and re-produced in a short time, and several different types of vaccines, for example, seasonal influenza vaccines and influenza vaccines, can be easily produced in parallel. Difficult decisions made by the health sector for this or other vaccine species due to capacity limitations of the egg-based vaccine manufacturer would not be required.

The present invention relates to recombinant virus-like particles comprising two or more, e.g. two, three, four or five, or multiples of three, e.g. six, nine or twelve different epitopes or different epitope-containing proteins selected from (a) different virus strains of the same virus and/or (b) different serotypes of the same virus and/or (c) different virus strains specific for different hosts. Preferred are recombinant virus-like particles comprising three or more, preferably four or more, different epitopes or different epitope-containing proteins. Also preferred are recombinant virus-like particles comprising multiples of three, e.g., six, nine or twelve different epitopes or different epitope-containing proteins. The epitope is selected from two different strains, serotypes or virus strains specific for different hosts, or from three different strains, serotypes or virus strains specific for different hosts, or from four different strains or serotypes. Preferably, the virus-like particle comprises several epitopes from three different strains or serotypes. Also preferred are virus-like particles comprising several epitopes from virus strains specific for two or three different hosts.

Furthermore the invention relates to a vector comprising two or more, e.g. two, three, four or five, or a multiple of three, e.g. six, nine or twelve, different polynucleotides encoding epitopes or different epitope-containing proteins selected from (a) different virus strains of the same virus and/or (b) different serotypes of the same virus and/or (c) different virus strains specific for different hosts. As used herein, "polynucleotide" may represent a strand of 12 to 3,000 nucleotides, including the oligonucleotides referred to generally, and may be viral genes or open reading frames from the various viral sources mentioned, particularly genes or open reading frames encoding viral surface proteins or viral membrane proteins.

Preferred are vectors encoding the preferred virus-like particles mentioned above.

Most preferred are polypeptides comprising a sequence selected from SEQ ID NOs: 1 to 5.

In a preferred embodiment, the virus-like particle of the invention comprises

(1) The same type of surface protein from two or three different strains or serotypes of the same virus;

(2) mixtures of more than two different surface proteins from different virus strains, for example, from influenza virus strains H5N1 and H1N 1;

(3) a mixture of different surface proteins combined from viruses specific for different hosts, e.g. from influenza viruses specific for a pig, human and/or avian host.

Viruses considered to be the source of the epitopes contained in the virus-like particle of the present invention are, for example, influenza virus, HPV, HIV, CMV, dengue, HCV and newcastle disease virus. Epitopes may be derived from other viruses and bacteria. Particularly preferred are influenza viruses. Also preferred is Human Papilloma Virus (HPV).

Contemplated vectors are DNA vectors, which may be plasmid vectors or viral vectors. Methods for assembling such vectors are standard methods in the field of molecular biology. A preferred method is MultiBac, for example as described in WO2005/085456 and in I.Berger et al, Nature Biotechnology 22, 1583, 2004, or Polybac methods, for example as described in WO2007/054250, with CAP^TMThe prior art in the technical and gene synthesis fields combines. This technique allows the assembly of multigene co-expression DNA vectors, which are suitable for expression in different host cells. Preferred DNA vectors of the present invention are baculovirus vectors.

The host cell for expression of the vector of the invention may be any prokaryotic (e.g., E.coli) or eukaryotic expression cell line. For expression of preferred baculovirus vectors, insect cell lines are preferred. Examples of insect cell lines are, for example, SF9, SF21, Hi-5, Express Sf + and S2 Schneider cells. For expression in eukaryotic systems, mammalian cells are preferred, in particular human cells, e.g., HeLa, Huh7, HEK293, HepG2, BHK, CHO, MT-2, bone marrow fibroblasts, primary neural cells or embryonic cells. For expression in yeast, Saccharomyces cerevisiae (S.cerevisiae)S. cerevisiae) Schizosaccharomyces pombe (Schizosaccharomyces pombe)S. pombe) Candida albicans (C.albicans) (C.albicans)C. albicans) Or Pichia pastoris (P. pastoris) A cell.

The culturing and propagation of host cells according to the invention may be carried out in any container, bioreactor or disposable unit that provides suitable conditions for the particular host cell.

The virus-like particles of the present invention can be used as vaccines. In addition, they can be used as antigens in diagnostic tools, antigens for antibody production, and as virus mimetics for research and development tools, e.g., virus entry studies and virus-host interaction studies.

The vaccine according to the invention comprises recombinant virus-like particles in an aqueous solution, optionally further comprising a viscosity modulating compound, a stabilizing compound and/or an immunogenicity enhancing adjuvant, as is known in the art.

In a specific embodiment, Berger et al, Nature Biotechnology, 2004, WO2005/085456 and WO2007/054250 and CAP are used^TMMethods of the art H3N2 influenza virus-like particles were constructed. At least one of M1 and M2 genes of H1N1 influenza strain A/Puerto Rico/834 was cloned into a transfer vector pFL (WO 2007/054250, FIG. 1) by PCR amplification together with HA gene of influenza A/Brisbane10/2007 and NA gene of influenza A/Brisbane 10/2007. The constructs were confirmed by DNA sequencing.

In another specific embodiment, the same cloning technique is used to introduce at least one L1 gene of both serotypes HPV16 and HPV18, or other serotypes from groups 2, 4, 6, 11, 31, 33-35, 39, 40-45, 51-53, 55-59, 62, 66, 68, 70, 73 and 77, into the transfer vector pFL (FIG. 1).

Experimental part

Baculovirus vectors designated MultiBac or YFPUlBac (WO 2005/085456) which permit propagation in E.coli cells, and CAP^TMTechniques were used for the generation of recombinant AcNPVs (Autographa californica nuclear polyhedrosis virus, baculovirus) using conventional systems (Fitzgerald et al, Nature Methods, 3, 1021, 2006). 10 ng of the multigenic vector was transformed into DH10MultiBac and/or DH10 YFPUltBac competent cells. Positive clones were selected by blue/white screening and PCR. Corresponding MultiBac bacmid DNA Using Birnboim&Doly method separation. Recombinant AcNPVs were transfected with 1. mu.g of multigene MultiBac bacmid using the transfection reagent Fugene (Roche) at 0.9X10 according to the manufacturer's protocol⁶Transfection in Sf21 (Invitrogen) cells. Viral amplification was performed as described earlier (Fitzgerald et al, Nature Methods, 3, 1021, 2006). Titers of all recombinant AcNPVs were determined by phage described in Bac-to-Bac-Manual (Invitrogen)Spot analysis. Protein production parameters such as multiplicity of infection (MOI), cell number (TOI) and time to harvest (TOH) were analyzed using small scale expression studies.

Example 1: generation of expression vector constructs

For the manufacture of the various constructs, the transfer vector pFL was used according to the method described in WO2005/085456 (WO 2005/085456 and CAP)^TMTechnique) of the amplification module M. The DNA of the epitope is obtained by isolation of viral RNA from the original virus and subsequent reverse transcription combined with PCR (for influenza virus epitopes), or by gene synthesis (provided by Geneart). Reverse transcription RevertAId was used according to the manufacturer's protocol^TMH Minus first strand cDNA synthesis kit (Fermentas). cDNA (2. mu.L) was used as template for the PCR reaction. The following conditions were used according to the manufacturer's protocol. For a 50. mu.L total volume reaction, 0.2 mM dNTP (NEB), 1.2% DMSO, 0.5. mu.M reverse and forward primers (Microsynth), 10. mu.l of 5 × Phusion GC reaction buffer, and 2U of Phusion Hot Start polymerase (Finnzyme) were used. For multigene assembly, appropriate restriction sites (BstZ 17I, SpeI, PmeI, AvrII) were introduced using PCR. The PCR fragment was cleaved with restriction enzymes followed by ligation and transformation procedures to integrate the amplification module into the transfer vector. Ligation was performed overnight at 4 ℃ using 500 ng of linearized transfer vector (pFL), 4. mu.L of PCR product and 1U T4-DNA lyase (Fermentas). To generate plasmids, 4 μ L of ligation solution was added to 50 μ L of competent DH5 α cells and incubated on ice for 30 min. After heat shock at 42 ℃ for 30 seconds and cold shock at 4 ℃ for 2 minutes, 200. mu.l of LB medium was added, followed by incubation at 37 ℃ and 220 rpm for 1 h. Thereafter, 80. mu.l of the cell suspension were plated out on LB agar plates containing the appropriate antibiotic, in this case 100. mu.g ampicillin and 100. mu.g gentamicin. The entire procedure is repeated until all epitopes have been introduced into the transfer vector. The influenza epitope is selected from the genes HA and NA, both selected from the H3N2/Brisbane10/2007 strain, and from M1 andthe epitope of M2 was selected from the H1N1/Puerto Rico/834 strain. The M1 epitope is controlled by the promoter p10, and all other epitopes are controlled by the polyhedrin promoter polh. All epitopes are present on the same vector construct (FIG. 1A, SEQ ID NO: 1). Influenza B/Florida/2006 isolates were selected to generate constructs with genes from genes HA, NA, M1 and M2 (FIG. 1C, SEQ ID NO: 3). The epitope of human papillomavirus is selected from the group consisting of the gene L1 from the cancer-associated serotypes HPV16 and HPV18, integrated in one vector construct. Both epitopes are controlled by the polyhedrin promoter polh (FIG. 1B, SEQ ID NO: 2). To improve expression yield, the p10 promoter (FIG. 1C, SEQ ID NO: 5) was deleted in further constructs. In other constructs, the HPV16 epitope was controlled by the p10 promoter, while the polyhedrin promoter polh was chosen for the HPV18 epitope (FIG. 1D, SEQ ID NO: 4).

Example 2: production of recombinant baculovirus

This virus contains multiple epitopes to produce virus-like particles or virus mimics that present these epitopes on their surface. The virus-like particles can be used for different applications, e.g. as vaccines in the field of influenza. The AcNPV-derived baculovirus contains multiple distinct epitopes from the 2008/2009-VLP vaccination sports virus strain suggested by WHO. According to the protocol of WO2005/085456, all genes of the transfer vector were translocated into the MultiBac cell by site-specific homologous recombination. 10 ng of transfer vector was added to 100. mu.l of MultiBac competent cells and incubated at 4 ℃ for 30 minutes. After heat shock at 42 ℃ for 45 seconds and cold shock at 4 ℃ for 2 minutes, 400. mu.l of LB medium was added and the cell solution was incubated at 37 ℃ and 220 rpm for 4 h. Different dilutions were plated on appropriate LB agar plates containing various antibiotic resistances. Several correct MultiBac clones were selected based on blue/white and PCR screening. Corresponding MultiBac bacmid DNA Using Birnboim&Doly method separation. Selection of at least four MultiBac bacmid clonesInitial transfection in insect cells such as Sf9 or Sf21 to generate recombinant AcNPV-derived baculoviruses. This was done by 1. mu.g of multigene MultiBacbacmid using the transfection reagent Fugene (Roche) at 0.9X10 according to the manufacturer's protocol⁶Transfection in Sf21 (Invitrogen) cells. Viral amplification was performed as described earlier (Fitzgerald et al, Nature Methods, 3, 1021, 2006; Bac-to-Bac-Manual, Invitrogen). Viruses were amplified to expand volume and increase infectious titer, as determined by plaque assay according to Bac-to-Bac-manual (invitrogen). The best expression constructs were determined by 50 mL small scale expression experiments followed by protein production assays by Bradford assay (ADV, cytocoelenton). Expression of the best expression was further verified by Western blot analysis with antibodies against a variety of different epitopes (fig. 2A).

Example 3: production and purification of multi-epitope influenza virus-like particles in insect cells

After determination of the best expression construct, biotechnological production parameters such as cell line, cell amount (TOI), amount of recombinant virus inoculum (multiplicity of infection, MOI) and time of harvest (TOH) were determined in a 50 mL bioreactor. Different matrices of TOI, MOI and TOH were designed according to Eibl, Riesen and John (Bioforum 03/2009) and Friesen J. (Bachelor thesis, University of Applied Science, Esslingen, Germany). Using daily sample collection, expression of secreted or intracellular polyepitopic virus-like particles is observed for six to eight days. For intracellular particles (e.g., HPV), cell pellets were lysed with 50 mM TrisCl, pH 7.6, 100 mM NaCl, 0.1% TritonX100 and centrifuged at 8000 Xg for 10 min at 4 ℃. The epitopes of the virus-like particles present in the supernatant were verified using a Dot-blot apparatus (Biometra) and subsequent Western blotting with specific antibodies. The conditions that yield the highest yield are defined as expression parameters with reference to a three to four day harvest time. According to these defined parameters, in shake flasks or wave bagsVirus-like particles were produced in Spodoptera frugiperda cells Sf9 and Sf 21. For multi-epitope influenza virus-like particle expression, Sf21 cells were selected using the following conditions: 1.5x10⁶Individual cells/mL, MOI 0.05 and harvest time four days post infection. Cells were propagated without carbon dioxide and fetal bovine serum supplementation at 27 ℃. Secreted virus-like particles were collected by centrifugation at 500-1000 Xg for 20 min at 4 ℃ according to defined harvest times. The volume of supernatant containing the particles was reduced for purification by tangential flow filtration using a 100 kDa sieve cut, using a cassette (Sartocon-Slice 200, Sartorius and CentramateOS, PALL). Purification of virus-like particles was performed using scalable chromatography methods and sucrose gradient ultracentrifugation. Chromatographic purification is a multi-step purification utilizing cation exchange, anion exchange and gel filtration chromatography. The supernatant was loaded onto a CaptoQ column connected to an FPLC-system (AEKTA purifier, GE Healthcare) in 50 mM phosphate buffer pH 7.4. The particles were eluted with increasing salt concentration in a linear gradient using 50 mM phosphate, 1M NaCl, pH 7.4. The fractions containing the particles were pooled and further purified by gel filtration chromatography (VLP from SEQ ID NO:1, FIG. 3). Purification was performed using a HighLoad Superdex 200 pg column in 50 mM phosphate, 150 mM NaCl pH 7.4 buffer. All chromatographic steps were analyzed by SDS-PAGE and subsequent Coomassie staining and immunoblotting.

Example 4: analysis of purified influenza Virus-like particles

To confirm the presence of the different epitopes, the purified material was analyzed by SDS-PAGE followed by Coomassie staining or Western blotting. Mu.l of the different chromatographic fractions were loaded on 4-12% Bis-Tris NuPAGE gels (Invitrogen), run at 150V for 15 min, at 175V for 45 min, and stained with SimplyBlueSafetasin (Invitrogen) Coomassie.

For immunoblotting, proteins were transferred onto nitrocellulose membranes (BioRAD) using a semi-dry device (BioRAD) at 19V for 40 minutes. After blocking non-specific binding sites with a 5% skim milk powder-TrisCl-Tween 20 (0.1%) solution for 30 min, the membranes were incubated overnight at 4 ℃ with antibodies against HA, NA and matrix proteins. Membranes were washed several times with TrisCl-Tween20 (0.1%) buffer. Depending on the source of the primary antibody, the secondary antibody is an anti-mouse or anti-rabbit antibody linked to alkaline phosphatase or horseradish peroxidase for detection. The co-localization of these proteins shows the assembly and production from one expression vector and one baculovirus (FIG. 3B, from SEQ ID NO: 1). This co-localization can also be shown for expression constructs containing the genes HA, NA and both matrix proteins M1 and M2, including their membrane anchors.

Example 5: functionality of influenza Virus Like Particles (VLPs)

To analyze whether VLPs correctly integrated the hemagglutinin protein (HA) on their surface, standard hemagglutination assays were performed using chicken-derived Red Blood Cells (RBC) (FIG. 4, VLP from SEQ ID NO: 3). Two-fold serial dilutions of purified VLPs were performed in V-shaped 96-well plates with PBS (1 ×). Equal amounts of red blood cells (1% solution) were added and incubated at 4 ℃ for 1 h. The appearance of RBC aggregates at the bottom of the reaction wells indicates no hemagglutination. Titers were expressed as the reciprocal of the highest dilution of purified VLP solution agglutinating RBCs. The results obtained show that VLPs are capable of agglutinating chicken erythrocytes, indirectly revealing the presence of HA on the surface of VLPs. The negative control (PBS) showed no agglutination.

Example 6: in vivo evaluation of influenza VLPs

Immunogenicity (stimulation of the immune system) of VLPs (prepared from SEQ ID NO: 3) was tested in vivo using two groups of mice, which were immunized subcutaneously at weeks 0 and 3, respectively, on a prime boost schedule. Immunization was performed using 50 or 100 μ l (50 or 100 ng) of VLP in suspension. To determine the quality of the immune response of individual VLPs, no adjuvant was used. Mice were bled at weeks 3 and 6 and sera analyzed for antibody responses against immunized VLPs. The results obtained show that VLPs are effective in stimulating antibody immune responses. The best results were obtained when immunisation was performed with 100 μ l, indicating a dose-dependent immune response. A clear increase in anti-VLP antibodies was observed after boosting. As expected, the first experimental animals did not show an immune response.

To reconfirm the specificity of the immune response, a hemagglutination inhibition test was performed at week 6 to analyze for the presence of specific anti-HA antibodies. The results show that specific anti-influenza HA antibodies are able to inhibit hemagglutination up to dilution 128 (mouse 6) and dilution 256 (mouse 8). No hemagglutination inhibition was observed using serum samples from mice used for the first time in the experiment. The newly produced multi-epitope influenza VLPs are able to stimulate the immune system in a dose-dependent manner. When the immune system is re-stimulated with boosting, the immune response is increased to at least 15-fold. The specificity of the elicited immune response was analyzed by ELISA and hemagglutination assays.

Example 7: production and purification of polyepitopic virus-like particles bearing epitopes of human papilloma virus in various cell lines

Biotechnological production parameters such as cell line, cell mass (TOI), amount of recombinant virus inoculum (multiplicity of infection, MOI) and harvest Time (TOH) were determined in a 50 mL bioreactor according to Eibl et al (Bioforum 3/2009) (fig. 7). According to these defined parameters, virus-like particles were produced in Spodoptera frugiperda cells Sf9 and Sf21 in shake flasks or wave bags. Polyepitopic papillomavirus-like particles 2x10 was used in Sf21 cells⁶Individual cells/mL, MOI 0.5 and harvest time three days post infection were determined from SEQ ID NO:2, and (3) expressing. The cells were free of carbon dioxide at 27 deg.CAnd proliferation in the presence of fetal bovine serum supplementation. Intracellular virus-like particles were collected by centrifugation at 500-. Cells were lysed using hypotonic phosphate buffer and subsequent sonication. After a centrifugation step at 4 ℃ and 2000 Xg, the supernatant was collected. Purification of virus-like particles was performed using scalable chromatography methods and sucrose gradient ultracentrifugation. Chromatographic purification is a multi-step purification utilizing cation exchange, anion exchange and gel filtration chromatography. The supernatant was loaded onto a CaptoDEAE column connected to an FPLC-system (AEKTA purifier, GE Healthcare) in 50 mM phosphate buffer pH 7.4. The particles were eluted with increasing salt concentration in a linear gradient using 50 mM phosphate, 1M NaCl, pH 7.4 (fig. 8A). Fractions containing particles were pooled and further purified using hydroxyapatite columns. Binding was performed in 20 mM phosphate buffer pH 7.0 followed by elution with a linear gradient of 500 mM phosphate, 150 mM NaCl, pH 7.0. To refine the polyepitopic particles, gel filtration chromatography was performed. Purification was performed using a HighLoad Superdex 200 pg column in 50 mM phosphate, 150 mM NaCl pH 7.4 buffer. All chromatographic steps were analyzed by SDS-PAGE and subsequent Coomassie staining and immunoblotting.

Example 8: analysis of purified chimeric human papilloma virus-like particles

To confirm the presence of different epitopes in the purified material, the epitope sequence was determined from SEQ ID NO:2 was analyzed by SDS-PAGE and subsequent coomassie staining (fig. 8B) and Western blot (fig. 8C). For immunoblotting, antibodies against the different serotypes of L1 protein were used. Mu.l of the different chromatographic fractions were loaded on 4-12% Bis-Tris NuPAGE gels (Invitrogen), run at 150V for 15 min, at 175V for 45 min, and stained with SimplyBlueSafetasin (Invitrogen) Coomassie. For immunoblotting, proteins were transferred onto nitrocellulose membranes (BioRAD) using a semi-dry device (BioRAD) at 19V for 40 minutes. After blocking non-specific binding sites with a 5% skim milk powder-TrisCl-Tween 20 (0.1%) solution for 30 minutes, the membranes were incubated overnight at 4 ℃ with antibodies against the L1 epitope. Camvir antibody (santa cruz) was used for HPV16 and anti-HPV 18ab (Abcam) was used for HPV18 detection. Membranes were washed several times with TrisCl-Tween20 (0.1%) buffer. The membrane was incubated with alkaline phosphatase-linked anti-mouse antibody for 1h for detection. Epitopes were detected by BCIP/NPT solution. The co-localization of these proteins shows the assembly and production from one expression vector and one baculovirus.

Example 9: functionality of chimeric human papilloma virus-like particles (VLPs)

For analysis of the peptide from SEQ ID NO:2 whether the prepared hpv-like particles correctly integrated the L1 protein on their surface, standard ELISA analyses were performed. Two-fold serial dilutions of purified VLPs were performed in V-shaped 96-well plates with PBS (1 ×). An equal amount of serotype-specific antibody (concentration 1: 1000) was added and incubated at 37 ℃ for 1 h. Suitable binding of the antibody to the L1 protein was detected using a secondary antibody with horseradish peroxidase and a chemiluminescent detection system. The results obtained show the binding of the antibody to the recombinantly expressed epitope in a dose-dependent manner. The negative control (PBS) showed no binding.

Sequence listing

<110> Redbiotec AG

John, Corinne

Schaub, Christian

Wellnitz, Sabine

<120> recombinant virus-like particle encoded by multigene vector

<130> P371A

<150> EP09159287.3

<151> 2009-05-01

<160> 5

<170> PatentIn version 3.5

<210> 1

<211> 10482

<212> DNA

<213> Artificial

<220>

<223> Multi-epitope type A influenza Virus-like particle

<400> 1

ttctctgtca cagaatgaaa atttttctgt catctcttcg ttattaatgt ttgtaattga 60

ctgaatatca acgcttattt gcagcctgaa tggcgaatgg gacgcgccct gtagcggcgc 120

attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct 180

agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg 240

tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga 300

ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt 360

ttttcgccct ttgacgttgg agtccacgtt cttaatagtg gactcttgtt ccaaactgga 420

acaacactca accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg 480

gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata 540

ttaacgctta caatttaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt 600

tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc 660

ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc 720

ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa 780

aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg 840

gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag 900

ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc 960

gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta 1020

cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg 1080

cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca 1140

acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac 1200

caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat 1260

taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg 1320

ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata 1380

aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta 1440

agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa 1500

atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag 1560

tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg 1620

tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact 1680

gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg 1740

taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc 1800

aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata 1860

ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta 1920

catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc 1980

ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg 2040

ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac 2100

agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg 2160

taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt 2220

atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct 2280

cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg 2340

ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata 2400

accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca 2460

gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc cttacgcatc 2520

tgtgcggtat ttcacaccgc atagaccagc cgcgtaacct ggcaaaatcg gttacggttg 2580

agtaataaat ggatgccctg cgtaagcggg tgtgggcgga caataaagtc ttaaactgaa 2640

caaaatagat ctaaactatg acaataaagt cttaaactag acagaatagt tgtaaactga 2700

aatcagtcca gttatgctgt gaaaaagcat actggacttt tgttatggct aaagcaaact 2760

cttcattttc tgaagtgcaa attgcccgtc gtattaaaga ggggcgtggc caagggcatg 2820

gtaaagacta tattcgcggc gttgtgacaa tttaccgaac aactccgcgg ccgggaagcc 2880

gatctcggct tgaacgaatt gttaggtggc ggtacttggg tcgatatcaa agtgcatcac 2940

ttcttcccgt atgcccaact ttgtatagag agccactgcg ggatcgtcac cgtaatctgc 3000

ttgcacgtag atcacataag caccaagcgc gttggcctca tgcttgagga gattgatgag 3060

cgcggtggca atgccctgcc tccggtgctc gccggagact gcgagatcat agatatagat 3120

ctcactacgc ggctgctcaa acttgggcag aacgtaagcc gcgagagcgc caacaaccgc 3180

ttcttggtcg aaggcagcaa gcgcgatgaa tgtcttacta cggagcaagt tcccgaggta 3240

atcggagtcc ggctgatgtt gggagtaggt ggctacgtct ccgaactcac gaccgaaaag 3300

atcaagagca gcccgcatgg atttgacttg gtcagggccg agcctacatg tgcgaatgat 3360

gcccatactt gagccaccta actttgtttt agggcgactg ccctgctgcg taacatcgtt 3420

gctgctgcgt aacatcgttg ctgctccata acatcaaaca tcgacccacg gcgtaacgcg 3480

cttgctgctt ggatgcccga ggcatagact gtacaaaaaa acagtcataa caagccatga 3540

aaaccgccac tgcgccgtta ccaccgctgc gttcggtcaa ggttctggac cagttgcgtg 3600

agcgcatacg ctacttgcat tacagtttac gaaccgaaca ggcttatgtc aactgggttc 3660

gtgccttcat ccgtttccac ggtgtgcgtc acccggcaac cttgggcagc agcgaagtcg 3720

aggcatttct gtcctggctg gcgaacgagc gcaaggtttc ggtctccacg catcgtcagg 3780

cattggcggc cttgctgttc ttctacggca aggtgctgtg cacggatctg cccttgcttc 3840

aggagatcgg tagacctcgg ccgtcgcggc gcttgccggt ggtgctgacc ccggatgaag 3900

tggttcgcat cctcggtttt ctggaaggcg agcatcgttt gttcgcccag gactctagct 3960

atagttctag tggttggcta cagctttgtt tgtactatca acaggttgaa ctgctgatca 4020

acagatcctc tacgcggccg cggtaccata acttcgtata gcatacatta tacgaagtta 4080

tctggtttaa acgtacccgt agtggctatg gcagggcttg ccgccccgac gttggctgcg 4140

agccctgggc cttcacccga acttgggggt tggggtgggg aaaaggaaga aacgcgggcg 4200

tattggtccc aatggggtct cggtggggta tcgacagagt gccagccctg ggaccgaacc 4260

ccgcgtttat gaacaaacga cccaacaccc gtgcgtttta ttctgtcttt ttattgccgt 4320

catagcgcgg gttccttccg gtattgtctc cttccgtgtt tcagttagcc tcccccatct 4380

cccggtaccg catgctatgc atgcggccgc tcacttgaac cgttgcatct gcacccccat 4440

tcgtttctga taggcctgca aattttcaag aagatcattt ttcagaccag cactggagct 4500

aggatgagtc ccaatggttc tcatcgcttg caccatttgc ctagcctgac tagcaacctc 4560

catggcctct gctgcttgct cactcgatcc agccatttgc tccatagcct tagctgtagt 4620

gctggctaaa accattctgt tctcatgtct gattagtgga ttggttgttg tcaccatttg 4680

cctatgagac cgatgctggg agtcagcaat ctgttcacag gttgcacata ccaggccaaa 4740

tgccacttca gtggtcacag cccccatcct gttgtatatg aggcccatac aactggcaag 4800

tgcaccagca gaataactga gtgagatttc tttggcccca tggaatgtta tctccctctt 4860

gagcttccta tacagtttaa ctgctttgtc catgttattt ggatccccgt tcccattaag 4920

ggcagtttgg acaaagcgtc tacgctgcag tcctcgctca ctgggcacgg tgagcgtgaa 4980

cacaaatcct aaaatcccct tagtcagagg tgacaggatt ggtcttgtct ttagccattc 5040

catgagaacc tcaagatcgg tgttcttccc tgcaaagaca tcttcaagtc tctgtgcgat 5100

ctcggctttg agggggcctg acgggatgat agagagaacg tacgtttcga cctcggttag 5160

aagactcatg gtggatccat cccgggtgat caagtcttcg tcgagtgatt gtaaataaaa 5220

tgtaatttac agtatagtat tttaattaat atacaaatga tttgataata attcttattt 5280

aactataata tattgtgttg ggttgaatta aaggtccgta tactagtatc gattcgcgac 5340

ctactccgga atattaatag atcatggaga taattaaaat gataaccatc tcgcaaataa 5400

ataagtattt tactgttttc gtaacagttt tgtaataaaa aaacctataa atattccgga 5460

ttattcatac cgtcccacca tcgggcgcgg atccaccatg aatccaaatc aaaagataat 5520

aacgattggc tctgtttctc tcaccatttc cacaatatgc ttcttcatgc aaattgccat 5580

cttgataact actgtaacat tgcatttcaa gcaatatgaa ttcaactccc ccccaaacaa 5640

ccaagtgatg ctgtgtgaac caacaataat agaaagaaac ataacagaga tagtgtatct 5700

gaccaacacc accatagaga aggaaatatg ccccaaacta gcagaataca gaaattggtc 5760

aaagccgcaa tgtgacatta caggatttgc acctttttct aaggacaatt cgattaggct 5820

ttccgctggt ggggacatct gggtgacaag agaaccttat gtgtcatgcg atcctgacaa 5880

gtgttatcaa tttgcccttg gacagggaac aacactaaac aacgtgcatt caaatgacac 5940

agtacgtgat aggacccctt atcggaccct attgatgaat gagttaggtg ttccttttca 6000

tctggggacc aagcaagtgt gcatagcatg gtccagctca agttgtcacg atggaaaagc 6060

atggctgcat gtttgtataa cgggggatga taaaaatgca actgctagct tcatttacaa 6120

tgggaggctt gtagatagta ttgtttcatg gtccaaagaa atcctcagga cccaggagtc 6180

agaatgcgtt tgtatcaatg gaacttgtac agtagtaatg actgatggga gtgcttcagg 6240

aaaagctgat actaaaatac tattcattga ggaggggaaa atcgttcata ctagcacatt 6300

gtcaggaagt gctcagcatg tcgaggagtg ctcctgctat cctcgatatc ctggtgtcag 6360

atgtgtctgc agagacaact ggaaaggctc caataggccc atcgtagata taaacataaa 6420

ggatcatagc actgtttcca gttatgtgtg ttcaggactt gttggagaca cacccagaaa 6480

aaacgacagc tccagcagta gccattgttt ggatcctaac aatgaagaag gtggtcatgg 6540

agtgaaaggc tgggcctttg atgatggaaa tgacgtgtgg atgggaagaa cgatcagcga 6600

gaagtcgcgc ttagggtatg aaaccttcaa agtcattgaa ggctggtcca accctaagtc 6660

caaattgcag ataaataggc aagtcatagt tgacagaggt aataggtccg gttattctgg 6720

tattttctct gttgaaggca aaagctgcat caatcggtgc ttttatgtgg agttgataag 6780

gggaagaaaa gaggaaactg aagtcttgtg gacctcaaac agtattgttg tgttttgtgg 6840

cacctcaggt acatatggaa caggctcatg gcctgatggg gcggacatca atctcatgcc 6900

tatataagta ctagaggatc ataatcagcc ataccacatt tgtagaggtt ttacttgctt 6960

taaaaaacct cccacacctc cccctgaacc tgaaacataa aatgaatgca attgttgttg 7020

ttaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca 7080

caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat 7140

cttatcatgt ctggatctga tcactgcttg agcctagaag atccggctgc taacaaagcc 7200

cgaaaggaag ctgagttggc tgctgtggct agcttatcta gaaatattaa tagatcatgg 7260

agataattaa aatgataacc atctcgcaaa taaataagta ttttactgtt ttcgtaacag 7320

ttttgtaata aaaaaaccta taaatattcc ggattattca taccgtccca ccatcgggcg 7380

caccatgagt cttctaaccg aggtcgaaac gcctatcaga aacgaatggg ggtgcagatg 7440

caacggttca agtgatcctc tcactattgc cgcaaatatc attgggatct tgcacttgac 7500

attgtggatt cttgatcgtc tttttttcaa atgcatttac cgtcgcttta aatacggact 7560

gaaaggaggg ccttctacgg aaggagtgcc aaagtctatg agggaagaat atcgaaagga 7620

acagcagagt gctgtggatg ctgacgatgg tcattttgtc agcatagagc tggagtaagt 7680

actagaggat cataatcagc cataccacat ttgtagaggt tttacttgct ttaaaaaacc 7740

tcccacacct ccccctgaac ctgaaacata aaatgaatgc aattgttgtt gttaacttgt 7800

ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag 7860

catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg 7920

tctggatctg atcactgctt gagcctagaa gatccggctg ctaacaaagc ccgaaaggaa 7980

gctgagttgg ctgctgccac cgctgagcaa taactatcat aaccggaata ttaatagatc 8040

atggagataa ttaaaatgat aaccatctcg caaataaata agtattttac tgttttcgta 8100

acagttttgt aataaaaaaa cctataaata ttccggatta ttcataccgt cccaccatcg 8160

ggcgcggatc caccatgaag actatcattg ctttgagcta cattctatgt ctggttttca 8220

ctcaaaaact tcccggaaat gacaacagca cggcaacgct gtgccttggg caccatgcag 8280

taccaaacgg aacgatagtg aaaacaatca cgaatgacca aattgaagtt actaatgcta 8340

ctgagctggt tcagagttcc tcaacaggtg aaatatgcga cagtcctcat cagatccttg 8400

atggagaaaa ctgcacacta atagatgctc tattgggaga ccctcagtgt gatggcttcc 8460

aaaataagaa atgggacctt tttgttgaac gcagcaaagc ctacagcaac tgttaccctt 8520

atgatgtgcc ggattatgcc tcccttaggt cactagttgc ctcatccggc acactggagt 8580

ttaacaatga aagcttcaat tggactggag tcactcaaaa cggaacaagc tctgcttgca 8640

taaggagatc taataacagt ttctttagta gattgaattg gttgacccac ttaaaattca 8700

aatacccagc attgaacgtg actatgccaa acaatgaaaa atttgacaaa ttgtacattt 8760

ggggggttca ccacccgggt acggacaatg accaaatctt cccgtatgct caagcatcag 8820

gaagaatcac agtctctacc aaaagaagcc aacaaactgt aatcccgaat atcggatcta 8880

gacccagagt aaggaatatc cccagcagaa taagcatcta ttggacaata gtaaaaccgg 8940

gagacatact tttgattaac agcacaggga atctaattgc tcctaggggt tacttcaaaa 9000

tacgaagtgg gaaaagctca ataatgagat cagatgcacc cattggcaaa tgcaattctg 9060

aatgcatcac tccaaacgga agcattccca atgacaaacc attccaaaat gtaaacagga 9120

tcacatacgg ggcctgtccc agatatgtta agcaaaacac tctgaaattg gcaacaggga 9180

tgcgaaatgt accagagaaa caaactagag gcatatttgg cgcaatcgcg ggtttcatag 9240

aaaatggttg ggagggaatg gtggatggtt ggtacggttt caggcatcaa aattctgagg 9300

gaataggaca agcagcagat ctcaaaagca ctcaagcagc aatcgatcaa atcaatggga 9360

agctgaatag gttgatcggg aaaaccaacg agaaattcca tcagattgaa aaagaattct 9420

cagaagtcga agggagaatt caggaccttg agaaatatgt tgaggacacc aaaatagatc 9480

tctggtcata caacgcggag cttcttgttg ccctggagaa ccaacataca attgatctaa 9540

ctgactcaga aatgaacaaa ctgtttgaaa aaacaaagaa gcaactgagg gaaaatgctg 9600

aggatatggg caatggttgt ttcaaaatat accacaaatg tgacaatgcc tgcataggat 9660

caatcagaaa tggaacttat gaccacgatg tatacagaga tgaagcatta aacaaccggt 9720

tccagatcaa gggcgttgag ctgaagtcag gatacaaaga ttggatccta tggatttcct 9780

ttgccatatc atgttttttg ctttgtgttg ctttgttggg gttcatcatg tgggcctgcc 9840

aaaaaggcaa cattaggtgc aacatttgca tttgagtact agaggatcat aatcagccat 9900

accacatttg tagaggtttt acttgcttta aaaaacctcc cacacctccc cctgaacctg 9960

aaacataaaa tgaatgcaat tgttgttgtt aacttgttta ttgcagctta taatggttac 10020

aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt 10080

tgtggtttgt ccaaactcat caatgtatct tatcatgtct ggatctgatc actgcttgag 10140

cctagaagat ccggctgcta acaaagcccg aaaggaagct gagttggctg ctgccaccgc 10200

tgagcaataa ctatcataac ccctaggaga tccgaaccag ataagtgaaa tctagttcca 10260

aactattttg tcatttttaa ttttcgtatt agcttacgac gctacaccca gttcccatct 10320

attttgtcac tcttccctaa ataatcctta aaaactccat ttccacccct cccagttccc 10380

aactattttg tccgcccaca gcggggcatt tttcttcctg ttatgttttt aatcaaacat 10440

cctgccaact ccatgtgaca aaccgtcatc ttcggctact tt 10482

<210> 2

<211> 8917

<212> DNA

<213> Artificial

<220>

<223> Polyepitope human papilloma virus-like particles

<400> 2

ttctctgtca cagaatgaaa atttttctgt catctcttcg ttattaatgt ttgtaattga 60

ctgaatatca acgcttattt gcagcctgaa tggcgaatgg gacgcgccct gtagcggcgc 120

attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct 180

agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg 240

tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga 300

ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt 360

ttttcgccct ttgacgttgg agtccacgtt cttaatagtg gactcttgtt ccaaactgga 420

acaacactca accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg 480

gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata 540

ttaacgctta caatttaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt 600

tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc 660

ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc 720

ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa 780

aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg 840

gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag 900

ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc 960

gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta 1020

cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg 1080

cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca 1140

acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac 1200

caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat 1260

taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg 1320

ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata 1380

aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta 1440

agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa 1500

atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag 1560

tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg 1620

tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact 1680

gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg 1740

taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc 1800

aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata 1860

ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta 1920

catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc 1980

ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg 2040

ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac 2100

agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg 2160

taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt 2220

atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct 2280

cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg 2340

ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata 2400

accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca 2460

gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc cttacgcatc 2520

tgtgcggtat ttcacaccgc atagaccagc cgcgtaacct ggcaaaatcg gttacggttg 2580

agtaataaat ggatgccctg cgtaagcggg tgtgggcgga caataaagtc ttaaactgaa 2640

caaaatagat ctaaactatg acaataaagt cttaaactag acagaatagt tgtaaactga 2700

aatcagtcca gttatgctgt gaaaaagcat actggacttt tgttatggct aaagcaaact 2760

cttcattttc tgaagtgcaa attgcccgtc gtattaaaga ggggcgtggc caagggcatg 2820

gtaaagacta tattcgcggc gttgtgacaa tttaccgaac aactccgcgg ccgggaagcc 2880

gatctcggct tgaacgaatt gttaggtggc ggtacttggg tcgatatcaa agtgcatcac 2940

ttcttcccgt atgcccaact ttgtatagag agccactgcg ggatcgtcac cgtaatctgc 3000

ttgcacgtag atcacataag caccaagcgc gttggcctca tgcttgagga gattgatgag 3060

cgcggtggca atgccctgcc tccggtgctc gccggagact gcgagatcat agatatagat 3120

ctcactacgc ggctgctcaa acttgggcag aacgtaagcc gcgagagcgc caacaaccgc 3180

ttcttggtcg aaggcagcaa gcgcgatgaa tgtcttacta cggagcaagt tcccgaggta 3240

atcggagtcc ggctgatgtt gggagtaggt ggctacgtct ccgaactcac gaccgaaaag 3300

atcaagagca gcccgcatgg atttgacttg gtcagggccg agcctacatg tgcgaatgat 3360

gcccatactt gagccaccta actttgtttt agggcgactg ccctgctgcg taacatcgtt 3420

gctgctgcgt aacatcgttg ctgctccata acatcaaaca tcgacccacg gcgtaacgcg 3480

cttgctgctt ggatgcccga ggcatagact gtacaaaaaa acagtcataa caagccatga 3540

aaaccgccac tgcgccgtta ccaccgctgc gttcggtcaa ggttctggac cagttgcgtg 3600

agcgcatacg ctacttgcat tacagtttac gaaccgaaca ggcttatgtc aactgggttc 3660

gtgccttcat ccgtttccac ggtgtgcgtc acccggcaac cttgggcagc agcgaagtcg 3720

aggcatttct gtcctggctg gcgaacgagc gcaaggtttc ggtctccacg catcgtcagg 3780

cattggcggc cttgctgttc ttctacggca aggtgctgtg cacggatctg cccttgcttc 3840

aggagatcgg tagacctcgg ccgtcgcggc gcttgccggt ggtgctgacc ccggatgaag 3900

tggttcgcat cctcggtttt ctggaaggcg agcatcgttt gttcgcccag gactctagct 3960

atagttctag tggttggcta cagctttgtt tgtactatca acaggttgaa ctgctgatca 4020

acagatcctc tacgcggccg cggtaccata acttcgtata gcatacatta tacgaagtta 4080

tctggtttaa acgtacccgt agtggctatg gcagggcttg ccgccccgac gttggctgcg 4140

agccctgggc cttcacccga acttgggggt tggggtgggg aaaaggaaga aacgcgggcg 4200

tattggtccc aatggggtct cggtggggta tcgacagagt gccagccctg ggaccgaacc 4260

ccgcgtttat gaacaaacga cccaacaccc gtgcgtttta ttctgtcttt ttattgccgt 4320

catagcgcgg gttccttccg gtattgtctc cttccgtgtt tcagttagcc tcccccatct 4380

cccggtaccg catgctatgc atatcccggg tgatcaagtc ttcgtcgagt gattgtaaat 4440

aaaatgtaat ttacagtata gtattttaat taatatacaa atgatttgat aataattctt 4500

atttaactat aatatattgt gttgggttga attaaaggtc cgtatactag tatcgattcg 4560

cgacctactc cggaatatta atagatcatg gagataatta aaatgataac catctcgcaa 4620

ataaataagt attttactgt tttcgtaaca gttttgtaat aaaaaaacct ataaatattc 4680

cggattattc ataccgtccc accatcgggc gcggatccgc caccatgtcc ctgtggctgc 4740

cctccgaggc taccgtgtac ctgccccccg tgcccgtgtc caaggtggtg tccaccgacg 4800

agtacgtggc tcgtaccaac atctactacc acgctggcac ctcccgtctg ctggctgtgg 4860

gtcaccccta cttccccatc aagaagccca acaacaacaa gatcctggtg cccaaggtgt 4920

ccggcctgca gtaccgtgtg ttccgtatcc acctgcccga ccccaacaag ttcggtttcc 4980

ccgacacctc cttctacaac cctgacaccc agcgcctcgt gtgggcttgc gtgggcgtgg 5040

aggtcggccg tggccagccc ctgggtgtcg gtatctccgg tcaccccctg ctgaacaagc 5100

tggacgacac cgagaacgct tccgcttacg ctgctaacgc tggtgtcgac aaccgcgagt 5160

gcatctccat ggactacaag cagacccagc tgtgcctgat cggttgcaag ccccccatcg 5220

gcgagcactg gggcaagggt tccccctgca ccaacgtggc tgtgaacccc ggcgactgcc 5280

cccctctcga gctgatcaac accgtgatcc aggacggcga catggtggac accggtttcg 5340

gtgctatgga cttcaccacc ctgcaggcta acaagtccga ggtgcccctg gacatctgca 5400

cctccatctg caagtacccc gactacatca agatggtgtc cgagccctac ggcgactccc 5460

tgttcttcta cctgcgtcgt gagcagatgt tcgtgcgtca cctgttcaac cgtgctggtg 5520

ctgtgggcga gaacgtgccc gacgacctgt acatcaaggg ttccggttcc accgctaacc 5580

tggcttccag caactacttc cctaccccct ccggttccat ggtcacctcc gacgctcaga 5640

tcttcaacaa gccctactgg ctgcagcgtg ctcagggtca caacaacggt atctgctggg 5700

gcaaccagct gttcgtgacc gtggtcgaca ccacccgttc caccaacatg tctctgtgcg 5760

ctgctatctc cacctccgag actacctaca agaacaccaa cttcaaggag tacctgcgtc 5820

acggcgagga gtacgacctg cagttcatct tccagctgtg caagatcacc ctgaccgctg 5880

acgtgatgac ctacatccac tccatgaact ccactatcct cgaagattgg aacttcggtc 5940

tgcagccccc tcccggtggc accctcgagg acacctaccg tttcgtcacc tcccaggcta 6000

tcgcttgcca gaagcacacc ccccctgctc ccaaggagga ccccctgaag aagtacacct 6060

tctgggaggt caacctgaag gagaagttct ccgctgacct ggaccagttc cccctgggtc 6120

gcaagttcct gctgcaggcc ggactgaagg ccaagcccaa gttcaccctg ggcaagcgca 6180

aggctacccc caccacctcc tccacctcca ccaccgctaa gaggaagaag cgcaagctgt 6240

aaaagcttgt cgagaagtac tagaggatca taatcagcca taccacattt gtagaggttt 6300

tacttgcttt aaaaaacctc ccacacctcc ccctgaacct gaaacataaa atgaatgcaa 6360

ttgttgttgt taacttgttt attgcagctt ataatggtta caaataaagc aatagcatca 6420

caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca 6480

tcaatgtatc ttatcatgtc tggatctgat cactgcttga gcctagaaga tccggctgct 6540

aacaaagccc gaaaggaagc tgagttggct gctgtggcta gctttgttta actttaagaa 6600

ggagatacat ctagaaatat taatagatca tggagataat taaaatgata accatctcgc 6660

aaataaataa gtattttact gttttcgtaa cagttttgta ataaaaaaac ctataaatat 6720

tccggattat tcataccgtc ccaccatcgg gcgcggatcc gccaccatgg ctctgtggcg 6780

tccctccgac aacaccgtgt acctgccccc tccctccgtg gctcgtgtgg tcaacaccga 6840

cgactacgtg acccgtacct ccatcttcta ccacgctggt tcctcccgtc tgctgaccgt 6900

gggcaacccc tacttccgtg tgcccgctgg cggtggcaac aagcaggaca tccccaaggt 6960

gtccgcttac cagtaccgtg tgttccgtgt gcagctgccc gaccccaaca agttcggtct 7020

gcccgacacc tccatctaca accccgagac tcagcgcctc gtgtgggctt gcgctggtgt 7080

cgagatcggt cgtggccagc ccctgggtgt cggcctgtcc ggtcacccct tctacaacaa 7140

gctggacgac accgagtcct cccacgctgc tacctccaac gtgtccgagg acgtgcgcga 7200

caacgtgtct gtggactaca agcagaccca gctgtgcatc ctgggttgcg ctcccgctat 7260

cggcgagcac tgggctaagg gcaccgcttg caagtcccgt cctctgtccc agggcgactg 7320

cccccctctc gagctgaaga acaccgtgct cgaggacggc gacatggtgg acaccggtta 7380

cggtgctatg gacttcagca ccctgcagga caccaagtgc gaggtgcccc tggacatctg 7440

ccagtccatc tgcaagtacc ccgactacct gcagatgtcc gctgacccct acggcgactc 7500

tatgttcttc tgcctgcgtc gtgagcagct gttcgctcgt cacttctgga accgtgctgg 7560

caccatgggt gacaccgtgc cccagtccct gtacatcaag ggcaccggca tgcgtgcttc 7620

ccccggttcc tgcgtgtact ccccttcccc ctccggttcc atcgtgacct ccgactccca 7680

gctgttcaac aagccctact ggctgcacaa ggctcagggt cacaacaacg gtgtctgctg 7740

gcacaaccag ctgttcgtga ccgtggtcga caccacccgt tccaccaacc tgaccatctg 7800

cgcttccacc cagtcccccg tgcccggcca gtacgacgct accaagttca agcagtactc 7860

ccgtcacgtg gaggagtacg acctgcagtt catcttccag ctctgcacta tcaccctgac 7920

cgctgacgtg atgtcctaca tccactccat gaactcctct atcctcgaag attggaactt 7980

cggtgtcccc cctcccccca ctacctccct ggtggacact taccgtttcg tgcagtccgt 8040

ggctatcacc tgccagaagg acgctgctcc cgctgagaac aaggacccct acgacaagct 8100

gaagttctgg aacgtggacc tgaaggagaa gttctccctg gacctggacc agtaccccct 8160

gggtcgcaag ttcctggtgc aggctggcct gaggcgcaag cccaccatcg gtccccgcaa 8220

gcgttccgct ccctccgcta ccacctcctc caagcccgcc aagcgtgtgc gtgtgcgcgc 8280

tcgcaagtaa gctagcaagc ttgtcgagaa gtactagagg atcataatca gccataccac 8340

atttgtagag gttttacttg ctttaaaaaa cctcccacac ctccccctga acctgaaaca 8400

taaaatgaat gcaattgttg ttgttaactt gtttattgca gcttataatg gttacaaata 8460

aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg 8520

tttgtccaaa ctcatcaatg tatcttatca tgtctggatc tgatcactgc ttgagcctag 8580

aagatccggc tgctaacaaa gcccgaaagg aagctgagtt ggctgctgcc accgctgagc 8640

aataactatc ataaccccta ggagatccga accagataag tgaaatctag ttccaaacta 8700

ttttgtcatt tttaattttc gtattagctt acgacgctac acccagttcc catctatttt 8760

gtcactcttc cctaaataat ccttaaaaac tccatttcca cccctcccag ttcccaacta 8820

ttttgtccgc ccacagcggg gcatttttct tcctgttatg tttttaatca aacatcctgc 8880

caactccatg tgacaaaccg tcatcttcgg ctacttt 8917

<210> 3

<211> 10595

<212> DNA

<213> Artificial

<220>

<223> Multi-epitope type B influenza Virus-like particle

<400> 3

ttctctgtca cagaatgaaa atttttctgt catctcttcg ttattaatgt ttgtaattga 60

ctgaatatca acgcttattt gcagcctgaa tggcgaatgg gacgcgccct gtagcggcgc 120

attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct 180

agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg 240

tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga 300

ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt 360

ttttcgccct ttgacgttgg agtccacgtt cttaatagtg gactcttgtt ccaaactgga 420

acaacactca accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg 480

gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata 540

ttaacgctta caatttaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt 600

tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc 660

ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc 720

ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa 780

aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg 840

gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag 900

ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc 960

gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta 1020

cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg 1080

cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca 1140

acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac 1200

caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat 1260

taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg 1320

ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata 1380

aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta 1440

agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa 1500

atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag 1560

tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg 1620

tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact 1680

gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg 1740

taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc 1800

aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata 1860

ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta 1920

catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc 1980

ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg 2040

ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac 2100

agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg 2160

taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt 2220

atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct 2280

cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg 2340

ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata 2400

accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca 2460

gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc cttacgcatc 2520

tgtgcggtat ttcacaccgc atagaccagc cgcgtaacct ggcaaaatcg gttacggttg 2580

agtaataaat ggatgccctg cgtaagcggg tgtgggcgga caataaagtc ttaaactgaa 2640

caaaatagat ctaaactatg acaataaagt cttaaactag acagaatagt tgtaaactga 2700

aatcagtcca gttatgctgt gaaaaagcat actggacttt tgttatggct aaagcaaact 2760

cttcattttc tgaagtgcaa attgcccgtc gtattaaaga ggggcgtggc caagggcatg 2820

gtaaagacta tattcgcggc gttgtgacaa tttaccgaac aactccgcgg ccgggaagcc 2880

gatctcggct tgaacgaatt gttaggtggc ggtacttggg tcgatatcaa agtgcatcac 2940

ttcttcccgt atgcccaact ttgtatagag agccactgcg ggatcgtcac cgtaatctgc 3000

ttgcacgtag atcacataag caccaagcgc gttggcctca tgcttgagga gattgatgag 3060

cgcggtggca atgccctgcc tccggtgctc gccggagact gcgagatcat agatatagat 3120

ctcactacgc ggctgctcaa acttgggcag aacgtaagcc gcgagagcgc caacaaccgc 3180

ttcttggtcg aaggcagcaa gcgcgatgaa tgtcttacta cggagcaagt tcccgaggta 3240

atcggagtcc ggctgatgtt gggagtaggt ggctacgtct ccgaactcac gaccgaaaag 3300

atcaagagca gcccgcatgg atttgacttg gtcagggccg agcctacatg tgcgaatgat 3360

gcccatactt gagccaccta actttgtttt agggcgactg ccctgctgcg taacatcgtt 3420

gctgctgcgt aacatcgttg ctgctccata acatcaaaca tcgacccacg gcgtaacgcg 3480

cttgctgctt ggatgcccga ggcatagact gtacaaaaaa acagtcataa caagccatga 3540

aaaccgccac tgcgccgtta ccaccgctgc gttcggtcaa ggttctggac cagttgcgtg 3600

agcgcatacg ctacttgcat tacagtttac gaaccgaaca ggcttatgtc aactgggttc 3660

gtgccttcat ccgtttccac ggtgtgcgtc acccggcaac cttgggcagc agcgaagtcg 3720

aggcatttct gtcctggctg gcgaacgagc gcaaggtttc ggtctccacg catcgtcagg 3780

cattggcggc cttgctgttc ttctacggca aggtgctgtg cacggatctg cccttgcttc 3840

aggagatcgg tagacctcgg ccgtcgcggc gcttgccggt ggtgctgacc ccggatgaag 3900

tggttcgcat cctcggtttt ctggaaggcg agcatcgttt gttcgcccag gactctagct 3960

atagttctag tggttggcta cagctttgtt tgtactatca acaggttgaa ctgctgatca 4020

acagatcctc tacgcggccg cggtaccata acttcgtata gcatacatta tacgaagtta 4080

tctggtttaa acgtacccgt agtggctatg gcagggcttg ccgccccgac gttggctgcg 4140

agccctgggc cttcacccga acttgggggt tggggtgggg aaaaggaaga aacgcgggcg 4200

tattggtccc aatggggtct cggtggggta tcgacagagt gccagccctg ggaccgaacc 4260

ccgcgtttat gaacaaacga cccaacaccc gtgcgtttta ttctgtcttt ttattgccgt 4320

catagcgcgg gttccttccg gtattgtctc cttccgtgtt tcagttagcc tcccccatct 4380

cccggtacct tataagtatt tcctcacaag agctgaattt cccatagagc tctgttttag 4440

cacttccatt acatctttgg caattccttc tccattcttt tgacttgctc ctagagatct 4500

caacactcca atgttgtttt gcagctcttc tgctagtttt tggacgtctt ctccctttcc 4560

cattccattc attgtctttg ctgtgttcat agctgaaacc atctgcattt ctcgtcttac 4620

tccaggtacc gaagaccttg ctgctctgct atgggctcta tgcgagtgcg atgcttgttt 4680

ctcgcataaa gcacagagcg ttcctagttt tacttgcatt gaatagtttt cagggtttag 4740

gtacatgacc ataagacaat atagtaatgc tgagctttcg tggccttctg ctatttcaaa 4800

tgcttcatga aagcttacac atcttctcat ttttctctca gctagaatta ggcctttctt 4860

ctttgttgct gttgttccca ttcctgacag gggctctgtg atgaatctcc tttttctttc 4920

ttggtctttg ggttttaaaa agcatataga ggcaccaatt agtgcttttt gtatatcagt 4980

taggcacctt ttgtttttta tccattccaa agcagaatct aggtcaaatt ctttcccacc 5040

gaaccaacag tgtaattttt cagctagttc tgctttgcct tctccatctt ctattagtga 5100

aagcaggtag gcaattgtgt ctccaaacag cgacatggtg gcatcccggg tgatcaagtc 5160

ttcgtcgagt gattgtaaat aaaatgtaat ttacagtata gtattttaat taatatacaa 5220

atgatttgat aataattctt atttaactat aatatattgt gttgggttga attaaaggtc 5280

cgtatactag tatcgattcg cgacctactc cggaatatta atagatcatg gagataatta 5340

aaatgataac catctcgcaa ataaataagt attttactgt tttcgtaaca gttttgtaat 5400

aaaaaaacct ataaatattc cggattattc ataccgtccc accatcgggc gcggatccac 5460

catgaaggca ataattgtac tactcatggt agtaacatcc aatgcagatc gaatctgcac 5520

tggaataaca tcttcaaact cacctcatgt ggtcaaaaca gccactcaag gggaggtcaa 5580

tgtgactggt gtgataccac taacaacaac accaacaaaa tcttattttg caaatctcaa 5640

aggaacaagg accagaggga aactatgccc agactgtctc aactgcacag atctggatgt 5700

ggctttgggc agaccaatgt gtgtggggac cacaccttcg gcgaaagctt caatactcca 5760

cgaagtcaaa cctgttacat ccgggtgctt tcctataatg cacgacagaa caaaaatcag 5820

gcaactaccc aatcttctca gaggatatga aaatatcagg ctatcaaccc aaaacgtcat 5880

cgatgcggaa aaggcaccag gaggacccta cagacttgga acctcaggat cttgccctaa 5940

cgctaccagt aagagcggat ttttcgcaac aatggcttgg gctgtcccaa aggacaacaa 6000

caaaaatgca acgaacccac taacagtaga agtaccatac atttgtacag aaggggaaga 6060

ccaaatcact gtttgggggt tccattcaga tgacaaaacc caaatgaaga acctctatgg 6120

agactcaaat cctcaaaagt tcacctcatc tgctaatgga gtaaccacac actatgtttc 6180

tcagattggc agcttcccag atcaaacaga agacggagga ctaccacaaa gcggcaggat 6240

tgttgttgat tacatgatgc aaaaacctgg gaaaacagga acaattgtct accaaagagg 6300

tgttttgttg cctcaaaagg tgtggtgcgc gagtggcagg agcaaagtaa taaaagggtc 6360

cttgccttta attggtgaag cagattgcct tcatgaaaaa tacggtggat taaacaaaag 6420

caagccttac tacacaggag aacatgcaaa agccatagga aattgcccaa tatgggtgaa 6480

aacacctttg aagcttgcca atggaaccaa atatagacct cctgcaaaac tattaaagga 6540

aaggggtttc ttcggagcta ttgctggttt cctagaagga ggatgggaag gaatgattgc 6600

aggctggcac ggatacacat ctcacggagc acatggagtg gcagtggcgg cggaccttaa 6660

gagtacgcaa gaagctataa acaagataac aaaaaatctc aattctttga gtgagctaga 6720

agtaaagaat cttcaaagac taagtggtgc catggatgaa ctccacaacg aaatactcga 6780

gctggatgag aaagtggatg atctcagagc tgacactata agctcgcaaa tagaacttgc 6840

agtcttgctt tccaacgaag gaataataaa cagtgaagat gagcatctat tggcacttga 6900

gagaaaacta aagaaaatgc tgggtccctc tgctgtagag ataggaaatg gatgcttcga 6960

aaccaaacac aagtgcaacc agacctgctt agacaggata gctgctggca cctttaatgc 7020

aggagaattt tctctcccca cttttgattc actgaacatt actgctgcat ctttaaatga 7080

tgatggattg gataaccata ctatactgct ctattactca actgctgctt ctagtttggc 7140

tgtaacattg atgctagcta tttttattgt ttatatggtc tccagagaca acgtttcatg 7200

ctccatctgt ctataagtcg acgtactaga ggatcataat cagccatacc acatttgtag 7260

aggttttact tgctttaaaa aacctcccac acctccccct gaacctgaaa cataaaatga 7320

atgcaattgt tgttgttaac ttgtttattg cagcttataa tggttacaaa taaagcaata 7380

gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca 7440

aactcatcaa tgtatcttat catgtctgga tctgatcact gcttgagcct agaagatccg 7500

gctgctaaca aagcccgaaa ggaagctgag ttggctgctg tggctagctt tgtttaactt 7560

taagaaggag atacatctag aaatattaat agatcatgga gataattaaa atgataacca 7620

tctcgcaaat aaataagtat tttactgttt tcgtaacagt tttgtaataa aaaaacctat 7680

aaatattccg gattattcat accgtcccac catcgggcgc ggatccacca tgctaccttc 7740

aactatacaa acgttaaccc tatttctcac atcaggggga gtgttattat cactatatgt 7800

gtcagcttca ttgtcatact tactatattc ggatatattg ctaaaatttt cacaaacaga 7860

aataactgca ccaataatgc cattggattg tgcaaacgca tcaaatgttc aggctgtgaa 7920

ccgttctgca gcaaaagggg tgacacttct tctcccagaa ccggagtgga cataccctcg 7980

tttatcttgc ccgggctcaa cctttcagaa agcactccta attagccccc atagattcgg 8040

agaaaccaaa ggaaactcag ctcccttgat aataagggaa ccttttattg cttgtggacc 8100

aacggaatgc aaacactttg ctctaaccca ttatgcagct caaccagggg gatactacaa 8160

tggaacaaga gaagacagaa acaagctgag gcatctaatt tcagtcaaat tgggcaaaat 8220

cccaacagta gaaaactcca ttttccatat ggcagcttgg agcgggtccg catgccatga 8280

tggtaaagaa tggacatata tcggagttga tggccccgac agtaatgcat tactcaaaat 8340

aaaatatgga gaagcatata ctgacacata ccattcctat gcaaaaaaca tcctaaggac 8400

acaagaaagt gcctgcaatt gcatcggggg agattgttat cttatgataa ctgatggccc 8460

agcttcaggg attagtgaat gcagattcct taagattcga gagggccgaa taataaaaga 8520

aatatttcca acaggaagag taaaacatac tgaggaatgc acatgcggat ttgccagcaa 8580

caaaaccata gaatgtgctt gtagagataa cagttacaca gcaaaaagac cctttgtcaa 8640

attaaatgtg gagactgata cagcggaaat aagattgatg tgcacagaga cttatttgga 8700

cacccccaga ccaaatgatg gaagcataac agggccttgc gaatctgatg gggacaaagg 8760

gagtggaggc atcaagggag gatttgttca tcaaagaatg gcatccaaga ttggaaggtg 8820

gtactctcga acgatgtcta aaactaaaag aatggggatg ggactgtatg taaagtatga 8880

tggagaccca tggactgaca gtgaagccct tgctcttagt ggagtaatgg tttcgatgga 8940

agaacctggt tggtattcct ttggcttcga aataaaagat aagaaatgtg atgtcccctg 9000

tattgggata gaaatggtac atgatggtgg gaaaacgact tggcactcag cagcaacagc 9060

catttactgt ttaatgggct caggacaact gctgtgggac actgtcacag gtgttgatat 9120

ggctctgtaa atcgatgaca agcttgtcga gaagtactag aggatcataa tcagccatac 9180

cacatttgta gaggttttac ttgctttaaa aaacctccca cacctccccc tgaacctgaa 9240

acataaaatg aatgcaattg ttgttgttaa cttgtttatt gcagcttata atggttacaa 9300

ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg 9360

tggtttgtcc aaactcatca atgtatctta tcatgtctgg atctgatcac tgcttgagcc 9420

tagaagatcc ggctgctaac aaagcccgaa aggaagctga gttggctgct gggtgcctaa 9480

tgagtgagct aactcacatt aattgcccgg aatattaata gatcatggag ataattaaaa 9540

tgataaccat ctcgcaaata aataagtatt ttactgtttt cgtaacagtt ttgtaataaa 9600

aaaacctata aatattccgg attattcata ccgtcccacc atcgggcgcg gatccaccat 9660

gctcgaacca tttcagattc tttcaatttg ttcttttatc ttatcagctc tccatttcat 9720

ggcttggaca atagggcatt tgaatcaaat aaaaagagga ataaacatga aaatacgaat 9780

aaaaggtcca aacaaagaga caataaacag agaggtatca attttgagac acagttacca 9840

aaaagaaatc caggccaaag aaacaatgaa ggaagtactc tctgacaaca tggaggtgtt 9900

gagtgaccac ataataattg aggggctttc tgccgaagag ataataaaaa tgggtgaaac 9960

agttttggag atagaagaat tgcattaagt actagaggat cataatcagc cataccacat 10020

ttgtagaggt tttacttgct ttaaaaaacc tcccacacct ccccctgaac ctgaaacata 10080

aaatgaatgc aattgttgtt gttaacttgt ttattgcagc ttataatggt tacaaataaa 10140

gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt 10200

tgtccaaact catcaatgta tcttatcatg tctggatctg atcactgctt gagcctagaa 10260

gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg ctgctgccac cgctgagcaa 10320

taactatcat aacccctagg agatccgaac cagataagtg aaatctagtt ccaaactatt 10380

ttgtcatttt taattttcgt attagcttac gacgctacac ccagttccca tctattttgt 10440

cactcttccc taaataatcc ttaaaaactc catttccacc cctcccagtt cccaactatt 10500

ttgtccgccc acagcggggc atttttcttc ctgttatgtt tttaatcaaa catcctgcca 10560

actccatgtg acaaaccgtc atcttcggct acttt 10595

<210> 4

<211> 8411

<212> DNA

<213> Artificial

<220>

<223> Polyepitope papillomavirus-like particles

<400> 4

ttctctgtca cagaatgaaa atttttctgt catctcttcg ttattaatgt ttgtaattga 60

ctgaatatca acgcttattt gcagcctgaa tggcgaatgg gacgcgccct gtagcggcgc 120

attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct 180

agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg 240

tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga 300

ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt 360

ttttcgccct ttgacgttgg agtccacgtt cttaatagtg gactcttgtt ccaaactgga 420

acaacactca accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg 480

gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata 540

ttaacgctta caatttaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt 600

tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc 660

ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc 720

ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa 780

aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg 840

gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag 900

ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc 960

gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta 1020

cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg 1080

cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca 1140

acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac 1200

caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat 1260

taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg 1320

ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata 1380

aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta 1440

agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa 1500

atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag 1560

tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg 1620

tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact 1680

gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg 1740

taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc 1800

aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata 1860

ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta 1920

catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc 1980

ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg 2040

ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac 2100

agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg 2160

taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt 2220

atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct 2280

cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg 2340

ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata 2400

accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca 2460

gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc cttacgcatc 2520

tgtgcggtat ttcacaccgc atagaccagc cgcgtaacct ggcaaaatcg gttacggttg 2580

agtaataaat ggatgccctg cgtaagcggg tgtgggcgga caataaagtc ttaaactgaa 2640

caaaatagat ctaaactatg acaataaagt cttaaactag acagaatagt tgtaaactga 2700

aatcagtcca gttatgctgt gaaaaagcat actggacttt tgttatggct aaagcaaact 2760

cttcattttc tgaagtgcaa attgcccgtc gtattaaaga ggggcgtggc caagggcatg 2820

gtaaagacta tattcgcggc gttgtgacaa tttaccgaac aactccgcgg ccgggaagcc 2880

gatctcggct tgaacgaatt gttaggtggc ggtacttggg tcgatatcaa agtgcatcac 2940

ttcttcccgt atgcccaact ttgtatagag agccactgcg ggatcgtcac cgtaatctgc 3000

ttgcacgtag atcacataag caccaagcgc gttggcctca tgcttgagga gattgatgag 3060

cgcggtggca atgccctgcc tccggtgctc gccggagact gcgagatcat agatatagat 3120

ctcactacgc ggctgctcaa acttgggcag aacgtaagcc gcgagagcgc caacaaccgc 3180

ttcttggtcg aaggcagcaa gcgcgatgaa tgtcttacta cggagcaagt tcccgaggta 3240

atcggagtcc ggctgatgtt gggagtaggt ggctacgtct ccgaactcac gaccgaaaag 3300

atcaagagca gcccgcatgg atttgacttg gtcagggccg agcctacatg tgcgaatgat 3360

gcccatactt gagccaccta actttgtttt agggcgactg ccctgctgcg taacatcgtt 3420

gctgctgcgt aacatcgttg ctgctccata acatcaaaca tcgacccacg gcgtaacgcg 3480

cttgctgctt ggatgcccga ggcatagact gtacaaaaaa acagtcataa caagccatga 3540

aaaccgccac tgcgccgtta ccaccgctgc gttcggtcaa ggttctggac cagttgcgtg 3600

agcgcatacg ctacttgcat tacagtttac gaaccgaaca ggcttatgtc aactgggttc 3660

gtgccttcat ccgtttccac ggtgtgcgtc acccggcaac cttgggcagc agcgaagtcg 3720

aggcatttct gtcctggctg gcgaacgagc gcaaggtttc ggtctccacg catcgtcagg 3780

cattggcggc cttgctgttc ttctacggca aggtgctgtg cacggatctg cccttgcttc 3840

aggagatcgg tagacctcgg ccgtcgcggc gcttgccggt ggtgctgacc ccggatgaag 3900

tggttcgcat cctcggtttt ctggaaggcg agcatcgttt gttcgcccag gactctagct 3960

atagttctag tggttggcta cagctttgtt tgtactatca acaggttgaa ctgctgatca 4020

acagatcctc tacgcggccg cggtaccata acttcgtata gcatacatta tacgaagtta 4080

tctggtttaa acgtacccgt agtggctatg gcagggcttg ccgccccgac gttggctgcg 4140

agccctgggc cttcacccga acttgggggt tggggtgggg aaaaggaaga aacgcgggcg 4200

tattggtccc aatggggtct cggtggggta tcgacagagt gccagccctg ggaccgaacc 4260

ccgcgtttat gaacaaacga cccaacaccc gtgcgtttta ttctgtcttt ttattgccgt 4320

catagcgcgg gttccttccg gtattgtctc cttccgtgtt tcagttagcc tcccccatct 4380

cccggtaccg catgctatgc ataagctttt acagcttgcg cttcttcctc ttagcggtgg 4440

tggaggtgga ggaggtggtg ggggtagcct tgcgcttgcc cagggtgaac ttgggcttgg 4500

ccttcagtcc ggcctgcagc aggaacttgc gacccagggg gaactggtcc aggtcagcgg 4560

agaacttctc cttcaggttg acctcccaga aggtgtactt cttcaggggg tcctccttgg 4620

gagcaggggg ggtgtgcttc tggcaagcga tagcctggga ggtgacgaaa cggtaggtgt 4680

cctcgagggt gccaccggga gggggctgca gaccgaagtt ccaatcttcg aggatagtgg 4740

agttcatgga gtggatgtag gtcatcacgt cagcggtcag ggtgatcttg cacagctgga 4800

agatgaactg caggtcgtac tcctcgccgt gacgcaggta ctccttgaag ttggtgttct 4860

tgtaggtagt ctcggaggtg gagatagcag cgcacagaga catgttggtg gaacgggtgg 4920

tgtcgaccac ggtcacgaac agctggttgc cccagcagat accgttgttg tgaccctgag 4980

cacgctgcag ccagtagggc ttgttgaaga tctgagcgtc ggaggtgacc atggaaccgg 5040

agggggtagg gaagtagttg ctggaagcca ggttagcggt ggaaccggaa cccttgatgt 5100

acaggtcgtc gggcacgttc tcgcccacag caccagcacg gttgaacagg tgacgcacga 5160

acatctgctc acgacgcagg tagaagaaca gggagtcgcc gtagggctcg gacaccatct 5220

tgatgtagtc ggggtacttg cagatggagg tgcagatgtc caggggcacc tcggacttgt 5280

tagcctgcag ggtggtgaag tccatagcac cgaaaccggt gtccaccatg tcgccgtcct 5340

ggatcacggt gttgatcagc tcgagagggg ggcagtcgcc ggggttcaca gccacgttgg 5400

tgcaggggga acccttgccc cagtgctcgc cgatgggggg cttgcaaccg atcaggcaca 5460

gctgggtctg cttgtagtcc atggagatgc actcgcggtt gtcgacacca gcgttagcag 5520

cgtaagcgga agcgttctcg gtgtcgtcca gcttgttcag cagggggtga ccggagatac 5580

cgacacccag gggctggcca cggccgacct ccacgcccac gcaagcccac acgaggcgct 5640

gggtgtcagg gttgtagaag gaggtgtcgg ggaaaccgaa cttgttgggg tcgggcaggt 5700

ggatacggaa cacacggtac tgcaggccgg acaccttggg caccaggatc ttgttgttgt 5760

tgggcttctt gatggggaag taggggtgac ccacagccag cagacgggag gtgccagcgt 5820

ggtagtagat gttggtacga gccacgtact cgtcggtgga caccaccttg gacacgggca 5880

cggggggcag gtacacggta gcctcggagg gcagccacag ggacatggtg gcggatccat 5940

cccgggtgat caagtcttcg tcgagtgatt gtaaataaaa tgtaatttac agtatagtat 6000

tttaattaat atacaaatga tttgataata attcttattt aactataata tattgtgttg 6060

ggttgaatta aaggtccgta tactagtatc gattcgcgac ctactccgga atattaatag 6120

atcatggaga taattaaaat gataaccatc tcgcaaataa ataagtattt tactgttttc 6180

gtaacagttt tgtaataaaa aaacctataa atattccgga ttattcatac cgtcccacca 6240

tcgggcgcgg atccgccacc atggctctgt ggcgtccctc cgacaacacc gtgtacctgc 6300

cccctccctc cgtggctcgt gtggtcaaca ccgacgacta cgtgacccgt acctccatct 6360

tctaccacgc tggttcctcc cgtctgctga ccgtgggcaa cccctacttc cgtgtgcccg 6420

ctggcggtgg caacaagcag gacatcccca aggtgtccgc ttaccagtac cgtgtgttcc 6480

gtgtgcagct gcccgacccc aacaagttcg gtctgcccga cacctccatc tacaaccccg 6540

agactcagcg cctcgtgtgg gcttgcgctg gtgtcgagat cggtcgtggc cagcccctgg 6600

gtgtcggcct gtccggtcac cccttctaca acaagctgga cgacaccgag tcctcccacg 6660

ctgctacctc caacgtgtcc gaggacgtgc gcgacaacgt gtctgtggac tacaagcaga 6720

cccagctgtg catcctgggt tgcgctcccg ctatcggcga gcactgggct aagggcaccg 6780

cttgcaagtc ccgtcctctg tcccagggcg actgcccccc tctcgagctg aagaacaccg 6840

tgctcgagga cggcgacatg gtggacaccg gttacggtgc tatggacttc agcaccctgc 6900

aggacaccaa gtgcgaggtg cccctggaca tctgccagtc catctgcaag taccccgact 6960

acctgcagat gtccgctgac ccctacggcg actctatgtt cttctgcctg cgtcgtgagc 7020

agctgttcgc tcgtcacttc tggaaccgtg ctggcaccat gggtgacacc gtgccccagt 7080

ccctgtacat caagggcacc ggcatgcgtg cttcccccgg ttcctgcgtg tactcccctt 7140

ccccctccgg ttccatcgtg acctccgact cccagctgtt caacaagccc tactggctgc 7200

acaaggctca gggtcacaac aacggtgtct gctggcacaa ccagctgttc gtgaccgtgg 7260

tcgacaccac ccgttccacc aacctgacca tctgcgcttc cacccagtcc cccgtgcccg 7320

gccagtacga cgctaccaag ttcaagcagt actcccgtca cgtggaggag tacgacctgc 7380

agttcatctt ccagctctgc actatcaccc tgaccgctga cgtgatgtcc tacatccact 7440

ccatgaactc ctctatcctc gaagattgga acttcggtgt cccccctccc cccactacct 7500

ccctggtgga cacttaccgt ttcgtgcagt ccgtggctat cacctgccag aaggacgctg 7560

ctcccgctga gaacaaggac ccctacgaca agctgaagtt ctggaacgtg gacctgaagg 7620

agaagttctc cctggacctg gaccagtacc ccctgggtcg caagttcctg gtgcaggctg 7680

gcctgaggcg caagcccacc atcggtcccc gcaagcgttc cgctccctcc gctaccacct 7740

cctccaagcc cgccaagcgt gtgcgtgtgc gcgctcgcaa gtaagctagc aagcttgtcg 7800

agaagtacta gaggatcata atcagccata ccacatttgt agaggtttta cttgctttaa 7860

aaaacctccc acacctcccc ctgaacctga aacataaaat gaatgcaatt gttgttgtta 7920

acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa 7980

ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt 8040

atcatgtctg gatctgatca ctgcttgagc ctagaagatc cggctgctaa caaagcccga 8100

aaggaagctg agttggctgc tgccaccgct gagcaataac tatcataacc cctaggagat 8160

ccgaaccaga taagtgaaat ctagttccaa actattttgt catttttaat tttcgtatta 8220

gcttacgacg ctacacccag ttcccatcta ttttgtcact cttccctaaa taatccttaa 8280

aaactccatt tccacccctc ccagttccca actattttgt ccgcccacag cggggcattt 8340

ttcttcctgt tatgttttta atcaaacatc ctgccaactc catgtgacaa accgtcatct 8400

tcggctactt t 8411

<210> 5

<211> 8445

<212> DNA

<213> Artificial

<220>

<223> Polyepitope papillomavirus-like particles

<400> 5

ttctctgtca cagaatgaaa atttttctgt catctcttcg ttattaatgt ttgtaattga 60

ctgaatatca acgcttattt gcagcctgaa tggcgaatgg gacgcgccct gtagcggcgc 120

attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct 180

agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg 240

tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga 300

ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt 360

ttttcgccct ttgacgttgg agtccacgtt cttaatagtg gactcttgtt ccaaactgga 420

acaacactca accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg 480

gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata 540

ttaacgctta caatttaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt 600

tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc 660

ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc 720

ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa 780

aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg 840

gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag 900

ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc 960

gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta 1020

cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg 1080

cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca 1140

acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac 1200

caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat 1260

taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg 1320

ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata 1380

aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta 1440

agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa 1500

atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag 1560

tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg 1620

tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact 1680

gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg 1740

taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc 1800

aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata 1860

ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta 1920

catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc 1980

ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg 2040

ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac 2100

agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg 2160

taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt 2220

atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct 2280

cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg 2340

ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata 2400

accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca 2460

gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc cttacgcatc 2520

tgtgcggtat ttcacaccgc atagaccagc cgcgtaacct ggcaaaatcg gttacggttg 2580

agtaataaat ggatgccctg cgtaagcggg tgtgggcgga caataaagtc ttaaactgaa 2640

caaaatagat ctaaactatg acaataaagt cttaaactag acagaatagt tgtaaactga 2700

aatcagtcca gttatgctgt gaaaaagcat actggacttt tgttatggct aaagcaaact 2760

cttcattttc tgaagtgcaa attgcccgtc gtattaaaga ggggcgtggc caagggcatg 2820

gtaaagacta tattcgcggc gttgtgacaa tttaccgaac aactccgcgg ccgggaagcc 2880

gatctcggct tgaacgaatt gttaggtggc ggtacttggg tcgatatcaa agtgcatcac 2940

ttcttcccgt atgcccaact ttgtatagag agccactgcg ggatcgtcac cgtaatctgc 3000

ttgcacgtag atcacataag caccaagcgc gttggcctca tgcttgagga gattgatgag 3060

cgcggtggca atgccctgcc tccggtgctc gccggagact gcgagatcat agatatagat 3120

ctcactacgc ggctgctcaa acttgggcag aacgtaagcc gcgagagcgc caacaaccgc 3180

ttcttggtcg aaggcagcaa gcgcgatgaa tgtcttacta cggagcaagt tcccgaggta 3240

atcggagtcc ggctgatgtt gggagtaggt ggctacgtct ccgaactcac gaccgaaaag 3300

atcaagagca gcccgcatgg atttgacttg gtcagggccg agcctacatg tgcgaatgat 3360

gcccatactt gagccaccta actttgtttt agggcgactg ccctgctgcg taacatcgtt 3420

gctgctgcgt aacatcgttg ctgctccata acatcaaaca tcgacccacg gcgtaacgcg 3480

cttgctgctt ggatgcccga ggcatagact gtacaaaaaa acagtcataa caagccatga 3540

aaaccgccac tgcgccgtta ccaccgctgc gttcggtcaa ggttctggac cagttgcgtg 3600

agcgcatacg ctacttgcat tacagtttac gaaccgaaca ggcttatgtc aactgggttc 3660

gtgccttcat ccgtttccac ggtgtgcgtc acccggcaac cttgggcagc agcgaagtcg 3720

aggcatttct gtcctggctg gcgaacgagc gcaaggtttc ggtctccacg catcgtcagg 3780

cattggcggc cttgctgttc ttctacggca aggtgctgtg cacggatctg cccttgcttc 3840

aggagatcgg tagacctcgg ccgtcgcggc gcttgccggt ggtgctgacc ccggatgaag 3900

tggttcgcat cctcggtttt ctggaaggcg agcatcgttt gttcgcccag gactctagct 3960

atagttctag tggttggcta cagctttgtt tgtactatca acaggttgaa ctgctgatca 4020

acagatcctc tacgcggccg cggtaccata acttcgtata gcatacatta tacgaagtta 4080

tctggtttcg acctactccg gaatattaat agatcatgga gataattaaa atgataacca 4140

tctcgcaaat aaataagtat tttactgttt tcgtaacagt tttgtaataa aaaaacctat 4200

aaatattccg gattattcat accgtcccac catcgggcgc ggatccgcca ccatgtccct 4260

gtggctgccc tccgaggcta ccgtgtacct gccccccgtg cccgtgtcca aggtggtgtc 4320

caccgacgag tacgtggctc gtaccaacat ctactaccac gctggcacct cccgtctgct 4380

ggctgtgggt cacccctact tccccatcaa gaagcccaac aacaacaaga tcctggtgcc 4440

caaggtgtcc ggcctgcagt accgtgtgtt ccgtatccac ctgcccgacc ccaacaagtt 4500

cggtttcccc gacacctcct tctacaaccc tgacacccag cgcctcgtgt gggcttgcgt 4560

gggcgtggag gtcggccgtg gccagcccct gggtgtcggt atctccggtc accccctgct 4620

gaacaagctg gacgacaccg agaacgcttc cgcttacgct gctaacgctg gtgtcgacaa 4680

ccgcgagtgc atctccatgg actacaagca gacccagctg tgcctgatcg gttgcaagcc 4740

ccccatcggc gagcactggg gcaagggttc cccctgcacc aacgtggctg tgaaccccgg 4800

cgactgcccc cctctcgagc tgatcaacac cgtgatccag gacggcgaca tggtggacac 4860

cggtttcggt gctatggact tcaccaccct gcaggctaac aagtccgagg tgcccctgga 4920

catctgcacc tccatctgca agtaccccga ctacatcaag atggtgtccg agccctacgg 4980

cgactccctg ttcttctacc tgcgtcgtga gcagatgttc gtgcgtcacc tgttcaaccg 5040

tgctggtgct gtgggcgaga acgtgcccga cgacctgtac atcaagggtt ccggttccac 5100

cgctaacctg gcttccagca actacttccc taccccctcc ggttccatgg tcacctccga 5160

cgctcagatc ttcaacaagc cctactggct gcagcgtgct cagggtcaca acaacggtat 5220

ctgctggggc aaccagctgt tcgtgaccgt ggtcgacacc acccgttcca ccaacatgtc 5280

tctgtgcgct gctatctcca cctccgagac tacctacaag aacaccaact tcaaggagta 5340

cctgcgtcac ggcgaggagt acgacctgca gttcatcttc cagctgtgca agatcaccct 5400

gaccgctgac gtgatgacct acatccactc catgaactcc actatcctcg aagattggaa 5460

cttcggtctg cagccccctc ccggtggcac cctcgaggac acctaccgtt tcgtcacctc 5520

ccaggctatc gcttgccaga agcacacccc ccctgctccc aaggaggacc ccctgaagaa 5580

gtacaccttc tgggaggtca acctgaagga gaagttctcc gctgacctgg accagttccc 5640

cctgggtcgc aagttcctgc tgcaggccgg actgaaggcc aagcccaagt tcaccctggg 5700

caagcgcaag gctaccccca ccacctcctc cacctccacc accgctaaga ggaagaagcg 5760

caagctgtaa aagcttgtcg agaagtacta gaggatcata atcagccata ccacatttgt 5820

agaggtttta cttgctttaa aaaacctccc acacctcccc ctgaacctga aacataaaat 5880

gaatgcaatt gttgttgtta acttgtttat tgcagcttat aatggttaca aataaagcaa 5940

tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc 6000

caaactcatc aatgtatctt atcatgtctg gatctgatca ctgcttgagc ctagaagatc 6060

cggctgctaa caaagcccga aaggaagctg agttggctgc tgtggctagc tttgtttaac 6120

tttaagaagg agatacatct agaaatatta atagatcatg gagataatta aaatgataac 6180

catctcgcaa ataaataagt attttactgt tttcgtaaca gttttgtaat aaaaaaacct 6240

ataaatattc cggattattc ataccgtccc accatcgggc gcggatccgc caccatggct 6300

ctgtggcgtc cctccgacaa caccgtgtac ctgccccctc cctccgtggc tcgtgtggtc 6360

aacaccgacg actacgtgac ccgtacctcc atcttctacc acgctggttc ctcccgtctg 6420

ctgaccgtgg gcaaccccta cttccgtgtg cccgctggcg gtggcaacaa gcaggacatc 6480

cccaaggtgt ccgcttacca gtaccgtgtg ttccgtgtgc agctgcccga ccccaacaag 6540

ttcggtctgc ccgacacctc catctacaac cccgagactc agcgcctcgt gtgggcttgc 6600

gctggtgtcg agatcggtcg tggccagccc ctgggtgtcg gcctgtccgg tcaccccttc 6660

tacaacaagc tggacgacac cgagtcctcc cacgctgcta cctccaacgt gtccgaggac 6720

gtgcgcgaca acgtgtctgt ggactacaag cagacccagc tgtgcatcct gggttgcgct 6780

cccgctatcg gcgagcactg ggctaagggc accgcttgca agtcccgtcc tctgtcccag 6840

ggcgactgcc cccctctcga gctgaagaac accgtgctcg aggacggcga catggtggac 6900

accggttacg gtgctatgga cttcagcacc ctgcaggaca ccaagtgcga ggtgcccctg 6960

gacatctgcc agtccatctg caagtacccc gactacctgc agatgtccgc tgacccctac 7020

ggcgactcta tgttcttctg cctgcgtcgt gagcagctgt tcgctcgtca cttctggaac 7080

cgtgctggca ccatgggtga caccgtgccc cagtccctgt acatcaaggg caccggcatg 7140

cgtgcttccc ccggttcctg cgtgtactcc ccttccccct ccggttccat cgtgacctcc 7200

gactcccagc tgttcaacaa gccctactgg ctgcacaagg ctcagggtca caacaacggt 7260

gtctgctggc acaaccagct gttcgtgacc gtggtcgaca ccacccgttc caccaacctg 7320

accatctgcg cttccaccca gtcccccgtg cccggccagt acgacgctac caagttcaag 7380

cagtactccc gtcacgtgga ggagtacgac ctgcagttca tcttccagct ctgcactatc 7440

accctgaccg ctgacgtgat gtcctacatc cactccatga actcctctat cctcgaagat 7500

tggaacttcg gtgtcccccc tccccccact acctccctgg tggacactta ccgtttcgtg 7560

cagtccgtgg ctatcacctg ccagaaggac gctgctcccg ctgagaacaa ggacccctac 7620

gacaagctga agttctggaa cgtggacctg aaggagaagt tctccctgga cctggaccag 7680

taccccctgg gtcgcaagtt cctggtgcag gctggcctga ggcgcaagcc caccatcggt 7740

ccccgcaagc gttccgctcc ctccgctacc acctcctcca agcccgccaa gcgtgtgcgt 7800

gtgcgcgctc gcaagtaagc tagcaagctt gtcgagaagt actagaggat cataatcagc 7860

cataccacat ttgtagaggt tttacttgct ttaaaaaacc tcccacacct ccccctgaac 7920

ctgaaacata aaatgaatgc aattgttgtt gttaacttgt ttattgcagc ttataatggt 7980

tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct 8040

agttgtggtt tgtccaaact catcaatgta tcttatcatg tctggatctg atcactgctt 8100

gagcctagaa gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg ctgctgccac 8160

cgctgagcaa taactatcat aacccctagg agatccgaac cagataagtg aaatctagtt 8220

ccaaactatt ttgtcatttt taattttcgt attagcttac gacgctacac ccagttccca 8280

tctattttgt cactcttccc taaataatcc ttaaaaactc catttccacc cctcccagtt 8340

cccaactatt ttgtccgccc acagcggggc atttttcttc ctgttatgtt tttaatcaaa 8400

catcctgcca actccatgtg acaaaccgtc atcttcggct acttt 8445

Claims (6)

1. A recombinant polynucleotide sequence selected from the group consisting of SEQ ID NO: 2. 4 and 5.

2. A recombinant virus-like particle comprising the nucleic acid sequence according to claim 1 selected from SEQ ID NO: 2. 4 and 5.

3. A vector comprising a sequence selected from SEQ ID NOs: 2. 4 and 5.

4. The vector according to claim 3, which is a baculovirus vector.

5. A host cell comprising a vector according to any one of claims 3 or 4.

6. A vaccine comprising the recombinant virus-like particle according to claim 2.