The present invention relates to a technology and method for making a virus like particle based vaccine with efficient epitope display and capable of inducing a strong and long-term protective immune response. The present invention solves the key challenge of obtaining a virus like particle which presents a larger antigen on the particle surface at high density, while being regularly spaced, and with consistent orientation; three critical factors for obtaining optimal activation of the immune system.

Background of invention

Vaccines have played, and still play, a major role in reducing the impact of infectious diseases on global health. The first generation of vaccines was based on attenuated or inactivated pathogens. These full-pathogen-based vaccines have proven extremely effective and, in some cases, have (e.g. small pox) led to the complete eradication of the target pathogen. There are however serious concerns associated with using full- pathogens for immunization as these have been seen to induce severe side effects at some frequency in populations, underscoring the need to develop safer vaccines (Plotkin SA et. al 2005). Along with the recent advances in recombinant DNA technology and genetic engineering, modern vaccine research has put effort into identifying critical antigenic targets of neutralizing antibodies with the aim of developing so called 'subunit vaccines' composed solely of well-defined, purified antigen components (Murray K. et al. 1988). The immunogenicity of subunit vaccines based on soluble protein is, unfortunately, low compared to that of full pathogen-based vaccines. To induce a high-titer antibody response it is thus often necessary to use high antigen doses, booster administrations, and co-administration of adjuvants and even so these subunit vaccines are generally not capable of inducing long-term protective immunity. This is indeed exemplified by the many vaccine failures observed with soluble proteins during the past several years and point to an important fact: that the size and the spatial assembly of the vaccine antigen component is critical for proper activation of the immune system, demonstrating the importance of qualitative immune responses beyond quantitative ones.

Virus-like particles (VLPs), which are both highly immunogenic and safe, represent a major advancement in the development of subunit vaccines, combining many of the advantages of full pathogen-based vaccines and recombinant subunit vaccines. VLPs are composed of one or several recombinantly expressed viral proteins which spontaneously assemble into macromolecular particulate structures mimicking the morphology of the native virus coat - but lacking infectious genetic material. The particulate nature and size of VLPs (22-150nm) appears to be optimal for efficient uptake by professional antigen presenting cells, particularly dendritic cells (DCs) as well as for entry into lymph vessels (Bachmann, MF, Jennings, GT. 2010).

Furthermore, surface structures presenting an antigen at high density, while being regularly spaced, and with consistent orientation are characteristic of microbial surface antigens for which the mammalian immune system has evolved to respond vigorously to. At the molecular level, the presentation of an epitope at high density, while being regularly spaced, and with consistent orientation enables efficient cross-linking of B-cell receptors (Bachmann, MF and Zinkernagel, RM. 1997) leading to strong B-cell responses, even in the absence of T-cell help (Bachmann, MF et al., 1993; Chackerian et al., 1999; Kouskoff, V. et al., 2000) and cumulative data from several studies indicate that B-cells, in fact, discriminate antigen patterns via the degree of surface Ig-cross- linking and use antigen repetitiveness as a self/nonself discriminator.

It has long been an attractive goal to exploit the VLPs as an immunogenicity-boosting platform for inducing immune responses against heterologous antigens by using them as molecular scaffolds for antigen presentation. Traditionally this has been achieved either by incorporation of antigenic epitopes into VLPs by genetic fusion (chimeric VLPs) or by conjugating antigens to preassembled VLPs. The chimeric VLP approach is to date the most common method for displaying heterologous epitopes on VLPs (Pumpens, P and Grens, E. 2001 ; Bachmann, MF and Jennings, GT, 2004a;

Chackerian, 2007; Grgacic, EVL. and Anderson, DA. 2006). However, this strategy is severely limited by both the size and nature of epitopes that can be inserted into VLPs, especially in their immunodominant regions, and it has in general not been possible to insert peptides longer than 20 amino acids without disrupting the fragile self-assembly process of the VLPs. In addition, this approach requires that critical epitopes have already been identified in the target antigen and that these can be presented in an immunodominant region on the VLP surface while maintaining its native conformation. Therefore, despite a still growing understanding of the VLP structure / assembly process, generating chimeric VLPs is still a trial-and-error process and it remains impossible to predict whether individual peptides will be compatible with VLP assembly or whether insertions will be immunogenic. Finally, due to the small size of inserted peptide sequences the induced antibody response will essentially be monoclonal, which in some cases will set a limit to the potency of protection.

On the other hand chemical conjugation e.g. through chemical biotinylation of exposed lysine residues allows the attachment of diverse kinds of target antigens (incl. nonprotein targets) to VLPs and this approach is generally not restricted by the size of the antigen (Raja KS. Et al. 2003 and others). However with this approach it is very challenging, if not impossible, to control the orientation and the total amount / stoichiometry of the coupled antigen, affecting both the density and regularity of displayed epitopes, and thus potentially limiting the immune response. In addition to this, chemical coupling procedures are rarely compatible with large scale vaccine production. As a result the current technologies are not sufficient to ensure VLP display of antigens at high density, while being regularly spaced, and with consistent orientation, which are three critical factors for obtaining strong and long lasting activation of the immune system.

• Induction of a strong and long lasting immune response to pathogens as well as disease associated antigens is very difficult to obtain with subunit vaccines.

• Virus like particle (VLP) presentation of antigens has proven to be very efficient in inducing the highly functional long-term immune responses.

• Coupling of an antigen onto the surface of a VLP, to ensure a high density display of regularly spaced epitopes, pose a major biotechnological challenge.

• Specifically, the main challenge of current VLP delivery platforms is to present an antigen on the surface of the VLP, at high density, and with a consistent orientation to enable a regular, high density, spacing of displayed epitopes, which is important for inducing long-term protective immunity. Summary of invention

The present invention solves the challenges of obtaining a VLP which presents densely and regularly spaced surface antigens with consistent orientation, capable of efficiently displaying epitopes and to induce long-term protective immunity in a subject. The general concept of the present invention is illustrated in figure 1. The present inventors have identified regions of the Papilloma Virus (PV) VLP where the inventors can insert a biotin acceptor site, without compromising the self-assembly of the particle. In addition, the inventors have managed to setup a system to produce antigens fused to a monovalent streptavidin, which ensures control of the orientation of the coupled antigen. Enzymatical biotinylation of the human papilloma virus (HPV) VLPs facilitate linkage to the monovalent streptavidin/antigen and ensure control of the overall amount/stoichiometry as well as display of antigens in a densely and repetitive ordered manner with consistent orientation which is important for yielding efficient epitope display and consequently a potent immune response. The described antigen display scaffold is unique as it for the first time enables coupling of virtually any antigen at high density on a VLP surface, thereby presenting ordered arrays of the particular antigens which are all held in the same orientation, thereby solving three key issues of mounting an efficient immune response. The system can both be used to target self-antigens (i.e. break tolerance) as well as to efficiently target infectious organisms.

The problems described above are solved by the aspects and embodiments of the present invention characterized in the claims. As illustrated in figure 1 , a main aspect of the present invention concerns a vaccine for use in the prophylaxis and/or treatment of a disease wherein the vaccine comprises:

i. a papilloma virus (PV) L1 protein containing a biotin acceptor site, and ii. a biotin molecule enzymatically conjugated to said biotin acceptor site, and

iii. an antigen fused to a monovalent streptavidin,

wherein the antigen and PV L1 protein are linked via the interaction between the monovalent streptavidin and the biotin molecule enzymatically conjugated to the biotin acceptor site of the PV L1 protein, and wherein i - iii form a virus like particle displaying said antigen.

In another aspect the present invention concerns a vector comprising at least one polynucleotide encoding

i. a PV L1 protein containing a biotin acceptor site, and

ii. a biotin ligase capable of biotinylating the biotin acceptor site, and iii. an antigen fused to a monovalent streptavidin as illustrated on figure 1

In another aspect the present invention concerns a host cell expressing at least one polypeptide encoded by said polynucleotide. In another aspect the present invention concerns a composition comprising said

A further aspect of the present invention concerns a method of manufacturing a pharmaceutical composition comprising said vaccine, wherein the method comprises the steps of

i. obtaining a first polypeptide; a PV L1 protein containing a biotin acceptor site, and

ii. obtaining a second polypeptide; a biotin ligase, capable of biotinylating the biotin acceptor site, and

iii. obtaining a third polypeptide; an antigen fused to a monovalent

streptavidin, and

iv. subjecting the first polypeptide to conditions which enable formation of virus like particles, and

v. enable enzymatic biotinylating of the biotin acceptor site of said virus like particles using said second polypeptide, and

vi. obtaining a vaccine by linkage of the third polypeptide and said virus like particles via the interaction between the monovalent streptavidin and the biotin molecule enzymatically conjugated to the biotin acceptor site of said virus like particles, and

vii. generating a composition comprising said vaccine,

thereby obtaining a pharmaceutical composition. Yet an aspect of the present invention concerns a method of administering said vaccine to treat and/or prevent a clinical condition in a subject in need thereof comprising the steps of:

i. obtaining a composition comprising at least one vaccine, and/or ii. administering said composition to a subject at least once for prophylaxis and/or treatment of a disease.

In another aspect the present invention concerns a kit of parts comprising

i. a composition comprising a vaccine, and

ii. a medical instrument or other means for administering the vaccine, and iii. instructions on how to use the kit of parts. An aspect of the invention relates to a method for inducing an immune response in a subject, the method comprising the steps of

i. obtaining a composition comprising at least one vaccine, and ii. administering said composition to a subject at least once for prophylaxis and/or treatment of a disease.

Description of Drawings

Figure 1. Schematic representation of the VAR2CSA VLP-vaccine components and the assembly system.

a. Structure of HPV16 L1 major capsid protein. The biotin acceptor sequence

(AviTag™) was successfully inserted into the protruding DE and HI loops and the H4- coil (blue boxes) of the HPV16 L1. Gray boxes represent regions (loops and coils) of the L1 protein where the AviTag™ could not be inserted (FG loop) or has not been investigated. The VAR2CSA antigen encompasses the extracellular ID1-ID2a (ID1 - Interdomain 1 , DBL2X - Duffy binding like 2X and ID2a - Interdomain 2a) domains of the full-length VAR2CSA fused at the N-terminus to a previously described monovalent streptavidin (mSA) (Lim et al., 2011). b. Production process of HPV16 Avi-L1 VLPs displaying consistently oriented mSA-VAR2 antigens in a high-density repetitive manner. The HPV16 Avi-L1 and the mSA-vaccine antigen are separately expressed. The HPV16 Avi-VLPs are subsequently in vitro biotinylated (the triangle represents BirA ligase) and finally mixed with the soluble mSA-vaccine antigen. The HPV16 Avi-L1 VLP is theoretically expected to bind one mSA- VAR2CSA antigen per Avi-L1.

Figure 2: Transmission electron microscopy of HPV AviTag-L1 virus-like particles. The TEM pictures shows non-aggregated VLPs (size range: 28-60nm) assembled from biotinylated HPV16 L1-Avi (SEQ ID NO. 1-3) constructs, respectively. Scale bar = 200nm.

Figure 3: VLP / mSA-antigen coupling efficiency. The figure shows SDS-PAGE gels (reduced conditions) with relative amounts of coupled mSA-antigens (mSA-ID1 ID2a- HIS SEQ ID NO. 21 ; mSA-IL-5(C63T/C105T) SEQ ID NO. 19; HIS-GMZ2T:ggsmSA

SEQ ID NO. 25; mSA- Her2-ECD|23-686 SEQ ID NO. 18) and AviTag-L1 protein (SEQ ID NO. 2). There is an estimated 0.8-1.0 mSA-antigen per AviTag-L1 protein in the VLP containing fractions.

Figure 4: Transmission electron microscopy of HPV AviTag-L1 virus-like particles coupled with different mSA-antigens (mSA-ID1 ID2a-HIS SEQ ID NO. 21 ; mSA-IL- 5(C63T/C105T) SEQ ID NO. 19; HIS-GMZ2T:ggsmSA SEQ ID NO. 25; mSA- Her2- ECD|23-686 SEQ ID NO. 18) and AviTag-L1 protein (SEQ ID NO. 2). The TEM pictures shows non-aggregated VLPs assembled from biotinylated HPV16 L1-Avi (SEQ ID NO. 2) constructs, respectively. The apparent average size of the mSA-antigen coupled VLPs seems (-5-1 Onm) larger than corresponding non-coupled VLPs. Scale bar = 200nm.

Figure 5. Verification of self-assembly and subsequent in vitro biotinylation of HPV16 Avi-L1 VLPs

a. Purification of HPV16 Avi-L1 VLPs (HI) VLPs was performed by ultracentrifugation (UC) on an iodixanol (Optiprep™) density-gradient (27%/33%/39%). Subsequent reduced SDS-PAGE analyses showed the presence of a 56kDa protein band

(theoretical size of Avi-L1) in the high-density UC fractions [4-6] containing particulate material, b. Transmission-electron microscopy (TEM) analysis of material representing UC fraction 4 post UC purification. To verify the integrity of the chimeric HPV16 Avi-L1 (HI) VLPs, an aliquot of diluted particles was placed on carbon-coated grids, negatively stained with 2% phosphotungstic acid (pH=7.0) and examined by transmission electron microscopy (TEM) using a CM 100 BioTWIN at magnification x 36,000 (A), scale bar 80 nm. c. Western blot analysis of fraction 4 post UC purification. The blot

demonstrates the presence of HPV16 Avi-L1 (56kDa) detected by Camvir-1 (lane one) and successful biotinylation of HPV16 Avi-L1 using Strep-HRP to detect biotin (lane two).

Figure 6. HPV16 Avi-L1 VLP coupled to mSA-ID1 -ID2a analyzed by

ultracentrifugation followed by SDS-PAGE, Western blot and TEM analysis a After coupling of the mSA-ID1-ID2a antigen to the HPV16 Avi-L1 VLPs, excess antigen was removed by UC over an Optiprep™ gradient (27%/33%/39%). Reducing SDS-PAGE analysis showed the presence of two protein bands corresponding to the size of HPV16 Avi-L1 (56kDa) and mSA-VAR2CSA (85kDa), respectively, in the high- density fractions [4-6] post UC purification. Excess unbound mSA-VAR2CSA was present in the higher UC fractions [12-14] containing soluble proteins, b Transmission electron microscopy (TEM) analysis of material from UC fraction 4 containing HPV16 Avi-L1 VLPs coupled with mSA-VAR2CSA. An aliquot of diluted particles was placed on carbon-coated grids, negatively stained with 2% phosphotungstic acid (pH=7.0) and examined by transmission electron microscopy (TEM) using a CM 100 BioTWIN at magnification x 36,000 (A). Black scale bar 200 nm, enhanced section white scale bar 40 nm. c Western blot analysis of fraction 4 post UC purification of mixed HPV16 Avi- L1 and mSA-VAR2CSA. The blot confirms the presence of HPV16 Avi-L1 and mSA- VAR2CSA detected by Camvir-1 and a-PENTA HIS-tag, respectively.

Figure 7. Other PV VLPs with AviTag™ inserted in DE-loop.

The HPV16 L1 VLP was used as VLP platform for proof of concept in this study.

However, the AviTag™ can also be inserted into the DE loop of the major capsid protein from other papilloma viruses while retaining the ability to self-assemble into VLPs, as demonstrated in this figure, a Multiple sequence alignment of the HPV16 L1 , HPV1 18 L1 and major capsid protein from European Elk (Alces alces) papilloma virus (PAPVE). b Purification of HPV1 18 Avi-L1 and PAPVE Avi-L1 VLPs were performed by UC over an Optiprep™ density gradient (27%/33%/39). Subsequent reduced SDS-

PAGE analysis of high-density UC fractions [3-5] show the presence of a protein band of 56 kDa corresponding to the full-length Avi-L1 protein. These fractions also contain an intense protein band of approximately 43kDa, which may represent a truncated Avi- L1 product.

Figure 8. Reactivity of sera from vaccinated mice by ELISA.

C57BL/6 mice were immunized three times with three-week intervals. Serum samples were collected two weeks after each immunization. Total VAR2CSA-specific immunoglobulin was measured in a serial dilution of mouse anti-sera by ELISA using recombinant VAR2CSA as the solid phase capturing antigen. HRP conjugated anti- mouse Ig antibodies were used for detection by measuring absorbance at OD 490 nm. Serum reactivity from individual mice vaccinated with mSA-VAR2CSA coupled HPV16 Avi-L1 (HI) VLPs (blue) or uncoupled mSA-VAR2CSA (red) are shown after first (a), second (b) and third (c) immunization where each line shows the reactivity of one animal. Green curves represent sera from mice vaccinated with soluble naked

VAR2CSA and is a pool of sera obtained after 2 ^nd and 3 ^rd bleed.

Figure 9. Display of VAR2CSA on HPV16 L1 -AviTag VLPs assessed by parasite inhibition assay.

The functional antibody response was assessed by measuring the capacity of mouse anti-sera to inhibit binding between native VAR2CSA expressed on parasitized erythrocytes and CSA in a static binding-assay. P. falciparum (FCR3 genotype)- infected red blood cells, expressing the native VAR2CSA, were first incubated with mouse anti-serum (4 fold dilution series, starting from 1 :50) and then allowed to incubate on decorin coated plates for 90 min. Unbound IE were washed away and the remaining lEs were quantified. Normalized parasite binding after incubation with pooled anti-sera from mice (n=5) vaccinated with mSA-VAR2CSA-coupled HPV16 Avi-L1 VLPs (blue) or soluble mSA-VAR2CSA (red) are shown after first (a), second (b) and third (c) immunization. The green piles in Figure 6c represent anti-sera from mice vaccinated with soluble naked VAR2CSA and is a pool of sera from 2 ^nd and 3 ^rd bleed. Figure 10. Control experiment where unbiotinylated VLPs were mixed with mSA- VAR2CSA. SDS PAGE shows that ultracentrifugation efficiently separated soluble mSA-VAR2CSA from unbiotinylated VLPs.

Figure 11. Immuno-tolerance to a cancer-associated self-antigen "Survivin". a. All the survivin-VLP (mSA-Survivin coupled to HPV Avi-L1 (HI-LOOP)) immunized mice (n=3) induced high-titre antibody responses against the mSA-survivin self-antigen, whereas the negative control vaccine was not able to break immune-tolerance in the immunized mice (no sero-conversion). b. The same anti-sera in ELISA using the mSA- survivin as the solid phase capturing antigen, showing that the negative control mice did produce antibodies against the 'foreign' mSA part. Detailed description of the invention

The present invention solves the challenge of conjugating larger proteins (e.g. full length antigens) at high density and in a consistent orientation onto the surface of a VLP, thereby obtaining VLPs presenting densely and repetitive arrays of heterologous epitopes. The solution of the present invention represents a novel approach for making a versatile VLP-based vaccine delivery platform capable of efficiently displaying antigen epitopes and of inducing long-term protective immunity.

A general aspect of the present invention is illustrated in figure 1 b. Definitions

The term "PV" refers to papillomavirus.

The term "L1 protein" refers to the major capsid L1 protein from papillomavirus, which has the ability to spontaneously self-assemble into virus-like particles (VLPs). The L1 proteins of the present invention are displaying antigens.

The term "virus like particle" or "VLP" refers to one or several recombinantly expressed viral proteins which spontaneously assemble into macromolecular particulate structures mimicking the morphology of a virus coat, but lacking infectious genetic material. The term "self-assembly" refers to a process in which a system of pre-existing components, under specific conditions, adopts a more organised structure through interactions between the components themselves. In the present context, self- assembly refers to the intrinsic capacity of L1 , the major capsid protein of

papillomavirus to self-assemble into virus-like particles in the absence of L2 or other papillomavirus proteins, when subjected to specific conditions. The self-assembly process may be sensitive and fragile and may be influenced by factors such as, but not limited to, choice of expression host, choice of expression conditions, and conditions for maturing the virus-like particles.

The term "consistent orientation", as used herein, refers to the orientation of the monomeric streptavidin/antigen constructs of the present invention and their spatial orientation to the major capsid protein of papillomavirus (PV L1) of the present invention. When linking an antigen fused to a monomeric streptavidin to a PV L1 protein VLP comprising a biotin acceptor site with a biotin molecule enzymatically conjugated to the biotin acceptor site, the monomeric streptavidin can only be linked to a single PV L1 protein, thus creating a uniform and/or consistent presentation of said antigen with a consistent orientation. In contrast a streptavidin homo-tetramer may crosslink several PV L1 proteins on the surface of a VLP, thus creating an irregular and non-consistent orientation of said antigen. Besides, it is highly challenging to use a homo-tetramer streptavidin as a bridging molecule e.g. for conjugating biotinylated antigens onto biotinylated VLPs, since the multiple biotin binding sites will allow cross- linking and aggregation of the biotinylated VLPs. The term "regularly spaced" as used herein, refers to antigens of the present invention which forms a pattern on the surface of a VLP. Such pattern may be symmetric, circlelike, and/or bouquet like pattern of antigens.

The term "treatment" refers to the remediation of a health problem. Treatment may also be preventive and/or prophylactic or reduce the risk of the occurrence of a disease and/or infection. Treatment may also be curative or ameliorate a disease and/or infection. The term "prophylaxis" refers to the reduction of risk of the occurrence of a disease and/or infection. Prophylaxis may also refer to the prevention of the occurrence of a disease and/or infection. The term "loop" refers to a secondary structure of a polypeptide where the polypeptide chain reverses its overall direction and may also be referred to as a turn.

The term "vaccine cocktail" refers to a mixture of antigens administered together. A vaccine cocktail may be administered as a single dose or as several doses

administered over a period of time. Time intervals may be, but not limited to

administration within the same year, month, week, day, hour and/or minute. Co- vaccination and vaccine cocktail may be used interchangeably.

The term "self-antigens" refers to endogenous antigens that have been generated within previously normal cells as a result of normal cell metabolism, or because of viral or intracellular bacterial infection.

The term "biotin acceptor site" refers to a peptide sequence capable of binding a biotin molecule. Biotin acceptor site and AviTag are interchangeably used herein.

The term "sequence variant" refers to a polypeptide and/or polynucleotide sequence with at least 70%, such as 75%, such as 80%, such as 85%, such as 90%, such as 95%, such as 96%, such as, 97%, such as 98%, such as 99%, such as 99,5%, such as 100% sequence identity to said polypeptide and/or polynucleotide sequence.

VLP based vaccine

The expression of viral structural proteins, such as Envelope or Capsid proteins, can result in the self-assembly of virus-like particles (VLPs). VLPs resemble viruses, but are non-infectious as they do not contain any viral genetic material. For the purpose of active immunization VLPs have proven highly immunogenic and provide a safer alternative to attenuated viruses since they lack genetic material. Besides, VLPs are a useful tool for the development of vaccines and can be used as molecular scaffolds for efficient antigen epitope display. This has been achieved by either genetic insertion or by chemical conjugation approaches. However, it has generally not been possible to incorporate peptides longer than 20 amino acids without disrupting the self-assembly process of the chimeric VLP. At the same time the current technologies using chemical conjugation are not sufficient to enable VLP-presentation of larger proteins at high density and with a consistent orientation to ensure an orderly, high density, display of repetitive antigen epitopes, which are critical factors for obtaining strong and long- lasting immune responses.

The present inventors have solved these problems by a novel approach to linking antigens to a PV L1 VLP using a biotin acceptor site, a biotin molecule and a monovalent streptavidin tag without disrupting the self-assembly of the VLP. Thus in a main aspect, as illustrated in figure 1 b, the present invention concerns a vaccine for use in the prophylaxis and/or treatment of a disease wherein the vaccine comprises:

i. a papilloma virus (PV) L1 protein containing a biotin acceptor site, and ii. a biotin molecule enzymatically conjugated to said biotin acceptor site, and

iii. an antigen fused to a monovalent streptavidin,

wherein the antigen and PV L1 protein are linked via the interaction between the monovalent streptavidin and the biotin molecule enzymatically conjugated to the biotin acceptor site of the PV L1 protein, and wherein i - iii form a virus like particle displaying said antigen.

In an embodiment the PV L1 protein containing a biotin acceptor site is able to form a virus like particle.

The inventors of the present invention have demonstrated formation of VLP's using insect cells, such as High Five of Sf9 cells incubated at 28°C. In vitro maturation may occur for 18 - 24 hours at 30°C - 37°C. Other conditions and expression hosts (such as Pichia Pastoris) may work as well.

In an embodiment the antigen is capable of eliciting an immune reaction in an animal, such as a mammal, such as a cow, pig, horse, sheep, goat, llama, mouse, rat, monkey, most preferably such as a human being; or bird such as a chicken, or fish such as a Salmon.

It has long been an attractive goal to exploit the VLPs as an immunogenicity-boosting platform for inducing immune responses against heterologous antigens by using them as molecular scaffolds for antigen display. Thus another aspect of the present invention relates to an antigen display scaffold, comprising an assembled virus-like particle comprising:

i. a papilloma virus (PV) L1 protein containing a biotin acceptor site, and ii. a biotin molecule enzymatically conjugated to said biotin acceptor site, and

iii. an antigen fused to a monovalent streptavidin,

wherein the antigen and PV L1 protein are linked via the interaction between the monovalent streptavidin and the biotin molecule enzymatically conjugated to the biotin acceptor site of the PV L1 protein, and wherein i - iii form an antigen display scaffold.

Another aspect of the present invention relates to a method of producing a non- naturally occurring, ordered and repetitive antigen array comprising

i. a papilloma virus (PV) L1 protein containing a biotin acceptor site, and ii. a biotin molecule enzymatically conjugated to said biotin acceptor site, and

iii. an antigen fused to a monovalent streptavidin,

wherein the antigen and PV L1 protein are linked via the interaction between the monovalent streptavidin and the biotin molecule enzymatically conjugated to the biotin acceptor site of the PV L1 protein, and wherein i - iii form a non-naturally occurring, ordered and repetitive antigen array.

Diseases and medical indications

The present invention is a novel, generic, and easy-to-use-approach to conjugate various antigens to a VLP. Depending on the antigen the VLP-based vaccines of the present invention can be used for prophylaxis and/or treatment of a wide range of diseases. The diseases which the present invention may be used for prophylaxis and/or treatment of include but are not limited to cancers, cardiovascular diseases, allergic diseases, and/or infectious diseases.

In an embodiment an antigen which is associated with at least one cancer disease is linked to the PV L1 protein via the interaction between the monovalent streptavidin and the biotin molecule enzymatically conjugated to the biotin acceptor site of the PV L1 protein. In a further embodiment the present VLP vaccine may be used for prophylaxis and/or treatment of the cancer and/or cancers which the antigen is associated with. In an embodiment an antigen which is associated with at least one cardiovascular disease is linked to the PV L1 protein via the interaction between the monovalent streptavidin and the biotin molecule enzymatically conjugated to the biotin acceptor site of the PV L1 protein. In a further embodiment the present VLP vaccine can be used for prophylaxis and/or treatment of the cardiovascular disease and/or cardiovascular diseases which the antigen is associated with.

In an embodiment an antigen which is associated with at least one allergic disease is linked to the PV L1 protein via the interaction between the monovalent streptavidin and the biotin molecule enzymatically conjugated to the biotin acceptor site of the PV L1 protein. In a further embodiment the present VLP vaccine can be used for prophylaxis and/or treatment of the allergic disease and/or allergic diseases which the antigen is associated with.

In an embodiment an antigen which is associated with at least one infectious disease is linked to the PV L1 protein via the interaction between the monovalent streptavidin and the biotin molecule enzymatically conjugated to the biotin acceptor site of the PV L1 protein. In a further embodiment the present VLP vaccine can be used for prophylaxis and/or treatment of the infectious disease and/or infectious diseases which the antigen is associated with.

A non-exhaustive list of antigens which may be used by the present invention is outlined in table 1 and table 2. In addition table 1 show examples of specific diseases the antigens are associated with as well as examples of patient groups which may be in need of prophylaxis and/or treatment using the antigen-VLP vaccines of the present invention.

Table 1 Non-exhaustive list of antigens or parts hereof that could be used in treatment of specific diseases/medical indications in various patient groups.

Examples of Examples of a specific

antigens (non- disease (non- Examples of patient group (non- exhaustive) exhaustive) exhaustive)

Her2/Neu

(ERBB2) Breast cancer Females overexpressing Her2 Her2/Neu Males and Females overexpressing (ERBB2) Gastric cancer Her2

Her2/Neu

(ERBB2) Ovarian cancer Females overexpressing Her2

Her2/Neu Uterine serous Postmenopausal Females

(ERBB2) carcinoma overexpressing Her2

Cancer types Males and non-pregnant Females

Survivin overexpressing Survivin overexpressing Survivin

PCSK9 cardiovascular disease Males and Females with dyslipidemia

PCSK9 cardiovascular disease Males and Females with atherosclerosis

Males and Females with

PCSK9 cardiovascular disease hypercholesterolemia

lnterleukin-5 Asthma Males and Females with eosinophilia lnterleukin-5 nasal polyposis Males and Females with eosinophilia lnterleukin-5 atopic dermatitis Males and Females with eosinophilia lnterleukin-5 eosinophilic esophagitis Males and Females with eosinophilia

Hypereosinophilic

lnterleukin-5 syndrome Males and Females with eosinophilia

Churg-Strauss

lnterleukin-5 syndrome Males and Females with eosinophilia

Ag85A Tuberculosis Males and Females with tuberculosis

PfRH5 Malaria Males and Females with malaria

VAR2CSA Malaria Females with malaria

PfEMPI , CIDRI a Malaria Males and Females with malaria

GLURP Malaria Males and Females with malaria

MSP3 Malaria Males and Females with malaria

Pfs25 Malaria Males and Females with malaria

CSP Malaria Males and Females with malaria

PfSEA-1 Malaria Males and Females with malaria

The disclosed antigens may as well be relevant for the use in other patient groups and/or against other specific or related diseases. In an embodiment at least two such as three, four, and/or five antigens may be combined. Table 2: Non-exhaustive list of diseases/medical indications and target antigen/organisms of the present VLP vaccine.

Disease: Target antigen/ Organism:

Cancer: Her2/Neu (ERBB2) / Homo Sapiens

Survivin (Baculoviral IAP repeat-containing protein 5) /

Homo Sapiens

Cardiovascular disease: PCSK9 (Proprotein convertase subtilisin/kexin type 9) /

Homo Sapiens

Asthma / Allergies: IL-5 (lnterleukin-5) / Homo Sapiens

Tuberculosis: Ag85A (Diacylglycerol cyltransferase/mycolyltransferase) /

Mycobacterium tuberculosis

Malaria: Reticulocyte-binding protein homologue 5 (PfRH5) /

Plasmodium falciparum

VAR2CSA (domain, ID1-ID2a) / Plasmodium falciparum CIDRI a domain of PfEMPI , Plasmodium falciparum Glutamate rich protein (GLURP) / Plasmodium falciparum

Merozoite surface protein 3 (MSP3) / Plasmodium falciparum

25 kDa ookinete surface antigen (Pfs25) / Plasmodium falciparum

Circumsporozoite protein (CSP) / Plasmodium falciparum

Schizont egress antigen-1 (PfSEA-1) / Plasmodium falciparum

The vaccine of the present invention may as well be used against other diseases and/or use other antigens.

In an embodiment of the present invention the medical indication is selected from the group consisting of a cancer, a cardiovascular disease, an allergy, and/or an infectious disease. In a particular embodiment the medical indication is an allergy. In another particular embodiment the medical indication is a cardiovascular disease. In a most preferred embodiment the medical indication is a cancer.

In another embodiment the antigen is a polypeptide, peptide and/or an antigenic fragment of a polypeptide associated with an abnormal physiological response such as a cardiovascular disease and/or an allergic reaction/disease. In a particular

embodiment the abnormal physiological response is a cancer.

In a further embodiment the antigen is a protein, peptide and/or an antigenic fragment associated with a medical indication disclosed in the present invention.

Cancer and associated antigens

In 2012 more than 14 million adults were diagnosed with cancer and there were more than 8 million deaths from cancer, globally. Consequently, there is a need for efficient cancer therapeutics.

One characteristic of cancer cells is abnormal expression levels of genes and proteins. One example of a cancer associated gene is HER2, which is overexpressed in 20% of all breast cancers and is associated with increased metastatic potential and poor patient survival. Although cancer cells express cancer associated antigens in a way that immunologically distinguishes them from normal cells, most cancer associated antigens are only weakly immunogenic because most cancer associated antigens are "self" proteins which are generally tolerated by the host. The present invention has solved this problem by an effective antigen-VLP based vaccine which is capable of activating the immune system to react against for example cancer associated antigens and overcome the immunological tolerance to such antigens. Different cancers are characterized by having different cancer associated antigens. Survivin is regarded to be overexpressed in most cancer cells and could also be used in the present invention. Therefore the present invention may be used in treatment/prophylaxis of most types of cancers that overexpress a tumor associated antigen.

The antigen is linked to the PV L1 protein of the present invention via the interaction between the monovalent streptavidin and the biotin molecule enzymatically conjugated to the biotin acceptor site of the PV L1 protein (see figure 1 b for the general concept of the present invention). Thereby the present invention provides effective antigen-VLP based vaccine which is capable of activating the immune system to react against for example cancer associated antigens and overcome immunological tolerance to such antigens. In an embodiment the VLP vaccine of the present invention can be used for prophylaxis and/or treatment of the cancer which the antigen is associated with. An embodiment of the present invention comprises a cancer associated antigen linked to the PV L1 protein via the interaction between the monovalent streptavidin and the biotin molecule enzymatically conjugated to the biotin acceptor site of the PV L1 protein. In a further embodiment the present VLP vaccine can be used for prophylaxis and/or treatment of the cancer which the antigen is associated with.

In another embodiment the present invention is used in treatment/prophylaxis of any type of cancer which overexpresses an antigen. The type of cancer which the invention may be used against is determined by the choice of antigen. It is known that oncovirus can cause cancer. Therefore in an embodiment the vaccine of the present invention comprises an oncovirus associated antigen linked to the PV L1 protein via the interaction between the monovalent streptavidin and the biotin molecule enzymatically conjugated to the biotin acceptor site of the PV L1 protein. In a further embodiment the present vaccine can be used for prophylaxis and/or treatment of the cancer which the antigen is associated with.

In an embodiment the antigen is a protein or peptide or an antigenic fragment of a polypeptide associated with a cancer selected from the group comprising of Adrenal Cancer, Anal Cancer, Bile Duct Cancer, Bladder Cancer, Bone Cancer, Brain/CNS Tumors in adults, Brain/CNS Tumors In Children, Breast Cancer, Breast Cancer In Men, Cancer in Adolescents, Cancer in Children, Cancer in Young Adults, Cancer of Unknown Primary, Castleman Disease, Cervical Cancer, Colon/Rectum Cancer, Endometrial Cancer, Esophagus Cancer, Ewing Family Of Tumors, Eye Cancer, Gallbladder Cancer, Gastrointestinal Carcinoid Tumors, Gastrointestinal Stromal

Tumor, Gestational Trophoblastic Disease, Hodgkin Disease, Kaposi Sarcoma, Kidney Cancer, Laryngeal and Hypopharyngeal Cancer, Leukemia, Acute Lymphocytic in Adults, Leukemia, Acute Myeloid Leukemia, Chronic Lymphocytic Leukemia, Chronic Myeloid Leukemia, Chronic Myelomonocytic Leukemia, Leukemia in Children, Liver Cancer, Lung Cancer, Non-Small Cell Lung Cancer, Small Cell Lung Cancer, Lung Carcinoid Tumor, Lymphoma, Lymphoma of the Skin, Malignant Mesothelioma, Multiple Myeloma, Myelodysplasia Syndrome, Nasal Cavity and Paranasal Sinus Cancer, Nasopharyngeal Cancer, Neuroblastoma, Non-Hodgkin Lymphoma, Non- Hodgkin Lymphoma In Children, Oral Cavity and Oropharyngeal Cancer,

Osteosarcoma, Ovarian Cancer, Pancreatic Cancer, Penile Cancer, Pituitary Tumors, Prostate Cancer, Retinoblastoma, Rhabdomyosarcoma, Salivary Gland Cancer, Adult Soft Tissue Cancer Sarcoma, Skin Cancer, Basal and Squamous Cell Skin Cancer, Melanoma Skin Cancer, Merkel Cell Skin cancer, Small Intestine Cancer, Stomach Cancer, Testicular Cancer, Thymus Cancer, Thyroid Cancer, Uterine

Sarcoma, Vaginal Cancer, Vulvar Cancer, Waldenstrom Macroglobulinemia, and Wilms Tumor.

In a preferred embodiment the cancer is selected from the group consisting of breast cancer, gastric cancer, ovarian cancer, and uterine serous carcinoma.

Linking the Her2/Neu (ERBB2) and/or Survivin or an antigenic fragment hereof to the VLP forms a VLP based vaccine which is capable of activating the immune system to react against for example cells with high Her2/Neu (ERBB2) and/or Survivin expression and overcome immunological tolerance. In an embodiment the Her2/Neu (ERBB2) and/or Survivin VLP vaccine of the present invention can be used for prophylaxis and/or treatment of the herein disclosed cancer disease and/or other cancer diseases. Using a similar reasoning other cancer disease associated antigen-VLP based vaccines may be used against any cancer disease. In an embodiment the antigen of the present invention is Her2/Neu (ERBB2) and/or Survivin or an antigenic fragment hereof, wherein the antigen is associated with and directed against at least one of the herein disclosed types of cancers.

In a most preferred embodiment the present invention concerns a vaccine for use in the prophylaxis and/or treatment of one of the herein disclosed cancers wherein the vaccine comprises:

i. a papilloma virus (PV) L1 protein containing a biotin acceptor site, and

ii. a biotin molecule enzymatically conjugated to said biotin

acceptor site, and iii. a cancer associated antigen such as Her2/Neu (ERBB2) and/or Survivin or an antigenic fragment of Her2/Neu (ERBB2) and/or Survivin fused to a monovalent streptavidin, wherein the antigen and the PV L1 protein are linked via the interaction between the monovalent streptavidin and the biotin molecule enzymatically conjugated to the biotin acceptor site of the PV L1 protein, and wherein i - iii form a virus like particle displaying said antigen.

In an embodiment the antigen fused to a monovalent streptavidin is selected from the group comprising SEQ ID NO: 18, SEQ ID NO: 40, SEQ ID NO: 41 , SEQ ID NO: 42,

SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, and/or SEQ ID NO: 47 and/or a sequence variant hereof.

Cardiovascular diseases and associated antigens

An estimated 17.3 million people died from cardiovascular diseases in 2008, representing 30% of all global deaths. Addressing risk factors such as tobacco use, unhealthy diet and obesity, physical inactivity, high blood pressure, diabetes and raised lipids are important for prevention of cardiovascular diseases. However, the need for preventive pharmaceutical measures is increasingly important. The present invention may be used in treatment/prophylaxis of most types of cardiovascular diseases. The type of cardiovascular disease which the invention may be used against is decided by the choice of antigen.

In an embodiment of the invention the antigen is a protein or peptide or an antigenic fragment of a polypeptide associated with a disease selected from the group comprising a lipid disorder such as hyperlipidemia, type I, type II, type III, type IV, or type V hyperlipidemia, secondary hypertriglyceridemia, hypercholesterolemia, familial hypercholesterolemia, xanthomatosis, cholesterol acetyltransferase deficiency, an ateriosclerotic condition (e.g., atherosclerosis), a coronary artery disease, a cardiovascular disease, and Alzheimer's disease.

In an embodiment of the invention the antigen is a protein or peptide or an antigenic fragment of a polypeptide associated with a cardiovascular disease. In a further embodiment the cardiovascular disease is selected from the group consisting of dyslipidemia, atherosclerosis, and hypercholesterolemia. One example of a polypeptide associated with a cardiovascular disease is PCSK9 which acts in cholesterol homeostasis. Blockage of PCSK9 has medical significance and can lower the plasma and/or serum low-density lipoprotein cholesterol (LDL-C) levels. Reducing LDL-C reduces the risk of for example heart attacks.

Linking the PCSK9 antigen to the VLP forms a PCSK9-VLP based vaccine which is capable of activating the immune system to react against for example cells with high PCSK9 expression and overcome immunological PCSK9 tolerance, thereby lowering the LDL-C levels and the risk of heart attacks. In an embodiment the PCSK9-VLP vaccine of the present invention can be used for prophylaxis and/or treatment of the herein disclosed cardiovascular disease and/or other cardiovascular diseases. Using a similar reasoning other cardiovascular disease associated antigen-VLP based vaccines may be used against any cardiovascular disease.

In a preferred embodiment the antigen comprises PCSK9 or an antigenic fragment hereof, wherein the antigen is associated with and directed against at least one of the herein disclosed cardiovascular disease and/or other cardiovascular diseases. In a most preferred embodiment the present invention concerns a vaccine for use in the prophylaxis and/or treatment of at least one of the herein disclosed cardiovascular diseases wherein the vaccine comprises:

i. a papilloma virus (PV) L1 protein containing a biotin acceptor site, and

ii. a biotin molecule enzymatically conjugated to said biotin

acceptor site, and

iii. a cardiovascular disease associated antigen such as PCSK9 or an antigenic fragment hereof fused to a monovalent streptavidin, wherein the antigen and the PV L1 protein are linked via the interaction between the monovalent streptavidin and the biotin molecule enzymatically conjugated to the biotin acceptor site of the PV L1 protein, and wherein i - iii form a virus like particle displaying said antigen.

In an embodiment the antigen fused to a monovalent streptavidin comprise SEQ ID NO: 20 and/or a sequence variant hereof. Asthma or allergy diseases and associated antigens

The prevalence of allergic diseases worldwide is rising dramatically in both developed and developing countries. According to World Health Organization statistics, hundreds of millions of subjects in the world suffer from allergic rhinitis and it is estimated that 300 million have asthma markedly affecting the quality of life of these individuals and negatively impacting the socio-economic welfare of society.

Interleukin 5 (IL-5) has been shown to play an instrumental role in eosinophilic inflammation in various types of allergies, including severe eosinophilic asthma.

Eosinophils are regulated in terms of their recruitment, activation, growth, differentiation and survival by IL-5 which, consequently, has identified this cytokine as a primary target for therapeutic interventions. Linking an IL-5 antigen or a fragment hereof to the VLP of the present invention forms an IL-5-VLP based vaccine which is capable of activating the immune system to react against IL-5. Consequently an IL-5-VLP based vaccine described in the present invention may be used in the treatment/prophylaxis of eosinophilic asthma or allergy. Other allergy-associated antigens (e.g. IgE) may be used by the present invention using a similar reasoning. The type of asthma or allergy disease which the invention may be used against is decided by the choice of antigen. In an embodiment the antigen is a protein or peptide or an antigenic fragment of a polypeptide associated with one or more asthma or allergy diseases disclosed herein. In a preferred embodiment the asthma or allergy is selected from the group consisting of eosinophilic asthma, allergy, nasal polyposis, atopic dermatitis, eosinophilic esophagitis, hypereosinophilic syndrome, and Churg-Strauss syndrome.

In a preferred embodiment the antigen comprises IL-5 or an antigenic fragment hereof, wherein the antigen is associated with and directed against at least one of the herein disclosed asthma or allergy diseases and/or other asthma or allergy diseases.

In a most preferred embodiment the present invention concerns a vaccine for use in the prophylaxis and/or treatment of one of the herein disclosed asthma or allergy diseases wherein the vaccine comprises:

i. a papilloma virus (PV) L1 protein containing a biotin acceptor site, and

ii. a biotin molecule enzymatically conjugated to said biotin

acceptor site, and iii. an allergy associated antigen such as IL-5 or an antigenic fragment hereof fused to a monovalent streptavidin,

wherein the antigen and the PV L1 protein are linked via the interaction between the monovalent streptavidin and the biotin molecule enzymatically conjugated to the biotin acceptor site of the PV L1 protein, and wherein i - iii form a virus like particle displaying said antigen.

In an embodiment the antigen fused to a monovalent streptavidin comprises SEQ ID NO: 19 and/or a sequence variant hereof.

Infectious diseases and associated antigens

Tuberculosis and malaria are two major infectious diseases. In 2012, an estimated 207 million cases of malaria occurred which resulted in more than 500.000 deaths. Also in 2012, an estimated 8.6 million people developed tuberculosis and 1.3 million died from the disease. The current methods of treatment are insufficient and some have resulted in drug resistance. Consequently there is a need for new and efficient drugs for treatment/prophylaxis of tuberculosis and malaria. Linking a malaria or tuberculosis associated-antigen or a fragment hereof to the VLP of the present invention forms a VLP based vaccine which is capable of activating the immune system to react against for example malaria or tuberculosis. Using a similar line of reasoning the present invention may be used in treatment/prophylaxis of most infectious disease. The type of infectious disease which the invention may be used against is decided by the choice of antigen. In an embodiment the antigen fused to the monovalent streptavidin of the present invention is a protein or peptide or an antigenic fragment of a polypeptide associated with an infectious disease such as tuberculosis and/or malaria.

In a further embodiment an antigen from Plasmodium falciparum is fused to the monovalent streptavidin of the present invention for use in treatment/prophylaxis of malaria.

In a further embodiment an antigen from Mycobacterium tuberculosis is fused to the monovalent streptavidin of the present invention for use in treatment/prophylaxis of tuberculosis. In a further embodiment the antigen is selected from the group consisting of Ag85A from Mycobacterium tuberculosis, PfRH5 from Plasmodium falciparum, VAR2CSA (domain, ID1-ID2a) from Plasmodium falciparum, CIDRI a domain, of PfEMPI from Plasmodium falciparum, GLURP from Plasmodium falciparum, MSP3 from

Plasmodium falciparum, Pfs25 from Plasmodium falciparum, CSP from Plasmodium falciparum, and PfSEA-1 from Plasmodium falciparum or an antigenic fragment of the disclosed antigens. In another embodiment the antigen comprises a fusion construct between MSP3 and GLURP (GMZ2) from Plasmodium falciparum.

In another embodiment the antigen of the present invention comprises a protein, or an antigenic fragment hereof, from the pathogenic organism which causes the infectious disease.

In a most preferred embodiment the present invention concerns a vaccine for use in the prophylaxis and/or treatment of one of the herein disclosed infectious diseases wherein the vaccine comprises:

i. a papilloma virus (PV) L1 protein containing a biotin acceptor site, and

ii. a biotin molecule enzymatically conjugated to said biotin

acceptor site, and

iii. an antigen associated with an infectious disease such as Ag85A from Mycobacterium tuberculosis, PfRH5 from Plasmodium falciparum, VAR2CSA (domain, ID1-ID2a) from Plasmodium falciparum, CIDRIa domain, of PfEMPI from Plasmodium falciparum, GLURP from Plasmodium falciparum, MSP3 from Plasmodium falciparum, Pfs25 from Plasmodium falciparum, CSP from Plasmodium falciparum, and/or PfSEA-1 from

Plasmodium falciparum or an antigenic fragment hereof fused to a monovalent streptavidin,

wherein the antigen and the PV L1 protein are linked via the interaction between the monovalent streptavidin and the biotin molecule enzymatically conjugated to the biotin acceptor site of the PV L1 protein, and wherein i - iii form a virus like particle displaying said antigen. In a preferred embodiment, the antigen is VAR2CSA and the vaccine is for use in the prophylaxis and/or treatment of placental malaria.

In another preferred embodiment, the antigen is surviving and the vaccine is for use in the prophylaxis and/or treatment of cancer.

In an embodiment the antigen fused to a monovalent streptavidin comprises SEQ ID NO: 21 , SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, or SEQ ID NO: 28 and/or a sequence variant hereof. In a preferred embodiment the antigen fused to a monovalent streptavidin comprises SEQ ID NO: 21. In a more preferrred embodiment the antigen fused to a monovalent streptavidin comprises SEQ ID NO: 24. In a most preferred embodiment the antigen fused to a monovalent streptavidin comprises SEQ ID NO: 28. Induction of an immune response in a subject

Active vaccination (immunization), by delivering small doses of an antigen into a subject, is a way to activate a subject's immune system to develop adaptive immunity to the antigen. This allows a subjects body to react quickly and efficiently to future exposures.

An aspect of the invention relates to a method for inducing an immune response in a subject, the method comprising the steps of

i. obtaining a composition comprising at least one vaccine of the present invention and/or

ii. administering said composition to a subject at least once for prophylaxis and/or treatment of a disease, thereby inducing an immune response in the subject.

Another aspect of the present invention relates to a method of immunizing a subject in need thereof, said method comprises the steps of:

i. obtaining a composition comprising at least one vaccine of the present invention, and/or

ii. administering said composition to a subject at least once for prophylaxis and/or treatment of a disease.

thereby immunizing the subject in need thereof. Another aspect of the present invention relates to a method for obtaining a strong and long-lasting immune response in a subject in need thereof, said method comprising the steps of:

i. obtaining composition comprising a vaccine of the present

invention, and/or

ii. administering the vaccine to treat and/or prevent a clinical

condition in an subject in need thereof,

wherein the vaccine obtain a strong and long-lasting immune response in the subject.

In an embodiment the method of inducing an immune response in a subject, immunizing a subject in need thereof, and/or obtaining a strong and long-lasting immune response further comprising at least one booster vaccine and/or a second active ingredient.

Origin of the PV L1 protein

An important element of the present VLP based vaccine is the major capsid L1 protein from papillomavirus (PV), which has the ability to spontaneously self-assemble into virus-like particles (VLPs). The use of papillomavirus L1 proteins in the present invention is illustrated in figure 1 b. Several hundred species of papillomaviruses, traditionally referred to as "types", are known to infect most mammals, as well as birds, snakes, and turtles. In principle the major capsid protein L1 from most papillomavirus, capable of forming a VLP, may be used to carry out the present invention. In an embodiment the PV L1 protein of the present invention is from a mammal such as but not limited to: Alces genus, Homo sapiens, cow, pig, horse, sheep, goat, llama, mouse, rat, monkey, and chicken.

In a most preferred embodiment the PV L1 protein of the present invention is from the human PV genotype 16 and/or 1 18. In yet a particular embodiment the PV L1 protein is from the Alces alces.

Biotin acceptor site and its position in the PV L1 protein

The biotin acceptor site of the present invention comprises a sequence of 15 amino acids encoded by a polynucleotide sequence. It is extremely difficult, if not impossible, to predict the consequence of modifications to VLP forming proteins, such as PV L1 proteins. Such modifications often lead to disruption of the delicate and sensitive self- assembly process of the VLPs. Successful modification of the VLP forming process is at least dependent on three factors 1) the amino acid sequence of the biotin acceptor site, 2) the insertion site, and 3) deletion/removal of amino acid residues to make room for the biotin acceptor site. The inventors have demonstrated several successful modifications to PV L1 loops without disrupting the delicate self-assembly process.

In an embodiment the biotin acceptor site is fused into a loop of the PV L1 protein.

In an embodiment the present invention concerns a vaccine for use in the prophylaxis and/or treatment of a disease wherein the vaccine comprises:

i. a papilloma virus (PV) L1 protein containing a biotin

acceptor site in a loop of the PV L1 protein, and ii. a biotin molecule enzymatically conjugated to said biotin acceptor site, and

iii. an antigen fused to a monovalent streptavidin,

wherein the antigen and PV L1 protein are linked via the interaction between the monovalent streptavidin and the biotin molecule enzymatically conjugated to the biotin acceptor site of the PV L1 protein, and wherein i - iii form a virus like particle displaying said antigen.

In another embodiment the loop is selected from the group consisting of the DE-loop, and the Hl-loop of the PV L1 protein. The PV L1 protein starts with a methionine residue which is cleaved off after translation. There are thus two ways of numbering the amino acid residues of the PV L1 protein: either the methionine residue is the first, or the methionine residue is not counted and the following serine residue is the first. The positions given in the present disclosure are numbered using the first numbering, i.e. where the methionine is counted.

In an embodiment the biotin acceptor site is inserted into the Hl-loop of the L1 protein from the human PV genotype 16 at position 351/352 (SEQ ID NO: 2). In another embodiment the biotin acceptor site, is inserted into the H4-loop of the L1 protein from the human PV genotype 16 at position 431/432 (SEQ ID NO: 1). In an embodiment the biotin acceptor site is inserted into the Hl-loop of the L1 protein from the human PV genotype 118 at position 360/365 where the residues 361-364 (AGKI) are deleted (SEQ ID NO: 6). In a most preferred embodiment the biotin acceptor site is inserted into the DE-loop of the L1 protein from the human PV genotype 16 at position 134/139 where the residues 135 - 137 (YAAN) are deleted (SEQ ID NO: 3).

In an embodiment the PV L1 protein containing the biotin acceptor site comprises the amino acid sequences selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 2, and SEQ ID NO: 6 or a biologically active sequence variant that has at least 95%, such as 96%, such as, 97%, such as 98%, such as 99%, such as 99,5%, such as 100% sequence identity to any of the herein disclosed PV L1 proteins containing the biotin acceptor site sequences. By "biological active" is meant the ability to form a virus like particle.

In an embodiment the biotin acceptor site of the present invention comprises the amino acid sequence GLNDIFEAQKIEWHE (SEQ ID NO 36).

Enzymatic biotinylation

In general, two approaches for biotinylation of proteins exists; chemical biotinylation and enzymatic biotinylation. Chemical biotinylation such as biotinylation of exposed lysine residues allows the attachment to VLPs of diverse kinds of target antigens (incl. non-protein targets) and this approach is generally not restricted by the size of the antigen (Raja KS. et al., 2003). However, with this strategy it is very challenging to control the overall amount/stoichiometry as well as the orientation of the coupled antigen. Finally, chemical coupling procedures are rarely compatible with large scale vaccine production. The main challenge of current VLP platforms is to present the antigen on the surface of the VLP, at high density, in a regularly spaced order with a consistent orientation for an efficient epitope display which are capable of inducing long-term protective immunity. In order to solve these challenges the VLPs of the present invention are made by enzymatic biotinylation. Enzymatic biotinylation ensures that only a single biotin molecule is conjugated to each L1 in the VLP thus controlling the overall

ratio/stoichiometry and placement of VLP proteins and biotin molecules. This control will enable display of an antigen at high density i.e. in theory one antigen per L1 protein, while being regularly spaced, and with consistent orientation on the surface of the VLPs of the present invention.

Thus, in an embodiment the biotin molecule is enzymatically conjugated to the biotin acceptor site. In another embodiment the biotin molecule is enzymatically conjugated to the biotin acceptor site by a biotin ligase from a prokaryote, such as from a bacteria, such as from E. coli. The biotin ligase is in a preferred embodiment BirA from E. coli.

Antigen fused to a monovalent streptavidin

Streptavidin from Streptomyces avidinii forms streptavidin homo-tetramers and binds up to four biotin molecules. The monovalent streptavidin of the present invention forms monomers and binds a single biotin molecule. Thus when linking the antigen fused to a monovalent streptavidin to the VLP of the present invention, the monovalent streptavidin ensures a uniform presentation of said antigens. Using monovalent streptavidin further ensures control of the overall amount/stoichiometry as well as the orientation of the coupled antigen. This control will enable display of an antigen at high density, while being regularly spaced, and with consistent orientation on the surface of a VLP, thus solving three major critical factors for obtaining prober activation of the immune system. In contrast a streptavidin homo-tetramer may crosslink several PV L1 proteins on the surface of a VLP, thus creating an irregular presentation of said antigen, which may hamper the immune response of an antigen. It is thus an aspect of the present invention to use monovalent streptavidin fused to the antigen for linking the antigen to the PV L1 protein via the interaction between the monovalent streptavidin and the biotin molecule enzymatically conjugated to the biotin acceptor site of the PV L1 protein.

In an embodiment the monovalent streptavidin used by the present invention comprises or consists of the amino acid sequence of SEQ ID NO: 37. In another embodiment each antigen is presented in immediate continuation of each PV L1 protein in a consistent orientation.

In another embodiment the ratio of PV L1 protein:biotin molecule:antigen is 1 :1 : 1. Changing the position where the monomeric streptavidin tag is fused to the antigen will allow changing the orientation of the antigen. This may be performed to enable the best possible display of the most immunogenic epitopes of the antigen. The best possible orientation may be different from antigen to antigen.

In another embodiment the antigen fused to a monovalent streptavidin further comprises a tag. Such tags may be used for purification techniques known to the skilled person. The tag may be selected from the group comprising polyhistidine tag, Calmodulin-tag, polyglutamate tag, E-tag, FLAG-tag, HA-tag, Myc-tag, S-tag, SBP-tag, Softag 1 , Softag 3, Strep-tag, Strep-tag II, TC tag, V5 tag, VSV-tag, and Xpress tag. Other peptide or non-peptide tags may be used instead or in combination with the above mentioned peptide tags. In a particular embodiment the tag is a polyhistidine tag, such as a 4xHis, 5xHis, 6xHis, 7xHis, 8xHis, 9xHis, or 10xHis tag. In an embodiment the monovalent streptavidin is fused to the antigen in any position. In another embodiment the monovalent streptavidin is fused to the antigen in the N- terminal, C-terminal, and/or is fused to the antigen into the coding sequence of the antigen. A person of skill will know how to fuse the antigen and the monovalent streptavidin, without introducing stop-codons or causing frame shift or any other mutations.

In another embodiment the monovalent streptavidin fused to the antigen comprises i. a polypeptide sequence selected from the group consisting of SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 , SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28, or

ii. a sequence variant of said polypeptide sequence, wherein the sequence variant has at least 70%, such as 75%, such as 80%, such as 85%, such as 90%, such as 95%, such as 96%, such as, 97%, such as 98%, such as 99%, such as 99,5%, such as 100% sequence identity to the sequences of the group comprising SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 , SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28. In a preferred embodiment the monovalent streptavidin fused to the antigen comprises i. a polypeptide sequence comprising SEQ ID NO: 19, and/or

ii. a sequence variant of said polypeptide sequence, wherein the sequence variant has at least 70%, such as 75%, such as 80%, such as 85%, such as 90%, such as 95%, such as 96%, such as, 97%, such as 98%, such as 99%, such as 99,5%, such as 100% sequence identity to SEQ ID NO: 19.

In a most preferred embodiment the monovalent streptavidin fused to the antigen comprises

i. a polypeptide sequence comprising SEQ ID NO: 18, and/or

ii. a sequence variant of said polypeptide sequence, wherein the sequence variant has at least 70%, such as 75%, such as 80%, such as 85%, such as 90%, such as 95%, such as 96%, such as, 97%, such as 98%, such as 99%, such as 99,5%, such as 100% sequence identity to SEQ ID NO: 18.

Vector and polynucleotide/polypeptide sequences

In molecular cloning, a vector is a DNA molecule used as a vehicle to artificially carry foreign genetic material into a cell, where it can be replicated and/or expressed. The four major types of vectors are plasmids, viral vectors, cosmids, and artificial chromosomes. The vector itself is generally a DNA sequence that consists of an insert (transgene) and a larger sequence that serves as the "backbone" of the vector. The purpose of a vector which transfers genetic information to another cell is typically to isolate, multiply, or express the insert in the target cell. Expression vectors (expression constructs) specifically are for the expression of transgenes in target cells, and generally have a promoter sequence that drives expression of the transgene.

The heterologous expression/production of the vaccine of the present invention comprises 3 peptide components 1) a PV L1 protein containing a biotin acceptor site 2) a biotin ligase capable of biotinylating the biotin acceptor site, and 3) an antigen fused to a monovalent streptavidin. Thus in an embodiment of the present invention each of the peptide components are encoded by a polynucleotide sequence and each of the polynucleotide sequences may be expressed on one, two, or three different plasmids. To enable heterologous expression/production of the vaccine one aspect of the present invention is a vector comprising at least one polynucleotide encoding

i. a PV L1 protein containing a biotin acceptor site, and/or

ii. a biotin ligase capable of biotinylating the biotin acceptor site, and/or iii. an antigen fused to a monovalent streptavidin.

In another embodiment the vector comprises at least two, such as at least three polynucleotides of the following polypeptides:

i. a PV L1 protein containing a biotin acceptor site, and/or

ii. a biotin ligase capable of biotinylating the biotin acceptor site, and/or iii. an antigen fused to a monovalent streptavidin.

In a further embodiment the PV L1 protein containing the biotin acceptor site is encoded by a polynucleotide sequence comprising:

i. a polynucleotide sequence selected from the group comprising SEQ ID 11 , SEQ ID 12, SEQ ID 13, SEQ ID 14, SEQ ID 15, SEQ ID 16, SEQ ID 17, and/or

ii. a sequence variant of said polynucleotide sequence, wherein the

sequence variant has at least 70%, such as 75%, such as 80%, such as 85%, such as 90%, such as 95%, such as 96%, such as, 97%, such as 98%, such as 99%, such as 99,5%, such as 100% sequence identity to said SEQ ID 1 1 , SEQ ID 12, SEQ ID 13, SEQ ID 14, SEQ ID 15, SEQ ID 16, SEQ ID 17, and/or

iii. a sequence variant of said polynucleotide, wherein the codon usage is altered.

In a preferred embodiment embodiment the PV L1 protein containing the biotin acceptor site is encoded by a polynucleotide sequence comprising:

i. a polynucleotide sequence selected from the group comprising, SEQ ID 12, and/or SEQ ID 13, and/or

ii. a sequence variant of said polynucleotide sequence, wherein the

sequence variant has at least 70%, such as 75%, such as 80%, such as 85%, such as 90%, such as 95%, such as 96%, such as, 97%, such as 98%, such as 99%, such as 99,5%, such as 100% sequence identity to said, SEQ ID 12, and/or SEQ ID 13, and/or iii. a sequence variant of said polynucleotide, wherein the codon usage is altered.

In a further embodiment the antigen fused to the monovalent streptavidin has a polynucleotide sequence comprising:

i. a polynucleotide sequence selected from the group consisting of SEQ ID 29, SEQ ID 30, SEQ ID 31 , SEQ ID 32, SEQ ID 33, SEQ ID 34, SEQ ID 35, and/or

ii. a sequence variant of said polynucleotide sequence, wherein the

sequence variant has at least 70%, such as 75%, such as 80%, such as

85%, such as 90%, such as 95%, such as 96%, such as, 97%, such as 98%, such as 99%, such as 99,5%, such as 100% sequence identity to said SEQ ID 1 1 , SEQ ID 12, SEQ ID 13, SEQ ID 14, SEQ ID 15, SEQ ID 16, SEQ ID 17, and/or

iii. a sequence variant of said polynucleotide, wherein the codon usage is altered.

In an embodiment the biotin acceptor site, comprises the nucleotide sequence SEQ ID NO: 39.

Another aspect of the present invention relates to polynucleotide sequences encoding i. a PV L1 protein containing a biotin acceptor site, and/or

ii. a biotin ligase capable of biotinylating the biotin acceptor site, and/or iii. an antigen fused to a monovalent streptavidin.

In a preferred embodiment the present invention relates to polynucleotide sequences encoding

i. a PV L1 protein containing a biotin acceptor site comprising SEQ ID NO:

2, and/or

ii. a biotin ligase capable of biotinylating the biotin acceptor site comprising

SEQ ID 38, and/or

iii. an antigen fused to a monovalent streptavidin comprising SEQ ID NO:

19. In a more preferred embodiment the present invention relates to polynucleotide sequences encoding

i. a PV L1 protein containing a biotin acceptor site comprising SEQ ID NO:

2, and/or

ii. a biotin ligase capable of biotinylating the biotin acceptor site comprising

SEQ ID 38, and/or

iii. an antigen fused to a monovalent streptavidin comprising SEQ ID NO:

20. In a most preferred embodiment the present invention relates to polynucleotide sequences encoding

i. a PV L1 protein containing a biotin acceptor site comprising SEQ ID NO:

2, and/or

ii. a biotin ligase capable of biotinylating the biotin acceptor site comprising SEQ ID 38, and/or

iii. an antigen fused to a monovalent streptavidin comprising SEQ ID NO:

18.

Host cell

The invention further relates to a host cell comprising a polynucleotide and/or a vector. The polynucleotide may have a sequence that is codon-optimised. Codon optimisation methods are known in the art and allow optimised expression in a heterologous host organism or cell. In an embodiment the host cell may be selected from the group comprising bacteria, yeast, fungi, plant, mammalian and/or insect cells.

Methods for expressing a first polypeptide: a PV L1 protein containing a biotin acceptor site, and/or a second polypeptide; a biotin ligase, capable of biotinylating the biotin acceptor site, and/or a third polypeptide: an antigen fused to a monovalent streptavidin in a host cell are known in the art. The first, second or third polypeptide may be heterologously expressed from corresponding polynucleotide sequences cloned into the genome of the host cell or they may be comprised within a vector. For example, a first and/or second and/or third polynucleotide coding for the first and/or second and/or third polypeptide is cloned into the genome, and a first and/or second and/or third polynucleotide coding for the first and/or second and/or third polypeptide is comprised within a vector transformed or transfected into the host cell. Alternatively, the first and/or second and/or third polynucleotide is comprised within a first vector and the first and/or second and/or third polynucleotide is comprised within a second vector and the first and/or second and/or third is comprised within a third vector.

Expression of the first, second, and third polypeptides in the host cell may occur in a transient manner. When the polynucleotide encoding one of the polypeptides is cloned into the genome, an inducible promoter may be cloned as well to control expression of the polypeptides. Such inducible promoters are known in the art. Alternatively, genes coding for suppressors of gene silencing may also be cloned into the genome or into a vector transfected within the host cell.

In a particular embodiment the host cell may be selected from the group comprising Escherichia coli, Spodoptera frugiperda (sf9), Trichoplusia ni (BTI-TN-5B1-4), Pichia Pastoris, Saccharomyces cerevisiae, Hansenula polymorpha, Drosophila Schneider 2 (S2), Lactococcus lactis, Chinese hamster ovary (CHO), Human Embryonic Kidney 293, Nicotiana tabacum cv. Samsun NN and Solarium tuberosum cv. Solara. Thus in an embodiment, the host cell is Escherichia coli. In another embodiment, the host cell is Spodoptera frugiperda. In another embodiment, the host cell is Pichia Pastoris. In another embodiment, the host cell is Saccharomyces cerevisiae. In another embodiment, the host cell is Hansenula polymorpha. In another embodiment, the host cell is Drosophila Schneider 2. In another embodiment, the host cell is Lactococcus lactis. In another embodiment, the host cell is Chinese hamster ovary (CHO). In another embodiment, the host cell is Human Embryonic Kidney 293. In another embodiment, the host cell isTrichoplusia ni (BTI-TN-5B1-4). In another embodiment, the host cell is Nicotiana tabacum cv. Samsun NN. In another embodiment, the host cell is Solanum tuberosum cv. Solara.

In another aspect the present invention relates to a host cell expressing at least one polypeptide encoded by any of the polynucleotides disclosed by the present invention. In an embodiment the host cell expresses:

i. a first polypeptide; a PV L1 protein containing a biotin acceptor site, and/or

ii. a second polypeptide; a biotin ligase, capable of biotinylating the biotin acceptor site, and/or iii. a third polypeptide; an antigen fused to a monovalent streptavidin,

wherein the cell is selected from the group comprising bacteria, yeast, fungi, plant, mammalian and/or insect cells.

In a further embodiment the host cell, expresses

i. a first polypeptide having a sequence at least 70%, such as 75%, such as 80%, such as 85%, such as 90%, such as 95%, such as 96%, such as, 97%, such as 98%, such as 99%, such as 99,5%, such as 100% identical to SEQ ID NO: 3 [DE-loop, HPV genotype 16], SEQ ID NO: 2 [Hl-loop, HPV genotype 16], SEQ ID NO: 6 [Hl-loop, HPV1 18]; and/or ii. a second polypeptide having a sequence at least 70%, such as 75%, such as 80%, such as 85%, such as 90%, such as 95%, such as 96%, such as, 97%, such as 98%, such as 99%, such as 99,5%, such as 100% identical to SEQ ID NO: 38, and/or

iii. a third polypeptide having a sequence at least 70%, such as 75%, such as 80%, such as 85%, such as 90%, such as 95%, such as 96%, such as, 97%, such as 98%, such as 99%, such as 99,5%, such as 100% identical to SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 , SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28, wherein the cell is selected from the group comprising bacteria, yeast, fungi, plant, mammalian and/or insect cells. In a preferred embodiment the host cell expresses:

i. a first polypeptide; a PV L1 protein containing a biotin acceptor site comprising SEQ ID NO: 2, and/or

ii. a second polypeptide; a biotin ligase, capable of biotinylating the biotin acceptor site comprising SEQ ID NO: 38, and/or iii. a third polypeptide; an antigen fused to a monovalent

streptavidin comprising SEQ ID NO: 18,

wherein the cell is selected from the group comprising bacteria, yeast, fungi, plant, mammalian and/or insect cells. The inventors of the present invention have demonstrated formation of VLP's using insect cells, such as High Five (Trichoplusia ni) of Sf9 cells incubated at 28°C. Other conditions and expression hosts may work as well. In a particular embodiment Trichoplusia ni cells are used as host cell for expression of any of the disclosed polynucleotides and/or polypeptides. In another embodiment Trichoplusia ni cells are used to express a polypeptide having a sequence at least 70%, such as 75%, such as 80%, such as 85%, such as 90%, such as 95%, such as 96%, such as, 97%, such as 98%, such as 99%, such as 99,5%, such as 100% identical any of the polypeptides disclosed herein.

Composition comprising the vaccine

The vaccine of the present invention is to be used in the prophylaxis and/or treatment of a disease. Thus, one aspect of the present invention relates to a composition comprising the vaccine of the present invention. Such compositions may further comprise for example an adjuvant, a buffer, and/or salts or a combination hereof.

An adjuvant is a pharmacological and/or immunological agent that modifies the effect of other agents. Adjuvants may be added to a vaccine to modify the immune response by boosting it such as to give a higher amount of antibodies and/or a longer lasting protection, thus minimizing the amount of injected foreign material. Adjuvants may also be used to enhance the efficacy of a vaccine by helping to subvert the immune response to particular cell types of the immune system, for example by activating the T cells instead of antibody-secreting B cells dependent on the type of the vaccine.

Thus in an embodiment the composition comprises at least one adjuvant. In an embodiment the adjuvant is aluminium based. Alluminumum adjuvants may be aluminum phosphate, aluminum hydroxide, amorphous aluminum hydroxyphosphate sulfate and/or a combination hereof. Other adjuvants may be included as well. In another embodiment the composition described above comprises at least one buffer. In an embodiment the buffer is PBS and/or histidine based. In another embodiment the buffer has a pH between pH 6 - pH 7.5. In an embodiment the buffer, is isotonic such as has 0,6 % - 1 ,8 % NaCI. An emulsifier (also known as an "emulgent") is a substance that stabilizes an emulsion by increasing its kinetic stability. One class of emulsifiers is known as "surface active agents", or surfactants. Polysorbates are a class of emulsifiers used in some pharmaceuticals and food preparation. Common brand names for polysorbates include Alkest, Canarcel, and Tween. Some examples of polysorbates are Polysorbate 20,

Polysorbate 40, Polysorbate 60, Polysorbate 80. In an embodiment the composition of the present invention comprises an emulsifier such as one of the above described polysorbates. In a particular embodiment the composition comprises 0.001-0.02% polysorbate 80. Other polysorbates or emulsifiers may be used in the present invention as well.

A pharmaceutical composition comprising the vaccine

The vaccine of the present invention is intended to be used in the prophylaxis and/or treatment of a disease. Accordingly, the present invention further provides a

pharmaceutical formulation, which comprises the vaccine of the present invention and a pharmaceutically acceptable carrier therefor. The pharmaceutical formulations may be prepared by conventional techniques, e.g. as described in Remington: The Science and Practice of Pharmacy 2005, Lippincott, Williams & Wilkins. The pharmaceutically acceptable carriers can be either solid or liquid. Solid form preparations include powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules. A solid carrier can be one or more excipients which may also act as diluents, flavoring agents, solubilizers, lubricants, suspending agents, binders, preservatives, wetting agents, tablet disintegrating agents, or an encapsulating material.

Also included are solid form preparations which are intended to be converted, shortly before use, to liquid form preparations for oral administration. Such liquid forms include solutions, suspensions, and emulsions. These preparations may contain, in addition to the active component, colorants, flavors, stabilizers, buffers, artificial and natural sweeteners, dispersants, thickeners, solubilizing agents, and the like. 3

The compounds of the present invention may be formulated for parenteral

administration and may be presented in unit dose form in ampoules, pre-filled syringes, small volume infusion or in multi-dose containers, optionally with an added preservative. The compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, for example solutions in aqueous polyethylene glycol. Examples of oily or non-aqueous carriers, diluents, solvents or vehicles include propylene glycol, polyethylene glycol, vegetable oils, and injectable organic esters, and may contain agents such as preserving, wetting, emulsifying or suspending, stabilizing and/or dispersing agents. Preferably, the formulation will comprise about 0.5% to 75% by weight of the active ingredient(s) with the remainder consisting of suitable pharmaceutical excipients as described herein. The vaccine of the invention may be administered concurrently, simultaneously, or together with a pharmaceutically acceptable carrier or diluent, especially and preferably in the form of a pharmaceutical composition thereof, whether by oral, rectal, or parenteral (including subcutaneous) route, in an effective amount. Thus, one aspect of the present invention relates to a pharmaceutical composition comprising a vaccine. Such pharmaceutical composition may comprise an adjuvant, a buffer, and/or salts or a combination hereof.

In an embodiment the pharmaceutical composition, further comprises a composition comprising a vaccine as described by the present invention.

A method of manufacture a pharmaceutical composition comprising a vaccine

The present invention further relates to a method of manufacturing a pharmaceutical composition comprising a vaccine. In one aspect the VLP based vaccine of the present invention, may at least be manufactured by the following steps:

i. obtaining a first polypeptide comprising: a PV L1 protein containing a biotin acceptor site, and/or

ii. obtaining a second polypeptide: a biotin ligase capable of biotinylating the biotin acceptor site, and/or

iii. obtaining a third polypeptide: an antigen fused to a monovalent

streptavidin, and

iv. subjecting the first polypeptide to conditions which enable formation of virus like particles, and/or

v. enabling enzymatic biotinylation of the biotin acceptor site of said virus like particles using said second polypeptide, and/or vi. obtaining a vaccine by linkage of the third polypeptide and said virus like particles via the interaction between the monovalent streptavidin and the biotin molecule enzymatically conjugated to the biotin acceptor site of said virus like particles, and/or

vii. generating a composition comprising said vaccine,

thereby obtaining a pharmaceutical composition.

In the manufacture of the pharmaceutical composition other steps may be included for example a) isolation/purification of the VLP to yield a high purity/quality product. This will be accomplished using different techniques for protein purification. For this purpose several separation steps will be carried out using the differences in for instance protein size, physico-chemical properties, binding affinity or biological activity b) formulation by adding stabilizers to prolong the storage life or preservatives to allow multi-dose vials to be used safely as needed c) all components that constitute the final vaccine are combined and mixed uniformly e.g. in a single vial or syringe d) the vaccine is put in recipient vessel (e.g. a vial or a syringe) and sealed with sterile stoppers.

All the processes described above will have to comply with the standards defined for Good Manufacturing Practices (GMP) that will involve several quality controls and an adequate infrastructure and separation of activities to avoid cross-contamination.

Finally, the vaccine may be labeled and distributed worldwide.

Method of administering a vaccine

Routes of administration

Systemic treatment. The main routes of administration are oral and parenteral in order to introduce the agent into the blood stream to ultimately target the sites of desired action. Appropriate dosage forms for such administration may be prepared by conventional techniques. Oral administration. Oral administration is normally for enteral drug delivery, wherein the agent is delivered through the enteral mucosa.

Parenteral administration. Parenteral administration is any administration route not being the oral/enteral route whereby the medicament avoids first-pass degradation in the liver. Accordingly, parenteral administration includes any injections and infusions, for example bolus injection or continuous infusion, such as intravenous administration, intramuscular administration, subcutaneous administration. Furthermore, parenteral administration includes inhalations and topical administration. Accordingly, the agent may be administered topically to cross any mucosal membrane of an animal to which the biologically active substance is to be given, e.g. in the nose, vagina, eye, mouth, genital tract, lungs, gastrointestinal tract, or rectum, preferably the mucosa of the nose, or mouth, and accordingly, parenteral administration may also include buccal, sublingual, nasal, rectal, vaginal and intraperitoneal administration as well as pulmonal and bronchial administration by inhalation or installation. Also, the agent may be administered topically to cross the skin.

The subcutaneous and intramuscular forms of parenteral administration are generally preferred.

Local treatment. The agent according to the invention may be used as a local treatment, ie. be introduced directly to the site(s) of action as will be described below.

Thus one agent may be applied to the skin or mucosa directly, or the agent may be injected into the diseased tissue or to an end artery leading directly to the diseased tissue.

Thus another aspect of the present invention relates to a method of administering a vaccine to treat and/or prevent a clinical condition in a subject in need thereof, comprising the steps of

i. obtaining a composition comprising at least one vaccine

according to the present invention, and/or ii. administering said composition to a subject at least once for prophylaxis and/or treatment of a disease.

A preferred embodiment relates to a method of administering a vaccine to treat and/or prevent cancer, as disclosed herein, in a subject in need thereof, comprising the steps of

i. obtaining a composition comprising at least one vaccine as disclosed herein, and/or ii. administering said composition to a subject intramuscular and/or intravenous at least once for prophylaxis and/or treatment of a cancer. A preferred embodiment relates to a method of administering a vaccine to treat and/or prevent a cardiovascular disease, as disclosed herein, in a subject in need thereof, comprising the steps of

i. obtaining a composition comprising at least one vaccine as disclosed herein, and/or

ii. administering said composition to a subject intramuscular and/or intravenous at least once for prophylaxis and/or treatment of a a cardiovascular disease.

In another embodiment the vaccine of the present invention is administered by any type of injections or infusions selected from the group of bolus injection, continuous infusion, intravenous administration, intramuscular administration, subcutaneous administration, inhalation or topical administration or a combination hereof. In a particular embodiment the vaccine is administered by intramuscular administration and/or intravenous administration.

In medicine, a booster dose is an extra administration of a vaccine after an earlier dose. After initial immunization, a booster injection or booster dose is a re-exposure to the immunizing antigen cell. It is intended to increase immunity against that antigen back to protective levels after it has been shown to have decreased or after a specified period. In an embodiment the vaccine of the present invention is administered any number of times from one, two, three, four times or more.

In a further embodiment the vaccine is boosted by administration in a form and/or body part different from the previous administration. In another embodiment the vaccine is administered to the area most likely to be the receptacle of a given disease or infection which the vaccine is intended to prevent/reduce the risk of.

In another embodiment the recipient of the vaccine (the subject) of the present invention is an animal, for example a mammal, such as a Homo sapiens, cow, pig, horse, sheep, goat, llama, mouse, rat, monkey, and/or chicken. In a particular embodiment the subject is a Homo sapiens.

Administration of more than one vaccine is known in the art and refers to this concept as co-vaccination or to give a vaccine cocktail. Thus, in an embodiment of the vaccine, is co-administered with any other vaccine. In another embodiment the vaccine forms a part of a vaccine cocktail.

A kit of parts

In another aspect of the present invention relates to a kit of parts comprising

i. a composition comprising a vaccine of the present invention, and/or ii. a medical instrument or other means for administering the vaccine, and/or

iii. instructions on how to use the kit of parts.

In an embodiment the kit of parts comprises a second active ingredient or vaccine component for therapeutic use in the treatment or prevention of one or more of the diseased disclosed in the present invention.

In an embodiment the vaccine of the invention is administered separate, sequential, or simultaneously with at least one other pharmaceutical active ingredient and/or vaccine component.

Dosages and dosing regimes

The dosage requirements will vary with the particular drug composition employed, the route of administration and the particular subject being treated. It will also be recognized by one of skill in the art that the optimal quantity and spacing of individual dosages of a compound will be determined by the nature and extent of the condition being treated, the form, route and site of administration, and the particular patient being treated, and that such optimums can be determined by conventional techniques. It will also be appreciated by one of skill in the art that the optimal course of treatment, i.e., the number of doses of a compound given per day for a defined number of days, can be ascertained using conventional course of treatment determination tests. The daily oral dosage regimen will preferably be from about 0.01 to about 80 mg/kg of total body weight. The daily parenteral dosage regimen about 0.001 to about 80 mg/kg of total body weight. The daily topical dosage regimen will preferably be from 0.1 mg to 150 mg, administered one to four, preferably two or three times daily. The daily inhalation dosage regimen will preferably be from about 0.01 mg/kg to about 1 mg/kg per day.

The term "unit dosage form" refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of a compound, alone or in combination with other agents, calculated in an amount sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier, or vehicle. The specifications for the unit dosage forms of the present invention depend on the particular compound or compounds employed and the effect to be achieved, as well as the pharmacodynamics associated with each compound in the host. The dose administered should be an "effective amount" or an amount necessary to achieve an "effective level" in the individual patient.

When the "effective level" is used as the preferred endpoint for dosing, the actual dose and schedule can vary, depending on inter-individual differences in pharmacokinetics, drug distribution, age, gender, size, health and metabolism. The "effective level" can be defined, for example, as the blood or tissue level desired in the patient that

corresponds to a concentration of one or more compounds according to the invention.

Examples

Modification of VLPs without disrupting the delicate and sensitive self-assembly process is challenging. The inventors have below shown several examples of successful introduction of a biotin acceptor site into various VLP loops without disrupting the self-assembly process. In addition the inventors have shown enzymatic biotinylation of the biotin acceptor site as well as coupled several antigen/monovalent streptavidin fusion proteins to the VLPs. Immunological testing of the VLP vaccines have been initiated. The examples below are non-limiting to the scope of the invention.

Example 1 - Design of chimeric HPV16 Avi-L1 coat proteins

The chimeric HPV16 Avi-L1 gene sequences were constructed by insertion of the biotin acceptor sequence (AviTag™), GLNDIFEAQKIEWHE, into the DE- (aa positions 134- 139), FG- (aa positions 279-286), HI- (aa positions 351-352) or Η4-β coil (aa positions 431-432) after having deleted any intervening amino acids (HPV16 L1 accession number DQ155283.1). Gene sequences were further modified to contain an EcoRV restriction site followed by a polyhedrin promotor sequence at the N-terminus and a stop-codon followed by a Notl restriction site at the C-terminus. The synthetic gene sequences were finally codon-optimized for recombinant expression in Trichoplusia ni cells and synthesized by Geneart (Life Technologies).

Example 2 - Expression and purification of chimeric HPV16 Avi-L1 - VLPs

The HPV16 Avi-L1 gene fragments were cloned into the EcoRV/Notl sites of the pAcGP67A vector (BD Biosciences) deleting the gp67 secretion signal sequence. To generate recombinant virus particles, linearized BakPak viral DNA (BD Biosciences) was co-transfected with pAcGP67A/Avi-L1 into Sf9 insect cells using Lipofectamine 2000 10 Reagent (Invitrogen, 11668-019) and incubated at 28°C for 3-5 days.

Recombinant Baculovirus was harvested from the supernatant and used to generate a high-titer virus stock, which was used for infection of High-Five insect cells. Infected High-Five cells were incubated for 48 hours at 28°C with shaking. In vitro maturation and purification of HPV16 Avi-L1 VLPs were performed as previously described (Buck et al., 2005, Buck et al., 2007). In brief, cell lysates were harvested and VLPs were allowed to mature in maturation-buffer (0.5% Triton-X-100, 0.1 % BenzonaseONuclease (Sigma-Aldrich), 25 mM (NH ₄ ) ₂ S0 ₄ and 4mM MgCI ₂ ) for 18h at 37°C. Matured VLPs were subsequently purified by ultracentrifugation through an Optiprep™ step gradient (27%/33%/39%) as previously described (22). VLPs were dialyzed in PBS (0.02% PS80) and incubated for 30 min at 30°C with biotin and Biotin ligase (BirA) according to instructions from Avidity (Aurora, CO). Excess biotin was removed by dialysis in PBS (0.32 M NaCI, 0.02% PS80) and protein concentration was determined using BCA analysis. Collected ultracentrifugation fractions were analyzed with NuPAGE® Bis-Tris Protein gels (Life Technologies) or blotted onto a nitrocellulose membrane (GE- Healthcare, RPN203E) for detection of L1 or biotin with CamVir-1 (AbD Serotec, Bio- Rad, 7135-2804) or Streptavidin - HRP (Life Technologies, 43-4323), respectively. Densitometric analysis of SDS-PAGE gels was done using ImageJ.

Example 3 - Gene design and recombinant expression of biotin-binding vaccine antigens

Heterologous vaccine antigens were genetically fused with a GGS linker at either their C- or N-terminus to a previously described(refs)/patented (US8586708 B2) engineered monovalent streptavidin (mSA) (SEQ ID NO: 37), thereby introducing biotin binding capability to the expressed mSA-antigen fusion proteins. mSA-antigen fusion genes expressed in E. coli were designed with a 6xHistidine tag and Ncol/BamHI restriction sites for subcloning into pET-15b vector. mSA-antigen fusion genes expressed in either S2 cells, Human Embryonic Kidney 293 (HEK293) cells or in Baculovirus infected insect cells were designed with flanking EcoRI/BamHI (N-terminal) and Notl (C- terminal) sites and a 6xHistidine tag and were subcloned into the pHP34s,

pcDNA™4/HisMax or pAcGP67A (BD Biosciences) vector, respectively.

Example 4 - Characterisation of particles

Electron microscopy - Negative staining

An aliquot of diluted VLPs was adsorbed to 200-mesh mica carbon-coated grids and negatively stained with 2% phosphotungstic acid (pH=7.0). The sample was examined with a CM 100 BioTWIN electron microscope (Phillips, Amsterdam) at an accelerating voltage of 80 kV. Photographic records were performed on an Olympus Veleta camera. Particle sizes were estimated using ImageJ. Representative pictures are shown in figure 2.

Example 5 - Particle size measurement by dynamic light scattering (DLS)

The hydrodynamic diameter (referred to as size) of the VLP was measured using a particle analyzer, DynaPro NanoStar (WYATT Technology), equipped with a 658 nm laser. VLP samples were diluted to 0.2 mg/mL with PBS (0.32 M NaCI, 0.02% PS80) and 70 μΙ of each VLP sample was loaded into a disposable low volume cuvette and mounted into the DLS chamber. After 1 min equilibration, the size distribution was obtained by DLS measurement at 25°C. Each sample was measured 2 times with 20 runs in each measurement. The most predominant average sizes of particles in the population were calculated from the measurements and recorded together with the % polydispersity (%Pd).

Example 6 - Determination of the Extent of Biotinylation of AviTag-VLPs

The degree of biotinylation of the AviTag-VLPs is estimated by comparison with known quantities of fully biotinylated C-terminal AviTag Maltose Binding Protein (MBP-AviTag protein) using a typical ELISA setup. Briefly, the standard MBP-AviTag protein and AviTag-VLPs are adsorbed to the wells of a 96-well maxisorb plate (Nunc 456537) at known protein quantities (1 - 45ng, 5 ng increments). Extraneous biotin is removed by washing with TBST buffer (10 mM Tris, pH 7.5, 150 mM NaCI, 0.05% Tween 20) and the plate is blocked by adding 300μΙ blocking solution (PBS plus 40 μg/ml BSA) to each well. The streptavidin-horseradish peroxidase solution is then added into each well and incubated with gentle shaking for 1 hour at room temperature. Finally, the biotin associated with the biotin acceptor site is detected by its interaction with streptavidin- conjugated horseradish peroxidase by monitoring the absorption at 490 nm after adding the developing solution (3 OPD tablets dissolved in 12 ml d-water with 10μΙ H202).

Example 7 - Verification of the mSA-antigen coupling onto (biotin acceptor site)- VLPs:

The overall amounts of antigen coupled onto the VLPs was estimated by comparing the relative amounts of antigen / HPV L1 protein on a reduced SDS-PAGE gel (Figure 3). The material for this analysis was made by firstly mixing biotinylated VLPs with an excess amount of the mSA-antigen and then separating the mSA-antigen/ VLP complexes from the excess mSA-antigen by density gradient ultracentrifugation.

The VLPs were also examined by transmission electron microscopy to assess their integrity after coupling of different mSA-antigens (Figure 4). Specifically, an aliquot of diluted particles (post mSA-coupling and removal of excess mSA-antigen) was placed on 200-mesh mica carbon-coated grids, negatively stained with 2% phosphotungstic acid (pH=7.0) and examined by transmission electron microscopy (TEM) using a CM 100 BioTWIN.

Example 8 - Coupling of Antigens to VLPs

The inventors have partially tested the coupling of 7 different VLPs with 12 different antigen/monovalent streptavidin fusion proteins. The results have been obtained as described above and are summarized in table 3.

Table 3: Coupling of VLP1 -7 and antigen/mSA A1 -A11. Sequences of the constructs are found in table 5.

VLP1 VLP2 VLP3 VLP4 VLP5 VLP6 VLP7

Al No Yes Yes No N/A No No coupling VLPs VLPs VLPs

A2 No Yes Yes No N/A No No coupling VLPs VLPs VLPs

A3 No N/A N/A No N/A No No coupling VLPs VLPs VLPs

A4 No Yes Yes No N/A No No coupling VLPs VLPs VLPs

A5 No Yes Yes No N/A No No coupling VLPs VLPs VLPs

A6 No Yes Yes No N/A No No coupling VLPs VLPs VLPs

A7 No Yes Yes No N/A No No coupling VLPs VLPs VLPs

A8 No Yes Yes No N/A No No coupling VLPs VLPs VLPs

A9 No Yes Yes No N/A No No coupling VLPs VLPs VLPs

A10 No Yes Yes No N/A No No coupling VLPs VLPs VLPs

All No Yes Yes No N/A No No coupling VLPs VLPs VLPs

A12 No Yes N/A No N/A No No coupling VLPs VLPs VLPs

Example 9 - Expression and purification of VAR2CSA and mSA-VAR2CSA

The chimeric mSA-VAR2CSA gene sequence was designed with a small Gly-Gly-Ser linker separating the monomeric Streptavidin (mSA) [GenBank: 4JNJ_A] from the ID1- ID2a domains of VAR2CSA [FCR3 strain, GenBank: GU249598] (amino terminus). Both the VAR2CSA and mSA-VAR2CSA gene fragments were further modified to contain a C-terminal 6xhistidine tag and flanking BamHI and Notl restriction sites used for sub-cloning into the Baculovirus vector, pAcGP67A (BD Biosciences). Linearized Bakpak6 Baculovirus DNA (BD Biosciences) was co-transfected with pAcGP67A- VAR2CSA or pAcGP67A-mSA-VAR2CSA into Sf9 insect cells for generation of recombinant virus particles. Histidine-tagged recombinant protein was purified on Ni ²⁺ sepharose columns from the supernatant of Baculovirus infected High-Five insect cells using an AKTAxpress purification system (GE-Healthcare).

Example 10 - Conjugation and purification of mSA-VAR2CSA to L1 -Avi VLPs

For production of the VLP-VAR2CSA vaccine, biotinylated HPV16 Avi-L1 VLPs were mixed with 3x molar excess of mSA-VAR2CSA and the sample was incubated at 4°C for 18-24 hours with gentle shaking. Unbound mSA-VAR2CSA was removed by ultracentrifugation over an Optiprep step gradient (27%, 33%, 39%) as previously described (Buck et al., 2004).

The VLP-VAR2CSA vaccine was dialyzed in PBS (0.32 M NaCI, 0.02% PS80) and the relative concentration of VLP-bound mSA-VAR2CSA was estimated by SDS-PAGE using a BSA standard dilution range. Post ultracentrifugation fractions were analyzed by SDS-PAGE or blotted onto a nitrocellulose membrane (GE-Healthcare RPN203E) for detection of L1 or mSA-VAR2CSA (via a 6xhistidine tag) with CamVir-1 (AbD Serotec, Bio-Rad, 7135-2804) or Penta His HRP (QIAGEN, 34460) respectively.

Example 11 - Anti-VAR2CSA antibody response measured by ELISA

Recombinant VAR2CSA (1 μg/ml in PBS) was coated on Nunc MaxiSorp plates overnight at 4°C. Plates were incubated with blocking buffer for 1 hour at room temperature (RT) to inhibit non-specific binding to the plate. Plates were washed three times in between different steps. Serum samples were diluted in blocking buffer (1 : 100), added to the wells in three fold dilutions, and incubated for 1 hour at RT.

Horseradish peroxidase (HRP)-conjugated polyclonal rabbit anti-mouse Ig (P260 DAKO, Denmark) was diluted 1 :3000 in blocking buffer and incubated for 1 hour.

Finally, color reactions were developed for 7 min by adding o-phenylenediamine substrate. The HRP enzymatic reaction was stopped by adding 2.5 M H ₂ S0 ₄ and the optical density was measured at 490 nm using an ELISA plate reader (VersaMax Molecular Devices).

Example 12 - Parasite culture

Parasites were maintained in culture as described (23). Briefly, parasites were cultured in 5% hematocrit of human blood (group 0+) in RPMI 1640 (Sigma) supplemented with 0.125 μg/ml Albumax II (Invitrogen) and 2% normal human serum. Atmospheric air was exchanged with a mixture of 1 % oxygen and 5 % carbon dioxide in nitrogen, whereafter incubation was done at 37°C under static conditions with ad hoc change of culture medium. The FCR3 isolate was selected for binding to CSA by panning on BeWo cells as described (Haase et al., 2006). Parasite isolates tested negative for mycoplasma (Lonza) and were regularly genotyped using nested GLURP and MSP-2 primers in a single PCR step, as described (Snounou et al., 1999). Example 13 - Inhibition of Binding assays

Parasite DNA was labeled with Tritium by overnight incorporation of titrated

hypoxanthine. A 96 well plate (Falcon) was coated with 2 μg/ml of Decorin (Sigma- Aldrich) overnight and blocked with 2% bovine serum albumin (Sigma) as described (Nielsen et al., 2009). Tritium labeled late-stage lEs were MACS purified and added to the 96 well plate in a concentration of 200,000 cells per well. Titrations of serum were added in a total volume of 100 μΙ in triplicate wells. After incubation for 90 min at 37°C, unbound lEs were washed away by a pipetting robot (Beckman-Coulter). The remaining lEs were harvested onto a filter plate (Perkin-Elmer). After addition of scintillation fluid (Perkin-Elmer) the counts per minute (CPM) recording the number of non-inhibited IE was determined by liquid scintillation counting on a Topcount NXT (Perkin-Elmer). Data were adjusted to percentage of binding by dividing test result with the mean value of wells with IE incubated without serum. Example 14 - generation of biotinylated HPV Avi-L1 VLPs

The biotin acceptor site (AviTag™) sequence was genetically inserted as described above at four different positions in the HPV16 L1 sequence, which based on the crystal structure of HPV16 L1 capsid protein, correspond to surface exposed loops in the assembled VLP structure. The chimeric Avi-L1 constructs were expressed in High-Five cells and allowed to assemble into VLPs (Fig. 1 b). Formation of VLPs was confirmed by performing density gradient ultracentrifugation followed by SDS-PAGE analysis. This analysis indicated that three of the recombinant proteins, Avi-L1 (HI), Avi-L1 (DE) and Avi-L1 (Η4-β coil) formed VLPs as the majority of the expressed protein were present in high molecular/density fractions 4-6 after ultracentrifugation (Fig. 5a). The Avi-L1 (FG) protein was exclusively found in the low density fractions containing non- particulate soluble protein, indicating that the AviTag™ insertion, in this case, prevented VLP assembly.

The identity of Avi-L1 proteins in the high-density fractions was confirmed by western blot analysis using a monoclonal anti-HPV16 L1 antibody (fig. 5c). VLP assembly of the Avi-L1 constructs was verified by transmission electron microscopy (TEM) as described above showing a heterogeneous population of VLPs of different sizes (28-60nm) (Fig. 5b), representing different icosahedral assembly symmetries of which roughly 30% had the size/appearance of native HPV16 L1 VLPs. The particles were subsequently biotinylated (Fig. 5c) and analyzed by TEM. .. .

51

Example 15 - Construction of the displayed antigen VAR2CSA

The shortest truncated VAR2CSA polypeptide sequence that is able to bind CSA covers the ID1-ID2a region of the full-length protein (Fig. 1 a). This construct (herein referred to as VAR2CSA) was genetically fused at the amino terminus to monomeric streptavidin (mSA) which can bind biotin with high affinity. To avoid VLP aggregation we used a monomeric form of streptavidin which is advantageous to native streptavidin as the latter contains four biotin binding sites. The chimeric construct expressed high levels of soluble protein and retained a structure capable of binding CSA (data not shown). Purified mSA-VAR2CSA was subsequently mixed with the biotinylated VLP

(1 :3 HPV L1/antigen), and examined by ultracentrifugation followed by SDS-PAGE and western blot analysis. High-density ultracentrifugation fractions [4-5] contained both mSA-VAR2CSA and Avi-L1 protein, which was estimated by densitometric analysis to be present at a 0.6:1 molar ratio. Excess mSA-VAR2CSA was present in the low- density fractions [12-14] (Fig. 6 a&c). The co-localization of Avi-L1 and mSA-

VAR2CSA, indicated that the mSA-VAR2CSA antigen was bound to the surface of Avi- L1 VLPs at high density and that these large protein complexes had been separated from the excess soluble mSA-VAR2CSA (Fig. 6a). To confirm that co-localization of mSA-VAR2CSA and Avi-L1 VLPs was caused by the specific interaction between mSA and biotin, the procedure was repeated using unbiotinylated VLPs. This control experiment showed that ultracentrifugation efficiently separated soluble mSA- VAR2CSA from unbiotinylated VLPs (Fig. 10).

Antigen-coupled VLPs were further examined by TEM, showing particles of a comparably larger size (-70 nm) than the non-coupled VLPs (-30-60 nm) (Fig. 6b). This observation was further examined by dynamic light scattering (DLS) analysis, which confirmed that mixing of mSA-VAR2CSA with biotinylated Avi-L1 VLPs resulted in measurably larger particles with an average diameter of -70 nm (12.9% Pd) compared to naked Avi-L1 VLPs (<60 nm, 23.9% Pd). Importantly, this analysis also confirmed that such large complexes were not formed after mixing unbiotinylated VLPs with mSA-VAR2CSA, demonstrating that mSA-VAR2CSA is, in fact, bound to the surface of Avi-L1 VLPs via the specific affinity interaction between mSA and biotin.

Together, these results indicate that the produced mSA-VAR2CSA VLP vaccine consists of non-aggregated HPV16 Avi-L1 VLPs displaying dense, repetitive arrays of the mSA-VAR2CSA antigen presented in a consistent orientation, as illustrated schematically in Figure 1 b.

Example 16 - Inserting AviTag™ into other coat proteins forming papilloma VLP The HPV16 L1 genotype, used as VLP platform in this study, constitutes one of the most prevalent oncogenic HPV types. This genotype is included in all licensed HPV vaccines, which are being administered to millions of young women every year. Thus, the immunogenicity of this VLP platform could be impeded by pre-existing immunity towards the HPV L1 major capsid protein. We therefore examined whether insertion of the AviTag™ sequence was compatible with VLP formation in other HPV genotypes as well as in non-human PV. The AviTag™ sequence was inserted in the L1 major capsid protein of the European elk PV as well as in HPV genotype 1 18 at a position corresponding to the fusion site in the HPV16 Avi-L1 (DE) (Fig. 7a). The two protein sequences were expressed and purified as previously described. For both constructs a L1 band of the expected protein size (56kDa) was found in the high-density ultracentrifugation fractions although the majority of protein present in these fractions was of a lower molecular size, possibly representing a L1 truncation (Fig. 7b).

These data indicate that insertion of the AviTag™ sequence into other PV types is a feasible strategy for avoiding the potential issue of pre-existing immunity.

Example 17 - Immunization of mice

Female C57BL/6 mice (Taconic, Denmark) were immunized by intramuscular injection with 5 μg VLP-coupled mSA-VAR2CSA, soluble mSA-VAR2CSA or soluble naked VAR2CSA on day 0 with no adjuvant. Two booster injections with 2.5 μg of the respective antigens were given on days 21 and 42. Immune-serum was collected on days 14, 35 and 56.

The immunogenicity of the mSA-VAR2CSA VLP vaccine was then tested. ELISA was used to measure total immunoglobulin (Ig) levels against VAR2CSA in sera obtained from mice immunized with mSA-VAR2CSA VLP, soluble mSA-VAR2CSA or soluble naked VAR2CSA (Fig. 8). After three immunizations the VAR2CSA specific Ig levels were higher in sera from mice immunized with the mSA-VAR2CSA Avi-L1 VLP vaccine than in sera from mice immunized with soluble naked VAR2CSA (Fig. 8c). After 1 ^st and 2 ^nd immunization sera from mSA-VAR2CSA Avi-L1 VLP immunized mice had statistically significantly higher Ig endpoint titers compared with sera from mice immunized with the soluble mSA-VAR2CSA vaccine (P = 0.014 and P = 0.018, respectively). This difference was, however, not statistically significant after the 3 ^r immunization (P = 0.058) (Table 4) where both vaccines seem to have reached a similar plateau.

Table 4. Serum endpoint titers (median {25 and 75 percentiles}) obtained with the different immunogens

a Endpoint titer, defined as the reciprocal of the highest serum dilution giving an OD measurement above the cutoff. The cutoff was set to be three standard deviations above the mean negative control reading.

b P values were calculated using Wilcoxon rank sum test.

Example 18 - Functionality of the mSA-VAR2CSA VLP vaccine induced anti- VAR2 antibodies

Antisera were examined for their ability to block the binding between native VAR2CSA expressed on the surface of parasite-infected erythrocytes and immobilized CSA. After first immunization, none of the three vaccines (mSA-VAR2CSA VLP, soluble mSA- VAR2CSA or soluble naked VAR2CSA) had induced efficient levels of functional binding-inhibitory antibodies, leading to full binding of parasites (Fig. 9a). However, after the second round of immunizations 1 :50 diluted serum from mSA-VAR2CSA VLP immunized mice inhibited the binding between IE and CSA by approximately 70%. In comparison, the soluble mSA-VAR2CSA vaccine only inhibited approximately 20%, while no inhibition was seen for the soluble naked VAR2CSA vaccine (Fig. 9b). After three immunizations, 1 :200 diluted serum from mice immunized with the mSA- VAR2CSA VLP vaccine showed roughly 90% binding-inhibition, while the sera from mice immunized with the soluble mSA-VAR2CSA vaccine inhibited parasite binding by approximately 60%. By contrast, the soluble naked VAR2CSA vaccine failed to induce any binding-inhibitory antibodies (Fig. 9c).

These data show that the mSA-VAR2CSA VLP vaccine displays higher ability to inhibit binding between IE and CSA.

Example 19 - Measurement of antibody avidity:

The avidity values of serum antibodies (Ab) were determined by measuring the resistance of Ab-target complexes to 8 M urea by ELISA. Analyzed sera were obtained after the 2 ^nd immunization. Antibody titres of individual mouse sera were firstly normalized by dilution. After the serum incubation, triplicate wells were treated with either PBS or 8 M urea for 5 minutes. Subsequently, the wells were washed with PBS, and the ELISA was performed as previously described. The avidity value was calculated as the ratio of the mean OD value of urea-treated wells to PBS control wells multiplied by 100. A non-parametric two-sample Wilcoxon rank-sum (Mann-Whitney) test showed that sera from mice immunized with the VAR2CSA AviL1 VLP vaccine had significantly higher avidity values compared to mice immunized with soluble mSA- VAR2CSA (P value: 0.0275).

Statistical analysis:

Two-sample Wilcoxon rank-sum (Mann-Whitney) test

group I obs rank sum expected

29

16 combined I 9 45 45 unadjusted variance 16.67

adjustment for ties 0.00 adjusted variance Ho: reducall(group==1) = reducall(group

z = 2.205

Prob > Izl = 0.0275 Example 20 - Survivin

We wanted to examine if our virus-like particle (VLP) antigen presentation platform was able to overcome immune-tolerance to a cancer-associated self-antigen "Survivin". This was tested by coupling monovalent streptavidin (mSA)-Survivin fusion proteins to the surface of biotinylated HPV Avi-L1 (Hl-loop) VLPs. Three mice were immunized with this VLP-based survivin vaccine. Negative control mice (n=2) were immunized with the same amount (5 μg) of mSA-Survivin, but mixed with unbiotinylated Avi-L1 (Hl- loop) VLPs. Both vaccines were formulated using ALUM adjuvant. Mice were immunized three times at three week intervals and sera were obtained two weeks after each immunization.

The obtained anti-sera were tested in ELISA using naked survivin (no mSA) as the solid phase capturing antigen. In this way we could directly test what effect the VLP- display had on the ability of the mice to induce humoral immunity against the self- antigen.

The results clearly show that all the survivin-VLP (mSA-Survivin coupled to HPV Avi-L1 (HI-LOOP)) immunized mice (n=3) induced high-titre antibody responses against the mSA-survivin self-antigen, whereas the negative control vaccine was not able to break immune-tolerance in the immunized mice (no sero-conversion) (Fig. 1 1 a). The ability to break immune-tolerance thus relies solely on the virus-like display and is not a result of e.g. fusing the self-antigen to mSA.

By testing the same anti-sera in ELISA using the mSA-survivin as the solid phase capturing antigen we could see the negative control mice did produce antibodies against the 'foreign' mSA part (Fig. 1 1 b).

This experiment shows that after three immunizations mouse 1-3 (immunized with Survivin displayed on VLP) were able to break self-tolerance, whereas mouse 4 and 5 are unable to generate a response against soluble survivin. Example 20 - Testing of VLP:avi platform to induce humoral immunity against self-antigens.

To demonstrate the capacity of the VLP:Avi platform to break immune tolerance to self- antigens, associated with both cardiovascular disease (PCSK9), allergy (IL-5) and cancer (Her2), we will genetically fuse the self-antigens to the monovalent mSA and coupled them onto biotinylated (biotin acceptor site)-VLPs, as previously described. In some cases (IL-5) we will at first use the mouse gene homologues for the immunization of mice. Specifically, our working procedure will be to firstly couple HER2 (mSA:Her2- ECD|23-686|), and PCSK9(PCSK9|31-692|:mSA-HIS) to the (biotin acceptor site)- VLPs, immunize, and measure seroconversion of the animals in group a) mice immunized with conjugated VLPs and b) mice immunized with the non-coupled soluble antigen. The antigen specific Ig and IgG titer will be estimated in a 2 fold dilution series of the sera. A positive seroconversion is defined as ELISA OD measurements above 2 x standard deviation of a mock immunized animal. Serum conversion and induction of specific antibodies to HER2 is further confirmed by western blotting using the sera and cell lysates from different cancerous cell lines (e.g. melanoma, prostate, breast and lung cancer).

Example 21 - Testing of VLP:avi platform to induce cancer inhibitory antibodies. We are establishing animal models to study the effect of immunizing animals with tumor antigens on the growth of an established subcutaneous tumor. 100.000 tumor cells expressing HER2 and/or Survivin are injected into the left flank. This is being done in both vaccinated animals and mock immunized animals, to study the

prophylactic effect of the vaccine. Tumor growth is monitored by measuring the size of the growing tumor as well as by scanning of the animal when using luciferase transfected cell lines. In another setup we are studying the therapeutic effect of the vaccine by immunizing animals with established tumors and monitoring tumor regression/progression by size measurements and/or by fluorescent scannings. Example 22 - Testing of VLP:avi platform to induce anti-PCSK9 antibodies capable of lowering plasma/serum cholesterol levels.

The goal of making a VLP-based vaccine based on the PCSK9 antigen is to induce a humoral response capable of lowering blood cholesterol. Therefore, to test the VLP:avi platform we measure cholesterol levels in plasma and serum samples obtained from VLP-PCSK9 immunized mice and compare against the levels measured in mice immunized with the non-coupled PCSK9 antigen, as previously described. Cholesterol levels in plasma and serum samples are measured using a WAKO Cholesterol E Assay kit (Cat# 439-17501) following the manufacturers' instructions. Dilutions of cholesterol standard or test plasma/serum samples (4μΙ volume) are added to wells of a 96-well plate and 196μΙ of prepared cholesterol reagent added. The plate is incubated for 5 minutes at 37°C and the absorbance of the developed color read at 600nm within 30 minutes.

Sequences

Table 5: Overview of the sequences disclosed in the present invention

>SEQ ID NO: 1 [H4-loop, HPV genotype 16] L1Avi1 Protein

MSLWLPSEATVYLPPVPVSKVVSTDEYVARTNIYYHAGTSRLLAVGHPYFPIKKPNN N KILVPKVSGLQYRVFRIHLPDPNKFGFPDTSFYNPDTQRLVWACVGVEVGRGQPLGV GISGHPLLNKLDDTENASAYAANAGVDNRECISMDYKQTQLCLIGCKPPIGEHWGKG SPCTNVAVNPGDCPPLELINTVIQDGDMVDTGFGAMDFTTLQANKSEVPLDICTSICK YPDYIKMVSEPYGDSLFFYLRREQMFVRHLFNRAGAVGENVPDDLYIKGSGSTANLA SSNYFPTPSGSMVTSDAQIFNKPYWLQRAQGHNNGICWGNQLFVTWDTTRSTNMS LCAAISTSETTYKNTNFKEYLRHGEEYDLQFIFQLCKITLTADVMTYIHSMNSTILEDWN FGLQPPPGGTLEDTYRFVTSQAIACQKHGLNDIFEAQKI EWHETPPAPKEDPLKKYTF WEVNLKEKFSADLDQFPLGRKFLLQAGLKAKPKFTLGKRKATPTTSSTSTTAKRKKRK LELSGR

> SEQ ID NO: 2 [Hl-loop, HPV genotype 16] L1Avi2 Protein MSLWLPSEATVYLPPVPVSKVVSTDEYVARTNIYYHAGTSRLLAVGHPYFPIKKPNNN KILVPKVSGLQYRVFRIHLPDPNKFGFPDTSFYNPDTQRLVWACVGVEVGRGQPLGV GISGHPLLNKLDDTENASAYAANAGVDNRECISMDYKQTQLCLIGCKPPIGEHWGKG SPCTNVAVNPGDCPPLELINTVIQDGDMVDTGFGAMDFTTLQANKSEVPLDICTSICK YPDYIKMVSEPYGDSLFFYLRREQMFVRHLFNRAGAVGENVPDDLYIKGXXWIYXXQ XLASSNYFPTPSGSMVTSDAQIFNKPYWLQRAQGHNNGICWGNQLFVTVVDTTRST NMSLCAAISTSGLNDIFEAQKIEWHEETTYKNTNFKEYLRHGEEYDLQFIFQLCKITLTA DVMTYIHSMNSTILEDWNFGLQPPPGGTLEDTYRFVTSQAIACQKHTPPAPKEDPLKK YTFWEVNLKEKFSADLDQFPLGRKFLLQAGLKAKPKFTLGKRKATPTTSSTSTTAKRK KRKLELSGR

> SEQ ID NO: 3 [DE-loop, HPV genotype 16] L1 avi3 Protein MSLWLPSEATVYLPPVPVSKVVSTDEYVARTNIYYHAGTSRLLAVGHPYFPIKKPNNN KILVPKVSGLQYRVFRIHLPDPNKFGFPDTSFYNPDTQRLVWACVGVEVGRGQPLGV GISGHPLLNKLDDTENASAGLNDIFEAQKIEWHEAGVDNRECISMDYKQTQLCLIGCK PPIGEHWGKGSPCTNVAVNPGDCPPLELINTVIQDGDMVDTGFGAMDFTTLQANKSE VPLDICTSICKYPDYIKMVSEPYGDSLFFYLRREQMFVRHLFNRAGAVGENVPDDLYIK GSGSTANLASSNYFPTPSGSMVTSDAQIFNKPYWLQRAQGHNNGICWGNQLFVTW DTTRSTNMSLCAAISTSETTYKNTNFKEYLRHGEEYDLQFIFQLCKITLTADVMTYIHS MNSTILEDWNFGLQPPPGGTLEDTYRFVTSQAIACQKHTPPAPKEDPLKKYTFWEVN LKEKFSADLDQFPLGRKFLLQAGLKAKPKFTLGKRKATPTTSSTSTTAKRKKRKLEL

> SEQ ID NO: 4 [FG-loop HPV genotype 16] L1avi4) Protein MSLWLPSEATVYLPPVPVSKVVSTDEYVARTNIYYHAGTSRLLAVGHPYFPIKKPNNN KILVPKVSGLQYRVFRIHLPDPNKFGFPDTSFYNPDTQRLVWACVGVEVGRGQPLGV GISGHPLLNKLDDTENASAAGVDNRECISMDYKQTQLCLIGCKPPIGEHWGKGSPCT NVAVNPGDCPPLELINTVIQDGDMVDTGFGAMDFTTLQANKSEVPLDICTSICKYPDYI KMVSEPYGDSLFFYLRREQMFVRHLFNRAGAVGENVPDDLYIKGLNDIFEAQKI EWH EASSNYFPTPSGSMVTSDAQIFNKPYWLQRAQGHNNGICWGNQLFVTVVDTTRSTN MSLCAAISTSETTYKNTNFKEYLRHGEEYDLQFIFQLCKITLTADVMTYIHSMNSTILED WNFGLQPPPGGTLEDTYRFVTSQAIACQKHTPPAPKEDPLKKYTFWEVNLKEKFSAD LDQFPLGRKFLLQAGLKAKPKFTLGKRKATPTTSSTSTTAKRKKRKLELSGRF

> SEQ ID NO: 5 g i 196271081 ref | N P_041332.11 major capsid L1 protein [Human papillomavirus type 16] Protein

MSLWLPSEATVYLPPVPVSKVVSTDEYVARTNIYYHAGTSRLLAVGHPYFPIKKPNN N KILVPKVSGLQYRVFRIHLPDPNKFGFPDTSFYNPDTQRLVWACVGVEVGRGQPLGV GISGHPLLNKLDDTENASAYAANAGVDNRECISMDYKQTQLCLIGCKPPIGEHWGKG SPCTNVAVNPGDCPPLELINTVIQDGDMVHTGFGAMDFTTLQANKSEVPLDICTSICK YPDYIKMVSEPYGDSLFFYLRREQMFVRHLFNRAGTVGENVPDDLYIKGSGSTANLA SSNYFPTPSGSMVTSDAQIFNKPYWLQRAQGHNNGICWGNQLFVTWDTTRSTNMS LCAAISTSETTYKNTNFKEYLRHGEEYDLQFIFQLCKITLTADVMTYIHSMNSTILEDWN FGLQPPPGGTLEDTYRFVTQAIACQKHTPPAPKEDDPLKKYTFWEVNLKEKFSADLD QFPLGRKFLLQAGLKAKPKFTLGKRKATPTTSSTSTTAKRKKRKL

> SEQ ID NO: 6 L1Avi2 (Hlloop, HPV1 18genotype) Protein

MAVWQAASGKVYLPPSTPVARVQSTDEYVERTNIYYHAFTDRLLTVGHPYFNIFNND GNKLEVPKVSGNQHRVFRLRLPDPNRFALADMSVYNPDKERLVWAITGLEIGRGQPL GVGTSGHPLFNKFNDTENGNKYTNTSTDDRQNISFDPKQLQMFIIGCTPCIGEHWDR APACVEDEQLGRCPPIELVNTFIQDDDMADIGYGNLNFKALQQNRSDVSLDIVDEICKY PDFLKMQNDVYGDACFFYARREQCYARRFFVRGGKPGDDIPAEQIDAGKLKNEFYIP AAGGQAQGQLGNSMYFPTVSGSLVSSDAQLFNRPFWLQRAQGHNNGILWGNQLFV TVLDNTRNTNFSIAVYSEGLNDIFEAQKI EWHEQDIANYDSSKSREYQRHVEEYEVSMI LQLCKIPLKPEVLAHINAMNPAILEDWQLGFIPTPDNPIHDTYRYIDSLATRCPDKVPAK EKEDPYGKYVFWNVDLSERLSLDLDQYPLGRKFLFQAGLRQKSVNGSVTRTVSRGA KRKRKSGR

> SEQ ID NO: 7 L1avi3 (DEIoop, HPV1 18genotype) Protein MAVWQAASGKVYLPPSTPVARVQSTDEYVERTNIYYHAFTDRLLTVGHPYFNIFNND GNKLEVPKVSGNQHRVFRLRLPDPNRFALADMSVYNPDKERLVWAITGLEIGRGQPL GVGTSGHPLFNKFNDTENGNGLNDIFEAQKI EWHETSTDDRQNISFDPKQLQMFIIGC TPCIGEHWDRAPACVEDEQLGRCPPIELVNTFIQDDDMADIGYGNLNFKALQQNRSD VSLDIVDEICKYPDFLKMQNDVYGDACFFYARREQCYARRFFVRGGKPGDDIPAEQID AGKLKNEFYIPAAGGQAQGQLGNSMYFPTVSGSLVSSDAQLFNRPFWLQRAQGHNN GILWGNQLFVTVLDNTRNTNFSIAVYSEQDIANYDSSKSREYQRHVEEYEVSMILQLC KIPLKPEVLAHINAMNPAILEDWQLGFIPTPDNPIHDTYRYIDSLATRCPDKVPAKEKED PYGKYVFWNVDLSERLSLDLDQYPLGRKFLFQAGLRQKSVNGSVTRTVSRGAKRKR KSGRF > SEQ ID NO: 8 gi|256807737|gb|ACV30153.11 L1 [Human papillomavirus type 118] Protein MAVWQAASGKVYLPPSTPVARVQSTDEYVERTNIYYHAFTDRLLTVGHPYFNIFNND GNKLEVPKVSGNQHRVFRLRLPDPNRFALADMSVYNPDKERLVWAITGLEIGRGQPL GVGTSGHPLFNKFNDTENGNKYTNTSTDDRQNISFDPKQLQMFIIGCTPCIGEHWDR APACVEDEQLGRCPPIELVNTFIQDDDMADIGYGNLNFKALQQNRSDVSLDIVDEICKY PDFLKMQNDVYGDACFFYARREQCYARRFFVRGGKPGDDIPAEQIDAGKLKNEFYIP AAGGQAQGQLGNSMYFPTVSGSLVSSDAQLFNRPFWLQRAQGHNNGILWGNQLFV TVLDNTRNTNFSIAVYSEAGKIQDIANYDSSKSREYQRHVEEYEVSMILQLCKIPLKPE VLAHINAMNPAILEDWQLGFIPTPDNPIHDTYRYIDSLATRCPDKVPAKEKEDPYGKYV FWNVDLSERLSLDLDQYPLGRKFLFQAGLRQKSVNGSVTRTVSRGAKRKRK > SEQ ID NO: 9 Avi3 Europasisk elg papillomavirus (DEIoop) Protein

MAFWQPSQRLYLPPTPVTKVLCSEQYIRRKDVFYHGETERMLTVGHPYYEIKQSGSG KTIPKVSPNQYRVFRILLPDPNQFALPDKAMYDPSKERLVWAVVGVQVSRGQPLGGS VSGHSYQNTLIDAENVSGLNDIFEAQKI EWHEQGTDDRKQGGMDVKQQQILLLGCTP AIGEYWTTARPCVTDRPETGSCPPIELKNKPIEDGDMMDIGFGAANFKELNATKSDLP LDIAKDICLYPDYLKMTEEAAGNSMFFFARKEQVYVRHIWSRGGTDKEMPPEAYFLKP KGGDQTQKMPSILFGVPSGSLVSTDGQLFNRPYWLFRAQGMNNGICWLNQLFVTVG DNTRGTTLTITVPTSGSPLTEYDTSKFNVFQRHVEEYKLAFVFQLCSVTLSPETVSHLQ GLMPSILEHWDINMQPPTSSILEDTYRYLESPATKCADNVTPMGPEDPYAGLKFWEV NLKERLSLDLDQFPLGRRFLAQQGLGCSTRKRVAPVPKVTEKRIVRKRRKGN > SEQ ID NO: 10 sp|P11326|VL1_PAPVE Major capsid protein L1 OS=European elk papillomavirus GN=L1 PE=3 SV=1 Protein

MAFWQPSQRLYLPPTPVTKVLCSEQYIRRKDVFYHGETERMLTVGHPYYEIKQSGSG KTIPKVSPNQYRVFRILLPDPNQFALPDKAMYDPSKERLVWAVVGVQVSRGQPLGGS VSGHSYQNTLIDAENVSKKVNAQGTDDRKQGGMDVKQQQILLLGCTPAIGEYWTTAR PCVTDRPETGSCPPIELKNKPIEDGDMMDIGFGAANFKELNATKSDLPLDIAKDICLYP DYLKMTEEAAGNSMFFFARKEQVYVRHIWSRGGTDKEMPPEAYFLKPKGGDQTQKM PSILFGVPSGSLVSTDGQLFNRPYWLFRAQGMNNGICWLNQLFVTVGDNTRGTTLTIT VPTSGSPLTEYDTSKFNVFQRHVEEYKLAFVFQLCSVTLSPETVSHLQGLMPSILEHW DINMQPPTSSILEDTYRYLESPATKCADNVTPMGPEDPYAGLKFWEVNLKERLSLDLD QFPLGRRFLAQQGLGCSTRKRVAPVPKVTEKRIVRKRRKGN > SEQ ID NO: 11 [H4-loop, HPV genotype 16] L1Avi1] DNA

GATATCATGGAGATAATTAAAATGATAACCATCTCGCAAATAAATAAGTATTTTACT GTTTTCGTAACAGTTTTGTAATAAAAAAACCTATAAATATTCCGGATTATTCATACC GTCCCACCATCGGGCGCGGATCTCTACTAGTATGTCTCTGTGGCTGCCCTCTGAA GCCACCGTCTACCTGCCCCCCGTCCCTGTCTCTAAGGTCGTCAGCACCGACGAAT ACGTCGCCAGGACCAACATCTACTACCACGCCGGAACCTCTAGGCTGCTGGCCG TCGGACACCCCTACTTCCCCATCAAGAAGCCCAACAACAACAAGATCCTGGTCCC CAAGGTGTCCGGACTGCAGTACAGGGTGTTCAGGATCCACCTCCCCGACCCCAA CAAGTTCGGATTCCCCGACACCTCTTTCTACAACCCCGACACCCAGAGGCTCGTC TGGGCCTGCGTCGGAGTCGAAGTCGGAAGGGGACAGCCCCTGGGAGTCGGAAT CTCTGGACACCCCCTGCTGAACAAGCTGGACGACACCGAAAACGCCTCTGCCTA CGCCGCCAACGCCGGTGTCGACAACAGGGAATGCATCTCTATGGACTACAAGCA GACCCAGCTGTGCCTGATCGGATGCAAGCCCCCCATCGGAGAACACTGGGGAAA GGGATCTCCCTGCACCAACGTCGCCGTCAACCCCGGCGACTGCCCCCCTCTGGA ACTGATCAACACCGTCATCCAGGACGGCGACATGGTCGACACCGGATTCGGAGC CATGGACTTCACCACCCTGCAGGCCAACAAGTCTGAAGTCCCCCTGGACATCTGC ACCTCTATCTGCAAGTACCCCGACTACATCAAGATGGTGTCTGAACCCTACGGCG ACTCTCTGTTCTTCTACCTGAGGCGCGAACAGATGTTCGTCAGGCACCTGTTCAA CCGCGCCGGTGCCGTCGGAGAAAACGTCCCCGACGACCTGTACATCAAGGGATC TGGATCTACCGCCAACCTGGCCTCTTCTAACTACTTCCCTACCCCTTCTGGATCTA TGGTCACCTCTGACGCCCAGATCTTCAACAAGCCCTACTGGCTGCAGAGGGCCC AGGGACACAACAACGGAATCTGCTGGGGAAACCAGCTGTTCGTCACCGTCGTCG ACACCACCAGGTCTACCAACATGTCCCTGTGCGCCGCCATCTCTACCTCTGAAAC CACCTACAAGAACACCAACTTCAAAGAATACCTGCGCCACGGCGAAGAATACGAC CTGCAGTTCATCTTCCAGCTGTGCAAGATCACCCTGACCGCCGACGTCATGACCT ACATCCACTCTATGAACTCTACCATCTTGGAGGACTGGAACTTCGGACTGCAGCC CCCTCCCGGTGGAACCCTCGAGGACACCTACCGCTTCGTCACCAGCCAGGCTAT CGCCTGCCAGAAGCACGGACTGAACGACATCTTCGAAGCCCAAAAGATCGAATG GCACGAAACCCCCCCTGCCCCCAAAGAGGACCCCCTGAAGAAGTACACCTTCTG GGAAGTCAACCTGAAAGAAAAGTTCTCTGCCGACCTGGACCAGTTCCCCCTGGGA CGCAAGTTCCTGCTGCAAGCCGGACTGAAGGCCAAGCCCAAGTTCACCCTGGGA AAGAGGAAGGCCACCCCCACCACCTCTTCTACCTCTACCACCGCCAAGAGGAAG AAGCGCAAGCTGGAACTGTAAAGCGGCCGC > SEQ ID NO: 12 L1avi2 (Hl-loop, HPV16) DNA

GATATCATGGAGATAATTAAAATGATAACCATCTCGCAAATAAATAAGTATTTTACT GTTTTCGTAACAGTTTTGTAATAAAAAAACCTATAAATATTCCGGATTATTCATACC GTCCCACCATCGGGCGCGGATCTCTACTAGTATGTCTCTGTGGCTGCCCTCTGAA GCCACCGTCTACCTGCCCCCCGTCCCTGTCTCTAAGGTCGTCAGCACCGACGAAT ACGTCGCCAGGACCAACATCTACTACCACGCCGGAACCTCTAGGCTGCTGGCCG TCGGACACCCCTACTTCCCCATCAAGAAGCCCAACAACAACAAGATCCTGGTCCC CAAGGTGTCCGGACTGCAGTACAGGGTGTTCAGGATCCACCTCCCCGACCCCAA CAAGTTCGGATTCCCCGACACCTCTTTCTACAACCCCGACACCCAGAGGCTCGTC TGGGCCTGCGTCGGAGTCGAAGTCGGAAGGGGACAGCCCCTGGGAGTCGGAAT CTCTGGACACCCCCTGCTGAACAAGCTGGACGACACCGAAAACGCCTCTGCCTA CGCCGCCAACGCCGGTGTCGACAACAGGGAATGCATCTCTATGGACTACAAGCA GACCCAGCTGTGCCTGATCGGATGCAAGCCCCCCATCGGAGAACACTGGGGAAA GGGATCTCCCTGCACCAACGTCGCCGTCAACCCCGGCGACTGCCCCCCTCTGGA ACTGATCAACACCGTCATCCAGGACGGCGACATGGTCGACACCGGATTCGGAGC CATGGACTTCACCACCCTGCAGGCCAACAAGTCTGAAGTCCCCCTGGACATCTGC ACCTCTATCTGCAAGTACCCCGACTACATCAAGATGGTGTCTGAACCCTACGGCG ACTCTCTGTTCTTCTACCTGAGGCGCGAACAGATGTTCGTCAGGCACCTCTTCAA CAGGGCCGGTGCCGTCGGAGAAAACGTCCCCGACGACCTGTACATCAAGGGATC TGGATCTACCGCCAACCTGGCCTCTTCTAACTACTTCCCTACCCCTTCTGGATCTA TGGTCACCTCTGACGCCCAGATCTTCAACAAGCCCTACTGGCTGCAGAGGGCCC AGGGACACAACAACGGAATCTGCTGGGGAAACCAGCTGTTCGTCACCGTCGTCG ACACCACCAGGTCTACCAACATGTCCCTGTGCGCCGCCATCTCTACCTCTGGACT GAACGACATCTTCGAGGCCCAAAAGATCGAATGGCACGAGGAAACCACCTACAAG AACACCAACTTCAAAGAATACCTGCGCCACGGCGAAGAATACGACCTGCAGTTCA TCTTCCAGCTGTGCAAGATCACCCTGACCGCCGACGTCATGACCTACATCCACTC TATGAACTCTACCATCTTGGAGGATTGGAACTTCGGACTGCAGCCCCCTCCCGGT GGAACCCTCGAGGACACCTACCGCTTCGTCACCAGCCAGGCTATCGCCTGCCAG AAGCACACCCCCCCTGCCCCCAAAGAGGACCCCCTGAAGAAGTACACCTTCTGG GAAGTCAACCTGAAAGAAAAGTTCTCTGCCGACCTGGACCAGTTCCCCCTGGGAC GCAAGTTCCTGCTGCAAGCCGGACTGAAGGCCAAGCCCAAGTTCACCCTGGGAA AGAGGAAGGCCACCCCCACCACCTCTTCTACCTCTACCACCGCCAAGAGGAAGA AGCGCAAGCTGGAACTGTAAAGCGGCCGC > SEQ ID NO: 13 L1avi3 (DE-loop, HPV16)

GATATCATGGAGATAATTAAAATGATAACCATCTCGCAAATAAATAAGTATTTTACT GTTTTCGTAACAGTTTTGTAATAAAAAAACCTATAAATATTCCGGATTATTCATACC GTCCCACCATCGGGCGCGGATCTCTACTAGTATGTCTCTGTGGCTGCCCTCTGAA GCCACCGTCTACCTGCCCCCCGTCCCTGTCTCTAAGGTCGTCAGCACCGACGAAT ACGTCGCCAGGACCAACATCTACTACCACGCCGGAACCTCTAGGCTGCTGGCCG TCGGACACCCCTACTTCCCCATCAAGAAGCCCAACAACAACAAGATCCTGGTCCC CAAGGTGTCCGGACTGCAGTACAGGGTGTTCAGGATCCACCTCCCCGACCCCAA CAAGTTCGGATTCCCCGACACCTCTTTCTACAACCCCGACACCCAGAGGCTCGTC TGGGCCTGCGTCGGAGTCGAAGTCGGAAGGGGACAGCCCCTGGGAGTCGGAAT CTCTGGACACCCCCTGCTGAACAAGCTGGACGACACCGAAAACGCCTCTGCCGG ACTGAACGACATCTTCGAGGCCCAAAAGATCGAATGGCACGAGGCCGGTGTCGA CAACAGGGAATGCATCTCTATGGACTACAAGCAGACCCAGCTGTGCCTGATCGGA TGCAAGCCCCCCATCGGAGAACACTGGGGAAAGGGATCTCCCTGCACCAACGTC GCCGTCAACCCCGGCGACTGCCCCCCTCTGGAACTGATCAACACCGTCATCCAG GACGGCGACATGGTCGACACCGGATTCGGAGCCATGGACTTCACCACCCTGCAG GCCAACAAGTCTGAAGTCCCCCTGGACATCTGCACCTCTATCTGCAAGTACCCCG ACTACATCAAGATGGTGTCTGAACCCTACGGCGACTCTCTGTTCTTCTACCTGAG GCGCGAACAGATGTTCGTCAGGCACCTCTTCAACAGGGCCGGTGCCGTCGGAGA AAACGTCCCCGACGACCTGTACATCAAGGGATCTGGATCTACCGCCAACCTGGCC TCTTCTAACTACTTCCCTACCCCTTCTGGATCTATGGTCACCTCTGACGCCCAGAT CTTCAACAAGCCCTACTGGCTGCAGAGGGCCCAGGGACACAACAACGGAATCTG CTGGGGAAACCAGCTGTTCGTCACCGTCGTCGACACCACCAGGTCTACCAACATG TCCCTGTGCGCCGCCATCTCTACCTCTGAAACCACCTACAAGAACACCAACTTCA AAGAATACCTGCGCCACGGCGAAGAATACGACCTGCAGTTCATCTTCCAGCTGTG CAAGATCACCCTGACCGCCGACGTCATGACCTACATCCACTCTATGAACTCTACC ATCTTGGAGGATTGGAACTTCGGACTGCAGCCCCCTCCCGGTGGAACCCTCGAG GACACCTACCGCTTCGTCACCAGCCAGGCTATCGCCTGCCAGAAGCACACCCCC CCTGCCCCCAAAGAGGACCCCCTGAAGAAGTACACCTTCTGGGAAGTCAACCTGA AAGAAAAGTTCTCTGCCGACCTGGACCAGTTCCCCCTGGGACGCAAGTTCCTGCT GCAAGCCGGACTGAAGGCCAAGCCCAAGTTCACCCTGGGAAAGAGGAAGGCCAC CCCCACCACCTCTTCTACCTCTACCACCGCCAAGAGGAAGAAGCGCAAGCTGGAA CTGTAAAGCGGCCGC > SEQ ID NO: 14 L1avi4 (FG-loop, HPV16)

GATATCATGGAGATAATTAAAATGATAACCATCTCGCAAATAAATAAGTATTTTACT GTTTTCGTAACAGTTTTGTAATAAAAAAACCTATAAATATTCCGGATTATTCATACC GTCCCACCATCGGGCGCGGATCTCTACTAGTATGTCTCTGTGGCTGCCCTCTGAA GCCACCGTCTACCTGCCCCCCGTCCCTGTCTCTAAGGTCGTCAGCACCGACGAAT ACGTCGCCAGGACCAACATCTACTACCACGCCGGAACCTCTAGGCTGCTGGCCG TCGGACACCCCTACTTCCCCATCAAGAAGCCCAACAACAACAAGATCCTGGTCCC CAAGGTGTCCGGACTGCAGTACAGGGTGTTCAGGATCCACCTCCCCGACCCCAA CAAGTTCGGATTCCCCGACACCTCTTTCTACAACCCCGACACCCAGAGGCTCGTC TGGGCCTGCGTCGGAGTCGAAGTCGGAAGGGGACAGCCCCTGGGAGTCGGAAT CTCTGGACACCCCCTGCTGAACAAGCTGGACGACACCGAAAACGCCTCTGCCTA CGCCGCCAACGCCGGTGTCGACAACAGGGAATGCATCTCTATGGACTACAAGCA GACCCAGCTGTGCCTGATCGGATGCAAGCCCCCCATCGGAGAACACTGGGGAAA GGGATCTCCCTGCACCAACGTCGCCGTCAACCCCGGCGACTGCCCCCCTCTGGA ACTGATCAACACCGTCATCCAGGACGGCGACATGGTCGACACCGGATTCGGAGC CATGGACTTCACCACCCTGCAGGCCAACAAGTCTGAAGTCCCCCTGGACATCTGC ACCTCTATCTGCAAGTACCCCGACTACATCAAGATGGTGTCTGAACCCTACGGCG ACTCTCTGTTCTTCTACCTGAGGCGCGAACAGATGTTCGTCAGGCACCTCTTCAA CAGGGCCGGTGCCGTCGGAGAAAACGTCCCCGACGACCTGTACATCAAGGGACT GAACGACATCTTCGAGGCCCAAAAGATCGAATGGCACGAGGCCTCTTCTAACTAC TTCCCTACCCCTTCTGGATCTATGGTCACCTCTGACGCCCAGATCTTCAACAAGCC CTACTGGCTGCAGAGGGCCCAGGGACACAACAACGGAATCTGCTGGGGAAACCA GCTGTTCGTCACCGTCGTCGACACCACCAGGTCTACCAACATGTCCCTGTGCGCC GCCATCTCTACCTCTGAAACCACCTACAAGAACACCAACTTCAAAGAATACCTGCG CCACGGCGAAGAATACGACCTGCAGTTCATCTTCCAGCTGTGCAAGATCACCCTG ACCGCCGACGTCATGACCTACATCCACTCTATGAACTCTACCATCTTGGAGGATT GGAACTTCGGACTGCAGCCCCCTCCCGGTGGAACCCTCGAGGACACCTACCGCT TCGTCACCAGCCAGGCTATCGCCTGCCAGAAGCACACCCCCCCTGCCCCCAAAG AGGACCCCCTGAAGAAGTACACCTTCTGGGAAGTCAACCTGAAAGAAAAGTTCTC TGCCGACCTGGACCAGTTCCCCCTGGGACGCAAGTTCCTGCTGCAAGCCGGACT GAAGGCCAAGCCCAAGTTCACCCTGGGAAAGAGGAAGGCCACCCCCACCACCTC TTCTACCTCTACCACCGCCAAGAGGAAGAAGCGCAAGCTGGAACTGTAAAGCGG CCGC > SEQ ID NO: 15 L1avi2 (Hl-loop, HPV1 18)

GATATCATGGAGATAATTAAAATGATAACCATCTCGCAAATAAATAAGTATTTTACT GTTTTCGTAACAGTTTTGTAATAAAAAAACCTATAAATATTCCGGATTATTCATACC GTCCCACCATCGGGCGCGGATCTCTACTAGTATGGCCGTCTGGCAGGCCGCCTC TGGAAAGGTCTACCTGCCCCCCTCTACCCCCGTCGCCAGGGTCCAGTCTACCGA CGAATACGTCGAAAGGACCAACATCTACTACCACGCCTTCACCGACAGGCTGCTG ACCGTCGGACACCCCTACTTCAACATCTTCAACAACGACGGAAACAAGCTCGAAG TCCCCAAGGTGTCCGGAAACCAGCACAGGGTGTTCAGGCTGAGGCTGCCCGACC CCAACCGCTTCGCCCTGGCCGACATGTCTGTCTACAACCCCGACAAAGAAAGGCT CGTCTGGGCCATCACCGGACTGGAAATCGGAAGGGGACAGCCCCTGGGAGTCG GAACCTCTGGACACCCCCTGTTCAACAAGTTCAACGACACCGAAAACGGCAACAA GTACACCAACACCTCCACCGACGACAGGCAGAACATCTCTTTCGACCCCAAGCAG CTGCAGATGTTCATCATCGGATGCACCCCCTGCATCGGAGAACACTGGGACAGG GCCCCTGCCTGCGTCGAGGACGAACAGCTGGGAAGGTGCCCCCCCATCGAACTG GTCAACACCTTCATCCAGGACGACGACATGGCCGACATCGGATACGGAAACCTGA ACTTCAAGGCCCTGCAGCAGAACCGCTCTGACGTGTCCCTGGACATCGTCGACG AAATCTGCAAGTACCCCGACTTCCTGAAGATGCAGAACGACGTCTACGGCGACGC CTGCTTCTTCTACGCTAGGCGCGAACAGTGCTACGCCAGGCGCTTCTTCGTCCGC GGAGGAAAGCCCGGCGACGACATCCCCGCCGAACAGATCGACGCCGGAAAGCT GAAGAACGAATTCTACATCCCTGCCGCCGGTGGACAGGCCCAGGGACAGCTCGG AAACTCTATGTACTTCCCCACCGTCAGCGGATCTCTCGTCAGCTCTGACGCCCAG CTGTTCAACAGGCCCTTCTGGCTGCAGCGCGCTCAGGGACACAACAACGGAATC CTGTGGGGAAACCAGTTGTTCGTCACCGTCCTGGACAACACCCGCAACACCAACT TCTCTATCGCCGTCTACTCTGAGGGACTGAACGACATCTTCGAAGCCCAAAAGAT CGAATGGCACGAGCAGGACATTGCCAACTACGACTCTTCTAAGTCTAGGGAATAC CAGCGCCACGTCGAAGAGTACGAAGTCTCTATGATCCTGCAGCTGTGCAAGATCC CCCTGAAGCCCGAAGTCCTGGCCCACATCAACGCCATGAACCCCGCCATCTTGG AGGACTGGCAGCTGGGATTCATCCCCACCCCCGACAACCCCATCCACGACACCT ACCGCTACATCGACTCCCTGGCCACCAGGTGCCCTGACAAGGTCCCCGCCAAAG AAAAAGAGGACCCCTACGGCAAATACGTGTTCTGGAACGTCGACCTGTCTGAAAG GCTGTCTCTGGACCTGGACCAGTACCCCCTGGGACGCAAGTTCCTGTTCCAAGC CGGACTGAGGCAGAAGTCTGTCAACGGATCTGTCACCAGGACCGTCAGCAGGGG AGCCAAGAGGAAGCGCAAGTAAAGCGGCCGC > SEQ ID NO: 16 L1avi3 (DE-loop, HPV1 18)

GATATCATGGAGATAATTAAAATGATAACCATCTCGCAAATAAATAAGTATTTTACT GTTTTCGTAACAGTTTTGTAATAAAAAAACCTATAAATATTCCGGATTATTCATACC GTCCCACCATCGGGCGCGGATCTCTACTAGTATGGCCGTCTGGCAGGCCGCCTC TGGAAAGGTCTACCTGCCCCCCTCTACCCCCGTCGCCAGGGTCCAGTCTACCGA CGAATACGTCGAAAGGACCAACATCTACTACCACGCCTTCACCGACAGGCTGCTG ACCGTCGGACACCCCTACTTCAACATCTTCAACAACGACGGAAACAAGCTCGAAG TCCCCAAGGTGTCCGGAAACCAGCACAGGGTGTTCAGGCTGAGGCTGCCCGACC CCAACCGCTTCGCCCTGGCCGACATGTCTGTCTACAACCCCGACAAAGAAAGGCT CGTCTGGGCCATCACCGGACTGGAAATCGGAAGGGGACAGCCCCTGGGAGTCG GAACCTCTGGACACCCCCTGTTCAACAAGTTCAACGACACCGAAAACGGCAACGG ACTGAACGACATCTTCGAAGCCCAAAAGATCGAATGGCACGAGACCTCCACCGAC GACAGGCAGAACATCTCTTTCGACCCCAAGCAGCTGCAGATGTTCATCATCGGAT GCACCCCCTGCATCGGAGAACACTGGGACAGGGCCCCTGCCTGCGTCGAGGAC GAACAGCTGGGAAGGTGCCCCCCCATCGAACTGGTCAACACCTTCATCCAGGAC GACGACATGGCCGACATCGGATACGGAAACCTGAACTTCAAGGCCCTGCAGCAG AACCGCTCTGACGTGTCCCTGGACATCGTCGACGAAATCTGCAAGTACCCCGACT TCCTGAAGATGCAGAACGACGTCTACGGCGACGCCTGCTTCTTCTACGCTAGGCG CGAACAGTGCTACGCCAGGCGCTTCTTCGTCCGCGGAGGAAAGCCCGGCGACGA CATCCCCGCCGAACAGATCGACGCCGGAAAGCTGAAGAACGAATTCTACATCCCT GCCGCCGGTGGACAGGCCCAGGGACAGCTCGGAAACTCTATGTACTTCCCCACC GTCAGCGGATCTCTCGTCAGCTCTGACGCCCAGCTGTTCAACAGGCCCTTCTGGC TGCAGCGCGCTCAGGGACACAACAACGGAATCCTGTGGGGAAACCAGTTGTTCG TCACCGTCCTGGACAACACCCGCAACACCAACTTCTCTATCGCCGTCTACTCTGA GGCCGGAAAGATCCAGGACATTGCCAACTACGACTCTTCTAAGTCTAGGGAATAC CAGCGCCACGTCGAAGAGTACGAAGTCTCTATGATCCTGCAGCTGTGCAAGATCC CCCTGAAGCCCGAAGTCCTGGCCCACATCAACGCCATGAACCCCGCCATCTTGG AGGACTGGCAGCTGGGATTCATCCCCACCCCCGACAACCCCATCCACGACACCT ACCGCTACATCGACTCCCTGGCCACCAGGTGCCCTGACAAGGTCCCCGCCAAAG AAAAAGAGGACCCCTACGGCAAATACGTGTTCTGGAACGTCGACCTGTCTGAAAG GCTGTCTCTGGACCTGGACCAGTACCCCCTGGGACGCAAGTTCCTGTTCCAAGC CGGACTGAGGCAGAAGTCTGTCAACGGATCTGTCACCAGGACCGTCAGCAGGGG AGCCAAGAGGAAGCGCAAGTAAAGCGGCCGC > SEQ ID NO: 17 PAPVE-L1 avi3

GATATCATGGAGATAATTAAAATGATAACCATCTCGCAAATAAATAAGTATTTTACT GTTTTCGTAACAGTTTTGTAATAAAAAAACCTATAAATATTCCGGATTATTCATACC GTCCCACCATCGGGCGCGGATCTCTACTAGTATGGCCTTCTGGCAGCCCTCTCAG CGTCTGTACCTGCCCCCCACCCCCGTCACCAAGGTCCTGTGCTCTGAACAGTACA TCAGGCGCAAGGACGTGTTCTACCACGGCGAAACCGAAAGGATGCTGACCGTCG GACACCCCTACTACGAAATCAAGCAGTCTGGATCTGGAAAGACCATCCCCAAGGT GTCCCCCAACCAGTACAGGGTGTTCAGGATCCTGCTGCCCGACCCTAACCAGTTC GCCCTGCCCGACAAGGCTATGTACGACCCCTCTAAGGAAAGGCTCGTCTGGGCC GTCGTCGGAGTCCAGGTGTCCCGTGGACAGCCTCTGGGAGGATCTGTCTCTGGA CACTCTTACCAGAACACCCTGATCGACGCCGAAAACGTGTCCGGACTGAACGACA TCTTCGAAGCCCAAAAGATCGAATGGCACGAACAGGGAACCGACGACCGCAAGC AGGGTGGAATGGACGTCAAGCAGCAGCAGATCCTGCTCCTGGGATGCACCCCCG CCATCGGAGAATACTGGACCACCGCCAGGCCCTGCGTCACCGACAGGCCCGAAA CCGGATCTTGCCCCCCCATCGAACTGAAGAACAAGCCCATCGAGGACGGCGACA TGATGGACATCGGATTCGGAGCCGCCAACTTCAAGGAACTGAACGCCACCAAGTC TGACCTGCCCCTGGACATCGCCAAGGACATCTGCCTGTACCCCGACTACCTGAAG ATGACCGAAGAAGCCGCCGGAAACTCTATGTTCTTCTTCGCCCGCAAGGAACAGG TCTACGTCCGCCACATCTGGTCCCGCGGAGGAACCGACAAGGAAATGCCCCCCG AAGCCTACTTCCTGAAGCCCAAGGGTGGCGACCAGACCCAGAAGATGCCCTCTAT CCTGTTCGGAGTCCCCTCTGGATCTCTCGTCAGCACCGACGGACAGCTGTTCAAC AGGCCCTACTGGCTGTTCAGGGCCCAGGGAATGAACAACGGAATCTGCTGGCTG AACCAGCTGTTCGTCACCGTCGGAGACAACACCAGGGGAACCACCCTGACCATC ACCGTCCCCACCTCTGGATCCCCCCTGACCGAATACGACACCTCCAAGTTCAACG TGTTCCAGAGGCACGTCGAAGAGTACAAGCTGGCCTTCGTGTTCCAGCTGTGCTC TGTCACCCTGTCTCCCGAAACCGTCAGCCACCTCCAGGGACTGATGCCTTCCATC CTGGAACACTGGGACATCAACATGCAGCCCCCCACCTCTTCTATCCTCGAGGACA CCTACCGCTACCTGGAATCTCCTGCCACCAAGTGCGCCGACAACGTCACCCCCAT GGGACCCGAGGACCCCTACGCCGGACTGAAGTTCTGGGAAGTCAACCTGAAGGA ACGCCTGTCCCTGGACCTGGACCAGTTCCCCCTGGGAAGGCGCTTCCTGGCCCA GCAGGGACTGGGATGCTCTACCCGCAAGAGGGTCGCCCCCGTCCCTAAGGTCAC CGAAAAGAGGATCGTCCGCAAGAGGCGCAAGGGAAACTAAAGCGGCCGCTAA > SEQ ID NO: 18 A1 >mSA- Her2-ECD|23-686

AEAGITGTWYNQHGSTFTVTAGADGNLTGQYENRAQGTGCQNSPYTLTGRYNGTKL EWRVEWNNSTENCHSRTEWRGQYQGGAEARINTQWNLTYEGGSGPATEQGQDTF TKVKGGSTQVCTGTDMKLRLPASPETHLDMLRHLYQGCQVVQGNLELTYLPTNASLS FLQDIQEVQGYVLIAHNQVRQVPLQRLRIVRGTQLFEDNYALAVLDNGDPLNNTTPVT GASPGGLRELQLRSLTEI LKGGVLIQRN PQLCYQDTI LWKDI FH KN NQLALTLI DTN RS RACHPCSPMCKGSRCWGESSEDCQSLTRTVCAGGCARCKGPLPTDCCHEQCAAG CTGPKHSDCLACLHFNHSGICELHCPALVTYNTDTFESMPNPEGRYTFGASCVTACP YNYLSTDVGSCTLVCPLHNQEVTAEDGTQRCEKCSKPCARVCYGLGMEHLREVRAV TSANIQEFAGCKKIFGSLAFLPESFDGDPASNTAPLQPEQLQVFETLEEITGYLYISAW PDSLPDLSVFQNLQVIRGRILHNGAYSLTLQGLGISWLGLRSLRELGSGLALIHHNTHL CFVHTVPWDQLFRNPHQALLHTANRPEDECVGEGLACHQLCARGHCWGPGPTQCV NCSQFLRGQECVEECRVLQGLPREYVNARHCLPCHPECQPQNGSVTCFGPEADQC VACAHYKDPPFCVARCPSGVKPDLSYMPIWKFPDEEGACQPCPINCTHSCVDLDDK GCPAEQRASPLTSIISAVVGILLWVLGWFGILIKRRQQKIRKYTHHHHHH

> SEQ ID NO: 19 A2 >mSA-IL-5(C63T/C105T)

AEAGITGTWYNQHGSTFTVTAGADGNLTGQYENRAQGTGCQNSPYTLTGRYNGTKL EWRVEWNNSTENCHSRTEWRGQYQGGAEARINTQWNLTYEGGSGPATEQGQDTF TKVKGGSIPTEIPTSALVKETLALLSTHRTLLIANETLRIPVPVHKNHQLTTEEIFQGIG TL ESQTVQGGTVERLFKNLSLIKKYIDGQKKKTGEERRRVNQFLDYLQEFLGVMNTEWII ES*SGRK

> SEQ ID NO: 20 A3 >PCSK9|31-692|:mSA:HIS

QEDEDGDYEELVLALRSEEDGLAEAPEHGTTATFHRCAKDPWRLPGTYWVLKEETH LSQSERTARRLQAQAARRGYLTKILHVFHGLLPGFLVKMSGDLLELALKLPHVDYIEED SSVFAQSIPWNLERITPPRYRADEYQPPDGGSLVEVYLLDTSIQSDHREIEGRVMVTD FENVPEEDGTRFHRQASKCDSHGTHLAGVVSGRDAGVAKGASMRSLRVLNCQGKG TVSGTLIGLEFIRKSQLVQPVGPLVVLLPLAGGYSRVLNAACQRLARAGVVLVTAAGN FRDDACLYSPASAPEVITVGATNAQDQPVTLGTLGTNFGRCVDLFAPGEDIIGASSDC STCFVSQSGTSQAAAHVAGIAAMMLSAEPELTLAELRQRLIHFSAKDVINEAWFPEDQ RVLTPNLVAALPPSTHGAGWQLFCRTVWSAHSGPTRMATAVARCAPDEELLSCSSF SRSGKRRGERMEAQGGKLVCRAHNAFGGEGVYAIARCCLLPQANCSVHTAPPAEAS MGTRVHCHQQGHVLTGCSSHWEVEDLGTHKPPVLRPRGQPNQCVGHREASIHASC CHAPGLECKVKEHGIPAPQEQVTVACEEGWTLTGCSALPGTSHVLGAYAVDNTCW RSRDVSTTGSTSEGAVTAVAICCRSRHLAQASQELQGGSAEAGITGTWYNQHGSTFT VTAGADGNLTGQYENRAQGTGCQNSPYTLTGRYNGTKLEWRVEWNNSTENCHSRT EWRGQYQGGAEARI NTQWN LTYEGGSGPATEQGQDTFTKVKH H H H H H

> SEQ ID NO: 21 A4 >mSA-ID1 ID2a-HIS AEAGITGTWYNQHGSTFTVTAGADGNLTGQYENRAQGTGCQNSPYTLTGRYNGTKL EWRVEWNNSTENCHSRTEWRGQYQGGAEARINTQWNLTYEGGSGPATEQGQDTF TKVKGGSNYIKGDPYFAEYATKLSFILNPSDANNPSGETANHNDEACNCNESGISSVG QAQTSGPSSNKTCITHSSIKTNKKKECKDVKLGVRENDKDLKICVIEDTSLSGVDNCC CQDLLGILQENCSDNKRGSSSNDSCDNKNQDECQKKLEKVFASLTNGYKCDKCKSG TSRSKKKWIWKKSSGNEEGLQEEYANTIGLPPRTQSLYLGNLPKLENVCEDVKDINFD TKEKFLAGCLIVSFHEGKNLKKRYPQNKNSGNKENLCKALEYSFADYGDLIKGTSIWD NEYTKDLELNLQNNFGKLFGKYIKKNNTAEQDTSYSSLDELRESWWNTNKKYIWTAM KHGAEMNITTCNADGSVTGSGSSCDDIPTIDLIPQYLRFLQEWVENFCEQRQAKVKDV ITNCKSCKESGNKCKTECKTKCKDECEKYKKFIEACGTAGGGIGTAGSPWSKRWDQI YKRYSKHIEDAKRNRKAGTKNCGTSSTTNAAASTDENKCVQSDIDSFFKHLIDIGLTTP SSYLSNVLDDNICGADKAPWTTYTTYTTTEKCNKERDKSKSQSSDTLVVVNVPSPLG NTPYRYKYACQCKIPTNEETCDDRKEYMNQWSCGSARTMKRGYKNDNYELCKYNG VDVKPTTVRSNSSKLDHHHHHH

> SEQ ID NO: 22 A5 >mSA-RO-HIS AEAGITGTWYNQHGSTFTVTAGADGNLTGQYENRAQGTGCQNSPYTLTGRYNGTKL EWRVEWNNSTENCHSRTEWRGQYQGGAEARINTQWNLTYEGGSGPATEQGQDTF TKVKGGSTSENRNKRIGGPKLRGNVTSNIKFPSDNKGKIIRGSNDKLNKNSEDVLEQS EKSLVSENVPSGLDIDDIPKESIFIQEDQEGQTHSELNPETSEHSKDLNNNGSKNESSD IISENNKSNKVQNHFESLSDLELLENSSQDNLDKDTISTEPFPNQKHKDLQQDLNDEPL EPFPTQIHKDYKEKNLINEEDSEPFPRQKH KKVDNHNEEKNVFHENGSANGNQGSLK LKSFDEHLKDEKIENEPLVHENLSIPNDPIEQILNQPEQETNIQEQLYNEKQNVEEKQN SQIPSLDLKEPTNEDILPNHNPLENIKQSESEINHVQDHALPKENIIDKLDNQKEHIDQS QHNINVLQENNINNHQLEPQEKPNIESFEPKNIDSEIILPENVETEEIIDDVPSPKHSNH E TFEEETSESEHEEAVSEKNAHETVEHEETVSQESNPEKADNDGNVSQNSNNELNEN EFVESEKSEHEARSKAKEASSYDYILGWEFGGGVPEHKKEENMLSHLYVSSKDKENI SKENDDVLDEKEEEAEETEEEELEEKNEEETESEISEDEEEEEEEEEKEEENDKKKEQ EKEQSNENNDQKKDMEAQNLISKNQNNNEKNVKEAAESIMKTLAGLIKGNNQIDSTLK DLVEELSKYFKNHRSHHHHHH > SEQ ID NO: 23 A6 >HIS-RO-mSA

GSTSENRNKRIGGPKLRGNVTSNIKFPSDNKGKIIRGSNDKLNKNSEDVLEQSEKSL V SENVPSGLDIDDIPKESIFIQEDQEGQTHSELNPETSEHSKDLNNNGSKNESSDIISEN NKSNKVQNHFESLSDLELLENSSQDNLDKDTISTEPFPNQKH KDLQQDLNDEPLEPFP TQIHKDYKEKNLINEEDSEPFPRQKHKKVDNHNEEKNVFHENGSANGNQGSLKLKSF DEHLKDEKIENEPLVHENLSIPNDPIEQILNQPEQETNIQEQLYNEKQNVEEKQNSQIP SLDLKEPTNEDILPNHNPLENIKQSESEINHVQDHALPKENIIDKLDNQKEHIDQSQHNI NVLQENNINNHQLEPQEKPNIESFEPKNIDSEIILPENVETEEIIDDVPSPKHSNHETFE E ETSESEHEEAVSEKNAHETVEHEETVSQESNPEKADNDGNVSQNSNNELNENEFVE SEKSEHEAGGSGAEAGITGTWYNQHGSTFTVTAGADGNLTGQYENRAQGTGCQNS PYTLTGRYNGTKLEWRVEWNNSTENCHSRTEWRGQYQGGAEARINTQWNLTYEGG SGPATEQGQDTFTKVK

> SEQ ID NO:24 A7 >HIS-GMZ2ggsmSA

GSTSENRNKRIGGPKLRGNVTSNIKFPSDNKGKIIRGSNDKLNKNSEDVLEQSEKSL V SENVPSGLDIDDIPKESIFIQEDQEGQTHSELNPETSEHSKDLNNNGSKNESSDIISEN NKSNKVQNHFESLSDLELLENSSQDNLDKDTISTEPFPNQKH KDLQQDLNDEPLEPFP TQIHKDYKEKNLINEEDSEPFPRQKHKKVDNHNEEKNVFHENGSANGNQGSLKLKSF DEHLKDEKIENEPLVHENLSIPNDPIEQILNQPEQETNIQEQLYNEKQNVEEKQNSQIP SLDLKEPTNEDILPNHNPLENIKQSESEINHVQDHALPKENIIDKLDNQKEHIDQSQHNI NVLQENNINNHQLEPQEKPNIESFEPKNIDSEIILPENVETEEIIDDVPSPKHSNHETFE E ETSESEHEEAVSEKNAHETVEHEETVSQESNPEKADNDGNVSQNSNNELNENEFVE SEKSEHEARSKAKEASSYDYILGWEFGGGVPEHKKEENMLSHLYVSSKDKENISKEN DDVLDEKEEEAEETEEEELEEKNEEETESEISEDEEEEEEEEEKEEENDKKKEQEKEQ SNENNDQKKDMEAQNLISKNQNNNEKNVKEAAESIMKTLAGLIKGNNQIDSTLKDLVE ELSKYFKNHGGSGAEAGITGTWYNQHGSTFTVTAGADGNLTGQYENRAQGTGCQN SPYTLTGRYNGTKLEWRVEWNNSTENCHSRTEWRGQYQGGAEARINTQWNLTYEG GSGPATEQGQDTFTKVK

> SEQ ID NO:25 A8 >HIS-GMZ2T:ggsmSA

GSTSENRNKRIGGPKLRGNVTSNIKFPSDNKGKIIRGSNDKLNKNSEDVLEQSEKSLV SENVPSGLDIDDIPKESIFIQEDQEGQTHSELNPETSEHSKDLNNNGSKNESSDIISEN NKSNKVQNHFESLSDLELLENSSQDNLDKDTISTEPFPNQKH KDLQQDLNDEPLEPFP TQIHKDYKEKNLINEEDSEPFPRQKHKKVDNHNEEKNVFHENGSANGNQGSLKLKSF DEHLKDEKIENEPLVHENLSIPNDPIEQILNQPEQETNIQEQLYNEKQNVEEKQNSQIP SLDLKEPTNEDILPNHNPLENIKQSESEINHVQDHALPKENIIDKLDNQKEHIDQSQHNI NVLQENNINNHQLEPQEKPNIESFEPKNIDSEIILPENVETEEIIDDVPSPKHSNHETFE E ETSESEHEEAVSEKNAHETVEHEETVSQESNPEKADNDGNVSQNSNNELNENEFVE SEKSEHEARSKTKEYAEKAKNAYEKAKNAYQKANQAVLKAKEASSYDYILGWEFGGG VPEHKKEENMLSHLYVSSKDKENISKENDDVLDEKEEEAEETEEEELEGGSGAEAGIT GTWYNQHGSTFTVTAGADGNLTGQYENRAQGTGCQNSPYTLTGRYNGTKLEWRVE WNNSTENCHSRTEWRGQYQGGAEARINTQWNLTYEGGSGPATEQGQDTFTKVK

> SEQ ID NO:26 A9 >mSA-PfRH5-HIS

AEAGITGTWYNQHGSTFTVTAGADGNLTGQYENRAQGTGCQNSPYTLTGRYNGTKL EWRVEWNNSTENCHSRTEWRGQYQGGAEARINTQWNLTYEGGSGPATEQGQDTF TKVKGGSLSFENAIKKTKNQENNLTLLPIKSTEEEKDDIKNGKDIKKEIDNDKENIKTNN AKDHSTYIKSYLNTNVNDGLKYLFIPSHNSFIKKYSVFNQINDGMLLNEKNDVKNNEDY KNVDYKNVNFLQYHFKELSNYNIANSIDILQEKEGHLDFVIIPHYTFLDYYKHLSYNSIY H KYSTYGKYIAVDAFIKKINETYDKVKSKCNDIKNDLIATIKKLEHPYDINNKNDDSYRYD I SEEIDDKSEETDDETEEVEDSIQDTDSNHTPSNKKKNDLMNRTFKKMMDEYNTKKKK LIKCIKNHENDFNKICMDMKNYGTNLFEQLSCYNNNFCNTNGIRFHYDEYIHKLILSVKS KNLNKDLSDMTNILQQSELLLTNLNKKMGSYIYIDTIKFIHKEMKHIFNRIEYHTKIIND KT KIIQDKI KLNIWRTFQKDELLKRILDMSNEYSLFITSDHLRQMLYNTFYSKEKHLNNIFHH LI YVLQM KFNDVPIKM EYFQTYKKN KPLTQHH H H H H > SEQ ID NO:27 A10 >mSA-Pfs25-HIS

AEAGITGTWYNQHGSTFTVTAGADGNLTGQYENRAQGTGCQNSPYTLTGRYNGTKL EWRVEWNNSTENCHSRTEWRGQYQGGAEARINTQWNLTYEGGSGPATEQGQDTF TKVKGGSNKLYSLFLFLFIQLSIKYNNAKVTVDTVCKRGFLIQMSGHLECKCENDLVLV NEETCEEKVLKCDEKTVNKPCGDFSKCIKIDGNPVSYACKCNLGYDMVNNVCIPNEC KNVTCGNGKCILDTSNPVKTGVCSCNIGKVPNVQDQNKCSKDGETKCSLKCLKENET CKAVDGIYKCDCKDGFIIDNESSICTAFSAYNILNLSIMFILFSVCFFIM

> SEQ ID NO:28 A1 1 >HIS-PfCSP(aa92-397)-mSA

KLKQPADGNPDPNANPNVDPNANPNVDPNANPNVDPNANPNANPNANPNANPNAN PNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNVDPNA NPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPN ANPNANPNANPNANPNKNNQGNGQGHNMPNDPNRNVDENANANSAVKNNNNEEP SDKHIKEYLNKIQNSLSTEWSPCSVTCGNGIQVRIKPGSANKPKDELDYANDIEKKICK MEKCSSVFNWNSSIGLIMVLSFLFLNGGSAEAGITGTWYNQHGSTFTVTAGADGNLT GQYENRAQGTGCQNSPYTLTGRYNGTKLEWRVEWNNSTENCHSRTEWRGQYQGG AEARINTQWNLTYEGGSGPATEQGQDTFTKVK

> SEQ ID NO:29 A3 >PCSK9|31-692|:mSA:HIS DNA TTTCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCT GGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTAC GTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGC GTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAA TGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACT CCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAA GCAGAGCTCTCTGGCTAACTAGAGAACCCACTGCTTACTGGCTTATCGAAATTAAT ACGACTCACTATAGGGAGACCCAAGCTGGCTAGCGTTTAAACTTAAGCTTAGCGC AGAGGCTTGGGGCAGCCGAGCGGCAGCCAGGCCCCGGCCCGGGCCTCGGTTCC AGAAGGGAGAGGAGCCCGCCAAGGCGCGCAAGAGAGCGGGCTGCCTCGCAGTC CGAGCCGGAGAGGGAGCGCGAGCCGCGCCGGCCCCGGACGGCCTCCGAAACC ATGCAGGAAGATGAGGACGGCGACTACGAGGAACTGGTGCTGGCCCTGCGGAG CGAAGAGGATGGACTGGCCGAGGCCCCTGAGCACGGCACCACCGCCACCTTCC ACAGATGCGCCAAGGACCCTTGGCGGCTGCCCGGCACATACGTGGTGGTGCTGA AAGAGGAAACCCACCTGAGCCAGAGCGAGCGGACCGCCAGAAGGCTGCAGGCC CAGGCCGCCAGAAGAGGCTACCTGACCAAGATCCTGCACGTGTTCCACGGCCTG CTGCCCGGCTTCCTGGTGAAAATGAGCGGCGACCTGCTGGAACTGGCCCTGAAG CTGCCCCACGTGGACTACATCGAAGAGGACAGCAGCGTGTTCGCCCAGAGCATC CCCTGGAACCTGGAACGGATCACCCCCCCCAGATACCGGGCCGACGAGTACCAG CCTCCTGACGGCGGCAGCCTGGTGGAAGTGTACCTGCTGGACACCAGCATCCAG AGCGACCACCGCGAGATCGAGGGCAGAGTGATGGTGACAGACTTCGAGAACGTG CCCGAAGAGGACGGCACCCGGTTCCACAGACAGGCCAGCAAGTGCGACAGCCA CGGCACACATCTGGCCGGCGTGGTGTCTGGCAGAGATGCCGGCGTGGCCAAGG GCGCCAGCATGAGAAGCCTGCGGGTGCTGAACTGCCAGGGCAAGGGCACCGTG TCCGGCACCCTGATCGGCCTGGAATTCATCCGGAAGTCCCAGCTGGTGCAGCCC GTGGGCCCTCTGGTGGTGCTGCTGCCTCTGGCTGGCGGCTACAGCAGAGTGCTG AACGCCGCCTGCCAGAGACTGGCCAGAGCTGGCGTGGTGCTGGTGACAGCCGC CGGAAACTTCCGGGACGACGCCTGCCTGTACAGCCCCGCCTCTGCCCCCGAAGT GATCACCGTGGGCGCCACCAACGCCCAGGACCAGCCTGTGACACTGGGCACCCT GGGCACAAACTTCGGCAGATGCGTGGACCTGTTCGCCCCTGGCGAGGACATCAT CGGCGCCAGCAGCGACTGCAGCACCTGTTTCGTGTCCCAGAGCGGCACCAGCCA GGCCGCTGCCCATGTGGCCGGAATCGCCGCCATGATGCTGAGCGCCGAGCCTG AGCTGACCCTGGCCGAGCTGCGGCAGCGGCTGATCCACTTCTCCGCCAAGGACG TGATCAACGAGGCCTGGTTCCCCGAGGACCAGAGAGTGCTGACCCCCAACCTGG TGGCCGCCCTGCCTCCTTCTACACACGGCGCTGGCTGGCAGCTGTTCTGCAGGA CAGTGTGGTCCGCCCACAGCGGCCCCACCAGAATGGCCACAGCCGTGGCCAGAT GCGCCCCTGATGAGGAACTGCTGAGCTGCAGCAGCTTCTCCAGAAGCGGCAAGC GGAGAGGCGAGCGGATGGAAGCCCAGGGCGGCAAGCTCGTGTGCAGAGCCCAC AATGCCTTCGGCGGCGAGGGCGTGTACGCCATTGCCAGATGCTGCCTGCTGCCT CAGGCCAACTGCAGCGTGCACACAGCCCCTCCAGCCGAGGCCAGCATGGGCAC CAGAGTGCACTGCCACCAGCAGGGCCACGTGCTGACCGGCTGTAGCAGCCACTG GGAGGTGGAAGATCTGGGCACCCACAAGCCCCCCGTGCTGAGGCCCAGAGGCC AGCCTAATCAGTGCGTGGGCCACAGAGAGGCCTCCATCCACGCCAGCTGTTGCC ACGCCCCTGGCCTGGAATGCAAAGTGAAAGAGCACGGCATCCCTGCCCCCCAGG AACAGGTCACAGTGGCCTGCGAGGAAGGCTGGACCCTGACAGGCTGTTCCGCCC TGCCAGGCACCTCTCACGTGCTGGGCGCCTACGCCGTGGACAATACCTGCGTCG TGCGCAGCCGGGACGTGTCCACAACCGGCTCTACAAGCGAGGGCGCCGTGACC GCCGTGGCCATCTGCTGCAGAAGCAGACACCTGGCCCAGGCCTCCCAGGAACTG CAGGGCGGATCTGCCGAGGCCGGCATCACCGGCACCTGGTACAATCAGCACGG CAGCACCTTCACCGTGACCGCTGGCGCCGACGGCAACCTGACCGGCCAGTACGA GAACAGAGCCCAGGGCACCGGCTGCCAGAACAGCCCTTACACCCTGACCGGCAG ATACAACGGCACCAAGCTGGAATGGCGGGTGGAATGGAACAACAGCACCGAGAA CTGCCACAGCCGGACCGAGTGGCGGGGACAGTATCAGGGCGGAGCCGAGGCCC GGATCAACACCCAGTGGAACCTGACCTACGAGGGCGGCTCTGGCCCTGCCACAG AGCAGGGACAGGACACCTTCACCAAAGTGAAGCACCACCACCATCACCACTAAGC GGCCGCTTTT

> SEQ ID NO:30 A4 >mSA-ID1 ID2a-HIS DNA

CCATGGGCGGTGCAGAAGCAGGTATTACCGGCACCTGGTATAATCAGCATGGTA GCACCTTTACCGTTACCGCAGGCGCAGATGGTAATCTGACAGGTCAGTATGAAAA TCGTGCACAGGGCACCGGTTGTCAGAATAGCCCGTATACCCTGACCGGTCGTTAT AATGGCACCAAACTGGAATGGCGTGTTGAATGGAATAATAGCACCGAAAATTGTC ATAGCCGTACCGAATGGCGTGGTCAGTATCAGGGTGGTGCAGAAGCCCGTATTAA TACCCAGTGGAATCTGACCTATGAAGGTGGTAGCGGTCCGGCAACCGAACAGGG TCAGGATACCTTTACCAAAGTTAAAGGTGGCAGCAACTATATCAAAGGCGATCCGT ATTTTGCAGAGTATGCAACCAAACTGAGCTTTATTCTGAATCCGAGTGATGCAAAT AATCCGAGCGGTGAAACCGCAAATCACAATGATGAAGCCTGTAATTGTAACGAAA GCGGTATTAGCAGCGTTGGTCAGGCACAGACCAGCGGTCCGAGCAGCAATAAAA CCTGTATTACCCATAGCAGCATTAAAACCAATAAAAAGAAAGAATGCAAAGATGTG AAACTGGGCGTGCGCGAAAATGATAAAGATCTGAAAATTTGCGTGATCGAGGATA CCAGCCTGAGCGGTGTTGATAATTGTTGTTGTCAGGATCTGCTGGGTATTCTGCA AGAAAATTGCAGCGATAATAAACGTGGTAGCAGCAGCAATGATAGCTGCGATAAC AAAAATCAGGATGAATGCCAGAAAAAACTGGAAAAAGTTTTTGCCAGCCTGACGAA TGGTTACAAATGCGATAAATGTAAAAGCGGCACCAGCCGCAGCAAAAAGAAATGG ATTTGGAAAAAAAGCAGCGGCAATGAAGAAGGTCTGCAAGAGGAATATGCAAATA CCATTGGTCTGCCTCCGCGTACCCAGAGCCTGTATCTGGGTAATCTGCCGAAACT GGAAAATGTGTGTGAAGATGTGAAAGATATCAATTTTGATACCAAAGAAAAATTTCT GGCAGGCTGCCTGATTGTGAGCTTTCATGAAGGTAAAAACCTGAAAAAACGCTAT CCGCAGAATAAAAACAGCGGTAACAAAGAAAATCTGTGCAAAGCACTGGAATACA GCTTTGCAGATTATGGCGATCTGATTAAAGGCACCAGCATTTGGGATAACGAGTAT ACCAAAGATCTGGAACTGAATCTGCAGAACAATTTCGGTAAACTGTTCGGCAAATA TATCAAAAAAAACAATACCGCAGAGCAGGATACCAGCTATAGCAGCCTGGATGAA CTGCGTGAAAGTTGGTGGAATACCAACAAAAAATACATTTGGACCGCCATGAAAC ATGGTGCCGAAATGAATATTACCACCTGTAATGCAGATGGTAGCGTTACCGGTAG CGGTAGCAGCTGTGATGATATTCCGACCATTGATCTGATTCCGCAGTATCTGCGTT TTCTGCAAGAATGGGTTGAAAACTTTTGTGAACAGCGTCAGGCGAAAGTGAAAGA TGTTATTACCAATTGCAAAAGCTGCAAAGAAAGCGGCAATAAATGCAAAACCGAGT GCAAAACCAAATGCAAAGACGAGTGCGAGAAATACAAAAAATTCATTGAAGCATGT GGTACAGCCGGTGGTGGTATTGGCACCGCAGGTAGCCCGTGGTCAAAACGTTGG GATCAGATCTATAAACGCTACAGCAAACACATCGAAGATGCCAAACGTAATCGTAA AGCAGGCACCAAAAATTGTGGCACCAGCAGCACCACCAATGCAGCAGCAAGCAC CGATGAAAACAAATGTGTTCAGAGCGATATCGATAGCTTCTTCAAACATCTGATTG ATATTGGTCTGACCACCCCGAGCAGCTATCTGAGCAATGTTCTGGATGATAACATT TGCGGTGCAGATAAAGCACCGTGGACCACCTATACCACATATACCACCACAGAAA AATGCAACAAAGAGCGCGATAAAAGCAAAAGCCAGAGCAGCGATACCCTGGTTGT TGTTAATGTTCCGAGTCCGCTGGGTAATACCCCGTATCGTTATAAGTATGCCTGCC AGTGTAAAATCCCGACCAATGAAGAAACCTGTGATGATCGCAAAGAATACATGAAT CAGTGGTCATGTGGTAGCGCACGTACCATGAAACGTGGCTATAAAAACGATAATT ATGAACTGTGCAAATATAACGGCGTGGATGTTAAACCGACCACCGTTCGTAGCAA TAGCAGCAAACTGGATCATCATCATCACCATCATTAAGGATCC > SEQ ID NO:31 A5 >mSA-RO-HIS DNA

CCATGGGCGGTGCAGAAGCAGGTATTACCGGCACCTGGTATAATCAGCATGGTA GCACCTTTACCGTTACCGCAGGCGCAGATGGTAATCTGACAGGTCAGTATGAAAA TCGTGCACAGGGCACCGGTTGTCAGAATAGCCCGTATACCCTGACCGGTCGTTAT AATGGCACCAAACTGGAATGGCGTGTTGAATGGAATAATAGCACCGAAAATTGTC ATAGCCGTACCGAATGGCGTGGTCAGTATCAGGGTGGTGCAGAAGCCCGTATTAA TACCCAGTGGAATCTGACCTATGAAGGTGGTAGCGGTCCGGCAACCGAACAGGG TCAGGATACCTTTACCAAAGTTAAAGGTGGCAGCACAAGTGAGAATAGAAATAAAC GAATCGGGGGTCCTAAATTAAGGGGTAATGTTACAAGTAATATAAAGTTCCCATCA GATAACAAAGGTAAAATTATAAGAGGTTCGAATGATAAACTTAATAAAAACTCTGAA GATGTTTTAGAACAAAGCGAAAAATCGCTTGTTTCAGAAAATGTTCCTAGTGGATT AGATATAGATGATATCCCTAAAGAATCTATTTTTATTCAAGAAGATCAAGAAGGTCA AACTCATTCTGAATTAAATCCTGAAACATCAGAACATAGTAAAGATTTAAATAATAA TGGTTCAAAAAATGAATCTAGTGATATTATTTCAGAAAATAATAAATCAAATAAAGT ACAAAATCATTTTGAATCATTATCAGATTTAGAATTACTTGAAAATTCCTCACAAGAT AATTTAGACAAAGATACAATTTCAACAGAACCTTTTCCTAATCAAAAACATAAAGAC TTACAACAAGATTTAAATGATGAACCTTTAGAACCCTTTCCTACACAAATACATAAA GATTATAAAGAAAAAAATTTAATAAATGAAGAAGATTCAGAACCATTTCCCAGACAA AAGCATAAAAAGGTAGACAATCATAATGAAGAAAAAAACGTATTTCATGAAAATGG TTCTGCAAATGGTAATCAAGGAAGTTTGAAACTTAAATCATTCGATGAACATTTAAA AGATGAAAAAATAGAAAATGAACCACTTGTTCATGAAAATTTATCCATACCAAATGA TCCAATAGAACAAATATTAAATCAACCTGAACAAGAAACAAATATCCAGGAACAATT GTATAATGAAAAACAAAATGTTGAAGAAAAACAAAATTCTCAAATACCTTCGTTAGA TTTAAAAGAACCAACAAATGAAGATATTTTACCAAATCATAATCCATTAGAAAATAT AAAACAAAGTGAATCAGAAATAAATCATGTACAAGATCATGCGCTACCAAAAGAGA ATATAATAGACAAACTTGATAATCAAAAAGAACACATCGATCAATCACAACATAATA TAAATGTATTACAAGAAAATAACATAAACAATCACCAATTAGAACCTCAAGAGAAAC CTAATATTGAATCGTTTGAACCTAAAAATATAGATTCAGAAATTATTCTTCCTGAAA ATGTTGAAACAGAAGAAATAATAGATGATGTGCCTTCCCCTAAACATTCTAACCAT GAAACATTTGAAGAAGAAACAAGTGAATCTGAACATGAAGAAGCCGTATCTGAAAA AAATGCCCACGAAACTGTCGAACATGAAGAAACTGTGTCTCAAGAAAGCAATCCT GAAAAAGCTGATAATGATGGAAATGTATCTCAAAACAGCAACAACGAATTAAATGA AAATGAATTCGTTGAATCGGAAAAAAGCGAGCATGAAGCAAGATCCAAAGCAAAA GAAGCTTCTAGTTATGATTATATTTTAGGTTGGGAATTTGGAGGAGGCGTTCCAGA ACACAAAAAAGAAGAAAATATGTTATCACATTTATATGTTTCTTCAAAGGATAAGGA AAATATATCTAAGGAAAATGATGATGTATTAGATGAGAAGGAAGAAGAGGCAGAAG AAACAGAAGAAGAAGAACTTGAAGAAAAAAATGAAGAAGAAACAGAATCAGAAATA AGTGAAGATGAAGAAGAAGAAGAAGAAGAAGAAGAAAAGGAAGAAGAAAATGACA AAAAAAAAGAACAAGAAAAAGAACAAAGTAATGAAAATAATGATCAAAAAAAAGATA TGGAAGCACAGAATTTAATTTCTAAAAACCAGAATAATAATGAGAAAAACGTAAAA GAAGCTGCTGAAAGCATCATGAAAACTTTAGCTGGTTTAATCAAGGGAAATAATCA AATAGATTCTACCTTAAAAGATTTAGTAGAAGAATTATCCAAATATTTTAAAAATCAT AGATCTCATCACCATCATCACCATTAGggatccttt

> SEQ ID NO:32 A6 >HIS-RO-mSA DNA

GGATCCACAAGTGAGAATAGAAATAAACGAATCGGGGGTCCTAAATTAAGGGGTA ATGTTACAAGTAATATAAAGTTCCCATCAGATAACAAAGGTAAAATTATAAGAGGTT CGAATGATAAACTTAATAAAAACTCTGAAGATGTTTTAGAACAAAGCGAAAAATCG CTTGTTTCAGAAAATGTTCCTAGTGGATTAGATATAGATGATATCCCTAAAGAATCT ATTTTTATTCAAGAAGATCAAGAAGGTCAAACTCATTCTGAATTAAATCCTGAAACA TCAGAACATAGTAAAGATTTAAATAATAATGGTTCAAAAAATGAATCTAGTGATATT ATTTCAGAAAATAATAAATCAAATAAAGTACAAAATCATTTTGAATCATTATCAGATT TAGAATTACTTGAAAATTCCTCACAAGATAATTTAGACAAAGATACAATTTCAACAG AACCTTTTCCTAATCAAAAACATAAAGACTTACAACAAGATTTAAATGATGAACCTT TAGAACCCTTTCCTACACAAATACATAAAGATTATAAAGAAAAAAATTTAATAAATG AAGAAGATTCAGAACCATTTCCCAGACAAAAGCATAAAAAGGTAGACAATCATAAT GAAGAAAAAAACGTATTTCATGAAAATGGTTCTGCAAATGGTAATCAAGGAAGTTT GAAACTTAAATCATTCGATGAACATTTAAAAGATGAAAAAATAGAAAATGAACCACT TGTTCATGAAAATTTATCCATACCAAATGATCCAATAGAACAAATATTAAATCAACC TGAACAAGAAACAAATATCCAGGAACAATTGTATAATGAAAAACAAAATGTTGAAG AAAAACAAAATTCTCAAATACCTTCGTTAGATTTAAAAGAACCAACAAATGAAGATA TTTTACCAAATCATAATCCATTAGAAAATATAAAACAAAGTGAATCAGAAATAAATC ATGTACAAGATCATGCGCTACCAAAAGAGAATATAATAGACAAACTTGATAATCAA AAAGAACACATCGATCAATCACAACATAATATAAATGTATTACAAGAAAATAACATA AACAATCACCAATTAGAACCTCAAGAGAAACCTAATATTGAATCGTTTGAACCTAAA AATATAGATTCAGAAATTATTCTTCCTGAAAATGTTGAAACAGAAGAAATAATAGAT GATGTGCCTTCCCCTAAACATTCTAACCATGAAACATTTGAAGAAGAAACAAGTGA ATCTGAACATGAAGAAGCCGTATCTGAAAAAAATGCCCACGAAACTGTCGAACAT GAAGAAACTGTGTCTCAAGAAAGCAATCCTGAAAAAGCTGATAATGATGGAAATGT ATCTCAAAACAGCAACAACGAATTAAATGAAAATGAATTCGTTGAATCGGAAAAAA GCGAGCATGAAGCAGGTGGTAGCGGTGCAGAAGCAGGTATTACCGGCACCTGGT ATAATCAGCATGGTAGCACCTTTACCGTTACCGCAGGCGCAGATGGTAATCTGAC AGGTCAGTATGAAAATCGTGCACAGGGCACCGGTTGTCAGAATAGCCCGTATACC CTGACCGGTCGTTATAATGGCACCAAACTGGAATGGCGTGTTGAATGGAATAATA GCACCGAAAATTGTCATAGCCGTACCGAATGGCGTGGTCAGTATCAGGGTGGTG CAGAAGCCCGTATTAATACCCAGTGGAATCTGACCTATGAAGGTGGTAGTGGTCC GGCAACCGAACAGGGTCAGGATACCTTTACCAAAGTGAAATAAcatatg > SEQ ID NO:33 A7 >HIS-GMZ2ggsmSA3

GGATCCACAAGTGAGAATAGAAATAAACGAATCGGGGGTCCTAAATTAAGGGGTA ATGTTACAAGTAATATAAAGTTCCCATCAGATAACAAAGGTAAAATTATAAGAGGTT CGAATGATAAACTTAATAAAAACTCTGAAGATGTTTTAGAACAAAGCGAAAAATCG CTTGTTTCAGAAAATGTTCCTAGTGGATTAGATATAGATGATATCCCTAAAGAATCT ATTTTTATTCAAGAAGATCAAGAAGGTCAAACTCATTCTGAATTAAATCCTGAAACA TCAGAACATAGTAAAGATTTAAATAATAATGGTTCAAAAAATGAATCTAGTGATATT ATTTCAGAAAATAATAAATCAAATAAAGTACAAAATCATTTTGAATCATTATCAGATT TAGAATTACTTGAAAATTCCTCACAAGATAATTTAGACAAAGATACAATTTCAACAG AACCTTTTCCTAATCAAAAACATAAAGACTTACAACAAGATTTAAATGATGAACCTT TAGAACCCTTTCCTACACAAATACATAAAGATTATAAAGAAAAAAATTTAATAAATG AAGAAGATTCAGAACCATTTCCCAGACAAAAGCATAAAAAGGTAGACAATCATAAT GAAGAAAAAAACGTATTTCATGAAAATGGTTCTGCAAATGGTAATCAAGGAAGTTT GAAACTTAAATCATTCGATGAACATTTAAAAGATGAAAAAATAGAAAATGAACCACT TGTTCATGAAAATTTATCCATACCAAATGATCCAATAGAACAAATATTAAATCAACC TGAACAAGAAACAAATATCCAGGAACAATTGTATAATGAAAAACAAAATGTTGAAG AAAAACAAAATTCTCAAATACCTTCGTTAGATTTAAAAGAACCAACAAATGAAGATA TTTTACCAAATCATAATCCATTAGAAAATATAAAACAAAGTGAATCAGAAATAAATC ATGTACAAGATCATGCGCTACCAAAAGAGAATATAATAGACAAACTTGATAATCAA AAAGAACACATCGATCAATCACAACATAATATAAATGTATTACAAGAAAATAACATA AACAATCACCAATTAGAACCTCAAGAGAAACCTAATATTGAATCGTTTGAACCTAAA AATATAGATTCAGAAATTATTCTTCCTGAAAATGTTGAAACAGAAGAAATAATAGAT GATGTGCCTTCCCCTAAACATTCTAACCATGAAACATTTGAAGAAGAAACAAGTGA ATCTGAACATGAAGAAGCCGTATCTGAAAAAAATGCCCACGAAACTGTCGAACAT GAAGAAACTGTGTCTCAAGAAAGCAATCCTGAAAAAGCTGATAATGATGGAAATGT ATCTCAAAACAGCAACAACGAATTAAATGAAAATGAATTCGTTGAATCGGAAAAAA GCGAGCATGAAGCAAGATCCAAAGCAAAAGAAGCTTCTAGTTATGATTATATTTTA GGTTGGGAATTTGGAGGAGGCGTTCCAGAACACAAAAAAGAAGAAAATATGTTAT C AC ATTTATATGTTTCTTC A A AG G ATA AG G AA AATATATCTA AG G AA AATG ATG ATG TATTAGATGAGAAGGAAGAAGAGGCAGAAGAAACAGAAGAAGAAGAACTTGAAGA AAAAAATGAAGAAGAAACAGAATCAGAAATAAGTGAAGATGAAGAAGAAGAAGAA GAAGAAGAAGAAAAGGAAGAAGAAAATGACAAAAAAAAAGAACAAGAAAAAGAAC AAAGTAATGAAAATAATGATCAAAAAAAAGATATGGAAGCACAGAATTTAATTTCTA AAAACCAGAATAATAATGAGAAAAACGTAAAAGAAGCTGCTGAAAGCATCATGAAA ACTTTAGCTGGTTTAATCAAGGGAAATAATCAAATAGATTCTACCTTAAAAGATTTA GTAG A AG A ATTATCC A A ATATTTTA AA AATC ATG GTGGTAGCGGTGCAGAAGCAGG TATTACCGGCACCTGGTATAATCAGCATGGTAGCACCTTTACCGTTACCGCAGGC GCAGATGGTAATCTGACAGGTCAGTATGAAAATCGTGCACAGGGCACCGGTTGTC AGAATAGCCCGTATACCCTGACCGGTCGTTATAATGGCACCAAACTGGAATGGCG TGTTGAATGGAATAATAGCACCGAAAATTGTCATAGCCGTACCGAATGGCGTGGT CAGTATCAGGGTGGTGCAGAAGCCCGTATTAATACCCAGTGGAATCTGACCTATG AAGGTGGTAGTGGTCCGGCAACCGAACAGGGTCAGGATACCTTTACCAAAGTGAA ATAAcatatg

> SEQ ID NO:34 A8 >HIS-GMZ2T:ggsmSA DNA GGATCCACAAGTGAGAATAGAAATAAACGAATCGGGGGTCCTAAATTAAGGGGTA ATGTTACAAGTAATATAAAGTTCCCATCAGATAACAAAGGTAAAATTATAAGAGGTT CGAATGATAAACTTAATAAAAACTCTGAAGATGTTTTAGAACAAAGCGAAAAATCG CTTGTTTCAGAAAATGTTCCTAGTGGATTAGATATAGATGATATCCCTAAAGAATCT ATTTTTATTCAAGAAGATCAAGAAGGTCAAACTCATTCTGAATTAAATCCTGAAACA TCAGAACATAGTAAAGATTTAAATAATAATGGTTCAAAAAATGAATCTAGTGATATT ATTTCAGAAAATAATAAATCAAATAAAGTACAAAATCATTTTGAATCATTATCAGATT TAGAATTACTTGAAAATTCCTCACAAGATAATTTAGACAAAGATACAATTTCAACAG AACCTTTTCCTAATCAAAAACATAAAGACTTACAACAAGATTTAAATGATGAACCTT TAGAACCCTTTCCTACACAAATACATAAAGATTATAAAGAAAAAAATTTAATAAATG AAGAAGATTCAGAACCATTTCCCAGACAAAAGCATAAAAAGGTAGACAATCATAAT GAAGAAAAAAACGTATTTCATGAAAATGGTTCTGCAAATGGTAATCAAGGAAGTTT GAAACTTAAATCATTCGATGAACATTTAAAAGATGAAAAAATAGAAAATGAACCACT TGTTCATGAAAATTTATCCATACCAAATGATCCAATAGAACAAATATTAAATCAACC TGAACAAGAAACAAATATCCAGGAACAATTGTATAATGAAAAACAAAATGTTGAAG AAAAACAAAATTCTCAAATACCTTCGTTAGATTTAAAAGAACCAACAAATGAAGATA TTTTACCAAATCATAATCCATTAGAAAATATAAAACAAAGTGAATCAGAAATAAATC ATGTACAAGATCATGCGCTACCAAAAGAGAATATAATAGACAAACTTGATAATCAA AAAGAACACATCGATCAATCACAACATAATATAAATGTATTACAAGAAAATAACATA AACAATCACCAATTAGAACCTCAAGAGAAACCTAATATTGAATCGTTTGAACCTAAA AATATAGATTCAGAAATTATTCTTCCTGAAAATGTTGAAACAGAAGAAATAATAGAT GATGTGCCTTCCCCTAAACATTCTAACCATGAAACATTTGAAGAAGAAACAAGTGA ATCTGAACATGAAGAAGCCGTATCTGAAAAAAATGCCCACGAAACTGTCGAACAT GAAGAAACTGTGTCTCAAGAAAGCAATCCTGAAAAAGCTGATAATGATGGAAATGT ATCTCAAAACAGCAACAACGAATTAAATGAAAATGAATTCGTTGAATCGGAAAAAA GCGAGCATGAAGCAAGATCCAAAACAAAAGAATATGCTGAAAAAGCAAAAAATGC TTATGAAAAGGCAAAAAATGCTTATCAAAAAGCAAACCAAGCTGTTTTAAAAGCAA AAGAAGCTTCTAGTTATGATTATATTTTAGGTTGGGAATTTGGAGGAGGCGTTCCA GAACACAAAAAAGAAGAAAATATGTTATCACATTTATATGTTTCTTCAAAGGATAAG GAAAATATATCTAAGGAAAATGATGATGTATTAGATGAGAAGGAAGAAGAGGCAGA AGAAACAGAAGAAGAAGAACTTGAAGGTGGTAGCGGTGCAGAAGCAGGTATTACC GGCACCTGGTATAATCAGCATGGTAGCACCTTTACCGTTACCGCAGGCGCAGATG GTAATCTGACAGGTCAGTATGAAAATCGTGCACAGGGCACCGGTTGTCAGAATAG CCCGTATACCCTGACCGGTCGTTATAATGGCACCAAACTGGAATGGCGTGTTGAA TGGAATAATAGCACCGAAAATTGTCATAGCCGTACCGAATGGCGTGGTCAGTATC AGGGTGGTGCAGAAGCCCGTATTAATACCCAGTGGAATCTGACCTATGAAGGTGG TAGTGGTCCGGCAACCGAACAGGGTCAGGATACCTTTACCAAAGTGAAATAAcatat 9

> SEQ ID NO:35 A9 >mSA-PfRH5-HIS DNA gaattcGGTGCAGAAGCAGGTATTACCGGCACCTGGTATAATCAGCATGGTAGCACC TTTACCGTTACCGCAGGCGCAGATGGTAATCTGACAGGTCAGTATGAAAATCGTG CACAGGGCACCGGTTGTCAGAATAGCCCGTATACCCTGACCGGTCGTTATAATGG CACCAAACTGGAATGGCGTGTTGAATGGAATAATAGCACCGAAAATTGTCATAGC CGTACCGAATGGCGTGGTCAGTATCAGGGTGGTGCAGAAGCCCGTATTAATACCC AGTGGAATCTGACCTATGAAGGTGGTAGCGGTCCGGCAACCGAACAGGGTCAGG ATACCTTTACCAAAGTTAAAGGTGGCAGCCTGTCCTTCGAGAACGCCATCAAGAA GACCAAGAACCAGGAAAACAACCTGACCCTGCTGCCCATCAAGTCCACCGAGGA AGAGAAGGACGACATCAAGAACGGCAAGGATATCAAGAAGGAAATCGACAACGA CAAGGAAAACATCAAGACCAACAACGCCAAGGACCACTCCACCTACATCAAGTCT TACCTGAACACCAACGTGAACGACGGCCTGAAGTACCTGTTCATCCCATCCCACA ACAGCTTCATCAAGAAGTACTCCGTTTTCAACCAGATCAACGACGGCATGCTGCT GAACGAGAAGAACGACGTGAAGAACAACGAGGACTACAAGAACGTCGACTACAA GAACGTGAACTTCCTGCAGTACCACTTCAAGGAACTGTCCAACTACAACATCGCC AACTCCATCGACATCCTGCAAGAAAAGGAAGGCCACCTGGACTTCGTGATCATCC CCCACTACACTTTCTTGGACTACTACAAGCACCTGTCCTACAACAGCATCTACCAC AAGTACAGCACCTACGGCAAGTACATCGCTGTGGACGCTTTCATCAAGAAGATCA ACGAGACTTACGACAAAGTGAAGTCCAAGTGTAACGATATCAAGAACGACCTGAT CGCCACCATCAAGAAGCTCGAGCACCCCTACGACATCAACAACAAGAACGACGAC AGCTACCGCTACGACATCTCCGAAGAGATCGACGACAAGTCCGAGGAAACCGAC GACGAGACTGAGGAAGTCGAGGACTCCATCCAGGACACCGACTCCAACCACACC CCCTCCAACAAGAAGAAGAACGATCTGATGAACCGCACCTTCAAGAAGATGATGG ACGAGTACAACACTAAGAAGAAGAAGCTGATCAAGTGCATCAAGAACCACGAGAA CGACTTCAACAAGATCTGCATGGACATGAAGAACTACGGCACCAACCTGTTCGAG CAGCTGTCCTGCTACAACAACAACTTCTGCAACACTAACGGCATCCGCTTCCACTA CGATGAGTACATCCACAAGCTGATCCTGTCCGTCAAGAGCAAGAACCTGAACAAG GACCTGAGCGACATGACCAACATCCTCCAGCAGTCCGAGCTGCTGCTGACCAACT TGAACAAGAAGATGGGCTCCTACATCTACATCGACACTATCAAGTTCATCCACAAG GAAATGAAGCACATCTTCAACCGCATCGAGTACCACACCAAGATCATCAACGATAA GACTAAGATCATCCAAGACAAGATCAAGCTGAACATCTGGCGCACTTTCCAAAAG GACGAACTGCTGAAGCGTATCCTGGACATGTCTAACGAGTACTCCCTCTTCATCA CCTCCGACCACCTGAGGCAGATGCTGTACAACACCTTCTACTCCAAGGAAAAGCA CCTCAACAACATCTTCCACCACCTGATCTACGTGCTGCAGATGAAGTTCAACGAC GTCCCCATCAAGATGGAATACTTCCAGACCTACAAGAAGAACAAGCCCCTGACCC AGCATCATCACCACCACCAC

> SEQ ID NO: 36 (biotin acceptor site) GLNDIFEAQKIEWHE

> SEQ ID NO: 37 monovalent streptavidin

AEAGITGTWYNQHGSTFTVTAGADGNLTGQYENRAQGTGCQNSPYTLTGRYNGTKL EWRVEWNNSTENCHSRTEWRGQYQGGAEARINTQWNLTYEGGSGPATEQGQDTF TKVK > SEQ ID NO: 38 BirA OS=Escherichia coli (strain K12) GN=birA PE=1 SV=1

MKDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRDWGVDVFTVPGKG Y SLPEPIQLLNAKQILGQLDGGSVAVLPVIDSTNQYLLDRIGELKSGDACIAEYQQAGRG RRGRKWFSPFGANLYLSMFWRLEQGPAAAIGLSLVIGIVMAEVLRKLGADKVRVKWP NDLYLQDRKLAGILVELTGKTGDAAQIVIGAGINMAMRRVEESWNQGWITLQEAGINL DRNTLAAMLIRELRAALELFEQEGLAPYLSRWEKLDNFINRPVKLIIGDKEIFGISRGID K QGALLLEQDGIIKPWMGGEISLRSAEK

> SEQ ID NO: 39 DNA sequence of the biotin binding site

GGTCTGAACGACATCTTCGAGGCTCAGAAAATCGAATGGCACGAA

> SEQ ID NO: 40 >Survivin:mSA (Homo Sapiens)

MGAPTLPPAWQPFLKDHRISTFKNWPFLEGCACTPERMAEAGFIHCPTENEPDLAQC FFCFKELEGWEPDDDPIEEHKKHSSGCAFLSVKKQFEELTLGEFLKLDRERAKNKIAK ETNNKKKEFEETAKKVRRAIEQLAAMDggsGAEAGITGTWYNQHGSTFTVTAGADGNL TGQYENRAQGTGCQNSPYTLTGRYNGTKLEWRVEWNNSTENCHSRTEWRGQYQG GAEARINTQWNLTYEGGSGPATEQGQDTFTKVK

> SEQ ID NO: 41 > mSA:Survivin (Homo Sapiens)

MAEAGITGTWYNQHGSTFTVTAGADGNLTGQYENRAQGTGCQNSPYTLTGRYNGT KLEWRVEWNNSTENCHSRTEWRGQYQGGAEARINTQWNLTYEGGSGPATEQGQD TFTKVKggsGAPTLPPAWQPFLKDHRISTFKNWPFLEGCACTPERMAEAGFIHCPTEN EPDLAQCFFCFKELEGWEPDDDPIEEHKKHSSGCAFLSVKKQFEELTLGEFLKLDRER AKNKIAKETNNKKKEFEETAKKVRRAIEQLAAMD

> SEQ ID NO: 42 >Survivin(F101A/L102A):mSA (Homo Sapiens)

MGAPTLPPAWQPFLKDHRISTFKNWPFLEGCACTPERMAEAGFIHCPTENEPDLAQC FFCFKELEGWEPDDDPIEEHKKHSSGCAFLSVKKQFEELTLGEAAKLDRERAKNKIAK ETNNKKKEFEETAKKVRRAIEQLAAMDggsGAEAGITGTWYNQHGSTFTVTAGADGNL TGQYENRAQGTGCQNSPYTLTGRYNGTKLEWRVEWNNSTENCHSRTEWRGQYQG GAEARINTQWNLTYEGGSGPATEQGQDTFTKVK

> SEQ ID NO: 43 > mSA:Survivin(F101A/L102A) (Homo Sapiens)

MAEAGITGTWYNQHGSTFTVTAGADGNLTGQYENRAQGTGCQNSPYTLTGRYNGT KLEWRVEWNNSTENCHSRTEWRGQYQGGAEARINTQWNLTYEGGSGPATEQGQD TFTKVKggsGAPTLPPAWQPFLKDHRISTFKNWPFLEGCACTPERMAEAGFIHCPTEN EPDLAQCFFCFKELEGWEPDDDPIEEHKKHSSGCAFLSVKKQFEELTLGEAAKLDRE RAKN KIAKETNNKKKEFEETAKKVRRAIEQLAAMD > SEQ ID NO: 44 > mSA:Survivin(F101A/L102A) (Mus Musculus)

MAEAGITGTWYNQHGSTFTVTAGADGNLTGQYENRAQGTGCQNSPYTLTGRYNGT KLEWRVEWNNSTENCHSRTEWRGQYQGGAEARINTQWNLTYEGGSGPATEQGQD TFTKVKGGSGAPALPQIWQLYLKNYRIATFKNWPFLEDCACTPERMAEAGFIHCPTEN EPDLAQCFFCFKELEGWEPDDNPIEEHRKHSPGCAFLTVKKQMEELTVSEAAKLDRQ RAKNKIAKETNNKQKEFEETAKTTRQSIEQLAASGRF

> SEQ ID NO: 45 >Survivin (F101A/L102A):mSA (Mus Musculus)

GAPALPQIWQLYLKNYRIATFKNWPFLEDCACTPERMAEAGFIHCPTENEPDLAQCF F CFKELEGWEPDDNPIEEHRKHSPGCAFLTVKKQMEELTVSEAAKLDRQRAKNKIAKE TNNKQKEFEETAKTTRQSIEQLAAggsAEAGITGTWYNQHGSTFTVTAGADGNLTGQY ENRAQGTGCQNSPYTLTGRYNGTKLEWRVEWNNSTENCHSRTEWRGQYQGGAEA RINTQWN LTYEGGSG PATEQGQDTFTKVK

> SEQ ID NO: 46 > mSA:Survivin (Mus Musculus)

MAEAGITGTWYNQHGSTFTVTAGADGNLTGQYENRAQGTGCQNSPYTLTGRYNGT KLEWRVEWNNSTENCHSRTEWRGQYQGGAEARINTQWNLTYEGGSGPATEQGQD TFTKVKGGSGAPALPQIWQLYLKNYRIATFKNWPFLEDCACTPERMAEAGFIHCPTEN EPDLAQCFFCFKELEGWEPDDNPIEEHRKHSPGCAFLTVKKQMEELTVSEFLKLDRQ RAKNKIAKETNNKQKEFEETAKTTRQSIEQLAASGRF

> SEQ ID NO: 47 >Survivin:mSA (Mus Musculus)

GAPALPQIWQLYLKNYRIATFKNWPFLEDCACTPERMAEAGFIHCPTENEPDLAQCFF CFKELEGWEPDDNPIEEHRKHSPGCAFLTVKKQMEELTVSEFLKLDRQRAKNKIAKET NNKQKEFEETAKTTRQSIEQLAAGGSAEAGITGTWYNQHGSTFTVTAGADGNLTGQY ENRAQGTGCQNSPYTLTGRYNGTKLEWRVEWNNSTENCHSRTEWRGQYQGGAEA RINTQWN LTYEGGSG PATEQGQDTFTKVK

> SEQ ID NO: 48 > mSA:Survivin(F101A/L102A) (Mus Musculus) DNA

ATGGCAGAAGCAGGTATTACCGGCACCTGGTATAATCAGCATGGTAGCACCTTTA CCGTTACCGCAGGCGCAGATGGTAATCTGACAGGTCAGTATGAAAATCGTGCACA GGGCACCGGTTGTCAGAATAGCCCGTATACCCTGACCGGTCGTTATAATGGCACC AAACTGGAATGGCGTGTTGAATGGAATAATAGCACCGAAAATTGTCATAGCCGTA CCGAATGGCGTGGTCAGTATCAGGGTGGTGCAGAAGCCCGTATTAATACCCAGT GGAATCTGACCTATGAAGGTGGTAGCGGTCCGGCAACCGAACAGGGTCAGGATA CCTTTACCAAAGTTAAAGGTGGCAGCGGTGCACCGGCACTGCCGCAGATTTGGC AGCTGTATCTGAAAAACTATCGTATCGCCACCTTTAAAAACTGGCCGTTTCTGGAA GATTGTGCATGTACACCGGAACGTATGGCAGAAGCAGGTTTTATTCATTGTCCGA CCGAAAATGAACCGGATCTGGCACAGTGTTTTTTTTGCTTTAAAGAACTGGAAGGT TGGGAGCCGGATGATAATCCGATTGAAGAACATCGTAAACATAGTCCGGGTTGTG CATTTCTGACCGTGAAAAAACAAATGGAAGAACTGACCGTTAGCGAGGCAGCAAA ACTGGATCGTCAGCGTGCCAAAAACAAAATTGCAAAAGAAACCAATAACAAACAGA AAGAATTCGAAGAAACCGCCAAAACCACCCGTCAGAGCATTGAACAGCTGGCAGC

Aagcggccgcttt > SEQ ID NO: 49 >Survivin (F101A/L102A):mSA (Mus Musculus) DNA

GGTGCACCGGCACTGCCGCAGATTTGGCAGCTGTATCTGAAAAACTATCGTATCG CCACCTTTAAAAACTGGCCGTTTCTGGAAGATTGTGCATGTACACCGGAACGTAT GGCAGAAGCAGGTTTTATTCATTGTCCGACCGAAAATGAACCGGATCTGGCACAG TGTTTTTTTTGCTTTAAAGAACTGGAAGGTTGGGAGCCGGATGATAATCCGATTGA AGAACATCGTAAACATAGTCCGGGTTGTGCATTTCTGACCGTGAAAAAACAAATGG AAGAACTGACCGTTAGCGAGGCAGCAAAACTGGATCGTCAGCGTGCCAAAAACAA AATTGCAAAAGAAACCAATAACAAACAGAAAGAATTCGAAGAAACCGCCAAAACCA CCCGTCAGAGCATTGAACAGCTGGCAGCAGGTGGCAGCGCAGAAGCAGGTATTA CCGGCACCTGGTATAATCAGCATGGTAGCACCTTTACCGTTACCGCAGGCGCAGA TGGTAATCTGACAGGTCAGTATGAAAATCGTGCACAGGGCACCGGTTGTCAGAAT AGCCCGTATACCCTGACCGGTCGTTATAATGGCACCAAACTGGAATGGCGTGTTG AATGGAATAATAGCACCGAAAATTGTCATAGCCGTACCGAATGGCGTGGTCAGTA TCAGGGTGGTGCAGAAGCCCGTATTAATACCCAGTGGAATCTGACCTATGAAGGT GGTAGCGGTCCGGCAACCGAACAGGGTCAGGATACCTTTACCAAAGTTAAA

> SEQ ID NO: 50 > mSA:Survivin (Mus Musculus) DNA

ATGGCAGAAGCAGGTATTACCGGCACCTGGTATAATCAGCATGGTAGCACCTTTA CCGTTACCGCAGGCGCAGATGGTAATCTGACAGGTCAGTATGAAAATCGTGCACA GGGCACCGGTTGTCAGAATAGCCCGTATACCCTGACCGGTCGTTATAATGGCACC AAACTGGAATGGCGTGTTGAATGGAATAATAGCACCGAAAATTGTCATAGCCGTA CCGAATGGCGTGGTCAGTATCAGGGTGGTGCAGAAGCCCGTATTAATACCCAGT GGAATCTGACCTATGAAGGTGGTAGCGGTCCGGCAACCGAACAGGGTCAGGATA CCTTTACCAAAGTTAAAGGTGGCAGCGGTGCACCGGCACTGCCGCAGATTTGGC AGCTGTATCTGAAAAACTATCGTATCGCCACCTTTAAAAACTGGCCGTTTCTGGAA GATTGTGCATGTACACCGGAACGTATGGCAGAAGCAGGTTTTATTCATTGTCCGA CCGAAAATGAACCGGATCTGGCACAGTGTTTTTTTTGCTTTAAAGAACTGGAAGGT TGGGAGCCGGATGATAATCCGATTGAAGAACATCGTAAACATAGTCCGGGTTGTG CATTTCTGACCGTGAAAAAACAAATGGAAGAACTGACCGTTAGCGAGTTTCTGAAA CTGGATCGTCAGCGTGCCAAAAACAAAATTGCAAAAGAAACCAATAACAAACAGAA AGAATTCGAAGAAACCGCCAAAACCACCCGTCAGAGCATTGAACAGCTGGCAGCA

agcggccgcttt

> SEQ ID NO: 51 >Survivin:mSA (Mus Musculus) DNA

GGTGCACCGGCACTGCCGCAGATTTGGCAGCTGTATCTGAAAAACTATCGTATCG CCACCTTTAAAAACTGGCCGTTTCTGGAAGATTGTGCATGTACACCGGAACGTAT GGCAGAAGCAGGTTTTATTCATTGTCCGACCGAAAATGAACCGGATCTGGCACAG TGTTTTTTTTGCTTTAAAGAACTGGAAGGTTGGGAGCCGGATGATAATCCGATTGA AGAACATCGTAAACATAGTCCGGGTTGTGCATTTCTGACCGTGAAAAAACAAATGG AAGAACTGACCGTTAGCGAGTTTCTGAAACTGGATCGTCAGCGTGCCAAAAACAA AATTGCAAAAGAAACCAATAACAAACAGAAAGAATTCGAAGAAACCGCCAAAACCA CCCGTCAGAGCATTGAACAGCTGGCAGCAGGTGGCAGCGCAGAAGCAGGTATTA CCGGCACCTGGTATAATCAGCATGGTAGCACCTTTACCGTTACCGCAGGCGCAGA TGGTAATCTGACAGGTCAGTATGAAAATCGTGCACAGGGCACCGGTTGTCAGAAT AGCCCGTATACCCTGACCGGTCGTTATAATGGCACCAAACTGGAATGGCGTGTTG AATGGAATAATAGCACCGAAAATTGTCATAGCCGTACCGAATGGCGTGGTCAGTA TCAGGGTGGTGCAGAAGCCCGTATTAATACCCAGTGGAATCTGACCTATGAAGGT GGTAGCGGTCCGGCAACCGAACAGGGTCAGGATACCTTTACCAAAGTTAAA

> SEQ ID NO: 52 >mSA:CIDR1a-HIS

GAEAGITGTWYNQHGSTFTVTAGADGNLTGQYENRAQGTGCQNSPYTLTGRYNGTK LEWRVEWNNSTENCHSRTEWRGQYQGGAEARINTQWNLTYEGGSGPATEQGQDT FTKVKGGSKITSFDEFFDFWVRKLLIDTIKWETELTYCINNTDVTDCNKCNKNCVCFDK WVKQKEDEWTNIMKLFTNKHDIPKKYYLNINDLFDSFFFQVIYKFNEGEAKWNELKEN LKKQIASSKANNGTKDSEAAIKVLFNHIKEIATICKDNNTN > SEQ ID NO: 53 > mSA:CIDR1a-HIS DNA

GCAGAAGCAGGTATTACCGGCACCTGGTATAATCAGCATGGTAGCACCTTT ACCGTTACCGCAGGCGCAGATGGTAATCTGACAGGTCAGTATGAAAATCGT GCACAGGGCACCGGTTGTCAGAATAGCCCGTATACCCTGACCGGTCGTTAT AATGGCACCAAACTGGAATGGCGTGTTGAATGGAATAATAGCACCGAAAAT TGTCATAGCCGTACCGAATGGCGTGGTCAGTATCAGGGTGGTGCAGAAGCC CGTATTAATACCCAGTGGAATCTGACCTATGAAGGTGGTAGCGGTCCGGCA ACCGAACAGGGTCAGGATACCTTTACCAAAGTTAAAGGTGGCAGCAAAATAAC GTCATTTGATGAATTTTTTGATTTTTGGGTTAGAAAATTATTAATAGACACTATAAAGTG GGAAACCGAA CTTACGTATTGTATAAATAATACTGATGTCACGGATTGTAATAAATGTAACAAAAATTGC GTATGTTTTG ACAAATGGGTTAAACAAAAAGAAGACGAATGGACAAATATAATGAAACTATTCACAAACA AACACGAT ATACCGAAAAAATATTATmAATATT TGATCmTTGATAGmmTTTCCAAGTTATATATAAGTTT AACGAAGGAGAAGCAAAATGGAATGAACTTAAAGAAAATTTAAAAAAGCAAATTGCGTCT TCCAAAGC AAATAACGGAACCAAAGATTCAGAAGCTGCAATAAAAGTGTTGTTTAATCACATAAAAGA AATTGCAA CAATATGCAAAGATAATAATACAAAC

References

Bachmann, MF. and Jennings, Gary T. Vaccine delivery: a matter of size, geometry, kinetics and molecular patterns. Nat Rev Immunol 10(1 1), 787-796. 2010.

Bachmann MF, Zinkernagel, RM. Neutralizing antiviral B cell responses. Annual review of immunology 15: 235-270. 1997.

Bachmann, MF. et al. The influence of antigen organization on B cell responsiveness. Science. 262(5138), 1448-1451. 1993.

Bachmann, M.F., Jennings, G.T., 2004a. Virus-like particles: combining innate and adaptive immunity for effective vaccination. In: Kaufmann, P.D.S.H.E. (Ed.),

Novel Vaccination Strategies. Wiley-VCH Verlag GmbH & Co, pp. 415^132.

Buck, Christopher B. and Thompson, Cynthia D. Production of Papillomavirus-Based

Gene Transfer Vectors. Current Protocols in Cell Biology. 2001.

Buck CB, Pastrana D V, Lowy DR, Schiller JT. Efficient intracellular assembly of papillomaviral vectors. J Virol. 2004;78(2):751-7.

Buck CB, Thompson CD, Pang Y-YS, Lowy DR, Schiller JT. Maturation of

papillomavirus capsids. J Virol. 2005;79(5):2839^16.

Buck CB, Thompson CD. Production of papillomavirus-based gene transfer vectors. Curr Protoc Cell Biol. 2007;Chapter 26:Unit 26.1. Chackerian, B. et al. Induction of autoantibodies to mouse CCR5 with recombinant papillomavirus particles. PNAS. (5) 2373-2378. 1999.

Chackerian, B. Virus-like particles: flexible platforms for vaccine development. Expert Review of Vaccines. 6(3), 381-390. 2007.

Grgacic, Elizabeth V. L. and Anderson, David A. Virus-like particles: Passport to immune recognition. Particle-based Vaccines. Methods 40(1), 60-65. 2006.

Kouskoff, V. et al. T Cell-Independent Rescue of B Lymphocytes from Peripheral Immune Tolerance. Science 287 (5462). 2501-2503. 2000.

Lim KH, Huang H, Pralle A, Park S. Engineered streptavidin monomer and dimer with improved stability and function. Biochemistry. 2011 ;50(40):8682-91.

Murray K. Application of recombinant DNA techniques in the development of viral vaccines. Vaccine. 6: 164-74.1988.

Nielsen MA, Pinto V V., Resende M, Dahlback M, Ditlev SB, Theander TG, et al.

Induction of adhesion-inhibitory antibodies against placental Plasmodium falciparum parasites by using single domains of VAR2CSA. Infect Immun. 2009;77(6):2482-7.

Haase RN, Megnekou R, Lundquist M, Ofori MF, Hviid L, Staalsoe T. Plasmodium falciparum parasites expressing pregnancy-specific variant surface antigens adhere strongly to the choriocarcinoma cell line BeWo. Infect Immun. 2006;74(5):3035-8.

Snounou G, Zhu X, Siripoon N, Jarra W, Thaithong S, Brown KN, et al. Biased distribution of msp1 and msp2 allelic variants in Plasmodium falciparum populations in

Thailand. Trans R Soc Trop Med Hyg. 1999;93(4):369-74.

Plotkin, SA. Vaccines: past, present and future. Nat Med . 5-4-2005.

Pumpens, P. and Grens, E. HBV Core Particles as a Carrier for B Cell/T Cell Epitopes.

Intervirology 44(2-3), 98-1 14. 2001.

Raja, Krishnaswami S. et al. Icosahedral Virus Particles as Polyvalent Carbohydrate

Display Platforms. ChemBioChem 4(12), 1348-1351. 2003.