US20230078675A1

US20230078675A1 - Virus-like particle with efficient epitope display

Info

Publication number: US20230078675A1
Application number: US17/968,115
Authority: US
Inventors: Adam Frederik Sander Bertelsen; Ali Salanti; Thor Theander; Susan Thrane; Christoph Mikkel Janitzek; Mette Ørskov Agerbaek; Morten Agertoug Nielsen; Jan Tobias Gustafsson
Original assignee: Københavns Universitet
Current assignee: Københavns Universitet
Priority date: 2015-01-15
Filing date: 2022-10-18
Publication date: 2023-03-16
Also published as: EA201791598A8; AU2016207099A1; EA201791598A1; WO2016112921A1; US10086056B2; CA3010975A1; US20180125954A1; US11497800B2; US20180362591A1; EP3244915A1; HK1246654A1; AU2016207099B2; US10526376B2; US20200115420A1; DE16703049T1; EA035378B1; AU2016207099C1

Abstract

The invention relates to a virus-like particle (VLP) based vaccine. The virus-like particle constitutes a non-naturally occurring, ordered and repetitive antigen array display scaffold which can obtain a strong and long-lasting immune response in a subject. The VLP-based vaccine may be used for the prophylaxis and/or treatment of a disease including, but is not limited to, cancer, cardiovascular, infectious, chronic, neurological diseases/disorders, asthma, and/or immune-inflammatory diseases/disorders.

Description

REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No. 16/691,897, filed Nov. 22, 2019, which is a divisional application U.S. patent application Ser. No. 15/542,623, filed Jul. 10, 2017, now U.S. Pat. No. 10,526,376, which is a U.S. national phase application of PCT/DK2016/050011, filed Jan. 15, 2016, which claims priority from Danish Patent Application No. PA 2015 70237, filed Apr. 22, 2015 and Danish Patent Application No. PA 2015 70019, filed Jan. 15, 2015; the entire content of each of which is incorporated herein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically as an XML file and is hereby incorporated by reference in its entirety. Said XML file, created on Oct. 18, 2022, is named Sequence_Listing_16COPE-H060505VA.xml and is 158,802 bytes in size.

FIELD OF THE INVENTION

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, with regular spacing, and with consistent orientation; three critical factors for obtaining optimal activation of the immune system.

BACKGROUND OF THE 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 S A 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 low valency 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 low valency soluble proteins during the past several years and have led to the conjecture that the size, valency, and the spatial assembly of the vaccine antigen component are critical parameters for optimal activation of the immune system.
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 simple 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 (22-150 nm) appears to be optimal for efficient uptake by professional antigen presenting cells, particularly dendritic cells (DCs) as well as for entry into lymph vessels and hence VLPs efficiently stimulate both the humoral and cellular arms of the immune system (Bachmann, M F, Jennings, G T. 2010). Furthermore, surface structures presenting an antigen at high density, with regular spacing, 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, M F and Zinkernagel, R M. 1997) leading to strong B-cell responses, even in the absence of T-cell help (Bachmann, M F 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. Antibodies are believed to be the primary effectors of all current prophylactic microbial vaccines and hence the main focus for developing VLP-based vaccines is to induce strong humoral responses, which is especially true when targeting self-antigens. 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, M F and Jennings, G T, 2004a; Chackerian, 2007; Grgacic, E V L. and Anderson, D A. 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 they can be presented in an immunodominant region on the VLP surface while maintaining their 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 functionally be essentially 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. non-protein targets) to VLPs and this approach is, in principle, not restricted by the size of the antigen (Raja K S. et al. 2003). However, so far only shorter peptides have successfully been coupled at high density and with consistent orientation to the surface of VLPs (Bachmann M F, Jennings G T. 2011) and in the case of larger antigens it remains highly challenging to control both 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, with regular spacing, and with consistent orientation, which are three critical factors for obtaining strong and long lasting activation of the immune system.
In brief:

- Induction of a strong and long lasting immune response to pathogens as well as disease associated antigens is very difficult to obtain with simple 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, at high density, and with a consistent orientation for optimal epitope display, poses a major biotechnological challenge.

SUMMARY OF THE INVENTION

The present invention solves the challenges of obtaining a VLP which presents densely and regularly spaced surface antigens with consistent orientation. Such VLPs are capable of efficiently displaying epitopes and are thus able to induce long-term protective immunity in a subject. A general concept of the present invention is illustrated in FIG. 1 . The inventors have identified bacteriophages (e.g. AP205) where a Spytag and/or a SpyCatcher can be fused to the capsid protein without compromising the self-assembly of the particle.
Surprisingly the inventors were able to fuse the entire 116 amino acid SpyCatcher to the N-terminal of the AP205 capsid protein. In addition, the inventors have managed to setup a system to produce antigens fused to a SpyCatcher and/or a SpyTag polypeptide, which ensures control of the orientation of the coupled antigen. The specific interaction between the SpyTag and SpyCatcher (Zakeri, B. et al. PNAS. 2012) ensures 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. Surprisingly, the present inventors have found that the large SpyCatcher protein, which comprises more than 100 amino acids, may be fused to a capsid protein without disrupting the spontaneous VLP self-assembly process. 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 SpyTag and SpyCatcher interact via a spontaneous isopeptide bond formation (Zakeri, B. et al. PNAS. 2012). This is a covalent interaction and ensures a high strength, one-to-one interaction between the SpyTag and SpyCatcher linked antigen. The flexibility of the isopeptide bond is limited by the conformation of the co-joined SpyTag-SpyCatcher polypeptide ensuring consistent orientation of the antigens thus displayed on the VLP.
The problems described above are solved by the aspects and embodiments of the present invention characterized in the claims. As illustrated in FIG. 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 virus capsid protein comprising a first peptide tag, and
- ii. an antigen fused to a second peptide tag,

wherein the antigen and virus capsid protein are linked via an isopeptide bond between the first and second peptide tag, and wherein i-ii form a virus-like particle displaying said antigen.
In a preferred embodiment the virus capsid protein comprises an AP205 capsid protein and/or phage fr capsid protein fused to a SpyCatcher, wherein the capsid protein—SpyCatcher fusion protein is capable of forming a virus-like particle. In a further embodiment SpyCatcher is fused to the N-terminus of the AP205 capsid protein optionally via a linker.
Another main aspect of the present invention concerns vaccine for use in the prophylaxis and/or treatment of a disease wherein the vaccine comprises:

- i. an AP205 capsid protein and/or a phage fr capsid protein comprising a SpyCatcher polypeptide insertion, and
- ii. an antigen fused to SpyTag,

wherein the antigen and AP205 capsid protein and/or fr capsid protein are linked via the interaction between the Spytag and the SpyCatcher, and wherein i-ii form a virus-like particle displaying said antigen.
In one embodiment, the vaccine for use in the prophylaxis and/or treatment of a disease comprises:

- iii. an AP205 capsid protein and/or a phage fr capsid protein comprising a SpyTag polypeptide insertion, and
- iv. an antigen fused to SpyCatcher,

wherein the antigen and AP205 capsid protein and/or fr capsid protein are linked via the interaction between the Spytag and the SpyCatcher, and wherein i-ii form a virus-like particle displaying said antigen.
The problems described above are solved by the aspects and embodiments of the present invention characterized in the claims. As illustrated in FIG. 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. an AP205 capsid protein and/or a phage fr capsid protein comprising a SpyCatcher polypeptide insertion such that the capsid protein can self-assemble into a VLP that displays the SpyCatcher in a context that it binds to SpyTag, and
- ii. an antigen fused to SpyTag,

wherein the antigen and AP205 capsid protein and/or phage fr capsid protein are linked via the interaction between the Spytag and the SpyCatcher, and wherein i-ii form a virus-like particle displaying said antigen.
In one embodiment, the vaccine for use in the prophylaxis and/or treatment of a disease comprises:

- i. an AP205 capsid protein and/or a phage fr capsid protein comprising a SpyTag polypeptide insertion such that the capsid protein can self-assemble into a VLP that displays the SpyTag in a context that it binds to SpyCatcher, and
- ii. an antigen fused to SpyCatcher·

wherein the antigen and AP205 capsid protein and/or phage fr capsid protein are linked via the interaction between the Spytag and the SpyCatcher, and wherein i-ii form a virus-like particle displaying said antigen.
In another aspect the present invention concerns a vector comprising at least one polynucleotide encoding

wherein the antigen and AP205 capsid protein and/or phage fr capsid protein are linked via the interaction between the Spytag and the SpyCatcher, and wherein i-ii form a virus-like particle displaying said antigen.
In one embodiment, the vector comprises at least one polynucleotide encoding

- i. an AP205 capsid protein and/or a phage fr capsid protein comprising a SpyTag polypeptide insertion, and
- ii. an antigen fused to SpyCatcher,

wherein the antigen and AP205 capsid protein and/or phage fr capsid protein are linked via the interaction between the Spytag and the SpyCatcher, and wherein i-ii form a virus-like particle displaying said antigen.
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 vaccine.
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; an AP205 capsid protein and/or a phage fr capsid protein comprising SpyCatcher, and
- ii. obtaining a second polypeptide; an antigen fused to SpyTag, and
- iii. subjecting the first polypeptide to conditions which enable formation of virus-like particles, and
- iv. obtaining a vaccine by linkage of the second polypeptide and said virus-like particles via the interaction between the SpyTag and SpyCatcher of said virus-like particles, and
- v. generating a composition comprising said vaccine,

thereby obtaining a pharmaceutical composition.
In one embodiment, the method of manufacturing a pharmaceutical composition comprising said vaccine comprises the steps of

- i. obtaining a first polypeptide; an AP205 capsid protein and/or a phage fr capsid protein comprising SpyTag, and
- ii. obtaining a second polypeptide; an antigen fused to SpyCatcher, and
- iii. subjecting the first polypeptide to conditions which enable formation of virus-like particles, and
- iv. obtaining a vaccine by linkage of the second polypeptide and said virus-like particles via the interaction between the SpyTag and SpyCatcher of said virus-like particles, and
- v. 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.

In 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.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 : A general aspect of the present invention. Production steps in making a fully biotinylated SpyTag-capsid protein coupled with a SpyCatcher-antigen. SpyC is an abbreviation of SpyCatcher.

FIG. 2 : A general aspect of the present invention. Production steps in making a SpyTag/KTag-capsid protein coupled with a SpyTag/KTag-antigen.

FIG. 3 : Transmission electron microscopy of SpyTag-AP205 virus-like particles. The TEM picture shows non-aggregated VLPs of approx. 30 nm, assembled from Spy-AP205 (SEQ ID NO: 62).

FIG. 4 : SpyTag-VLP/SpyCatcher-antigen coupling efficiency. The figure (SDS-PAGE gels) shows collected fraction after density gradient ultracentrifugation of a mixture of SpyTag-AP205 VLPs (SEQ ID NO: 62) and two different spycatcher-antigen fusions (SEQ ID NO: 19 (FIG. 4A) and 21 (FIG. 4B), respectively). The molar relationship between conjugated spy-AP205 capsid protein and spycatcher-antigen as compared to the amount of unconjugated spy-AP205 capsid protein can be used to estimate the antigen-VLP coupling efficiency. From this experiment it is estimated that 80-100% of the spy-AP205 capsid protein is conjugated to a spyCatcher-antigen.

FIG. 5 : Transmission electron microscopy of SpyTag-AP205 virus-like particles coupled with SpyCatcher-IL-5 (C63T/C105T) (SEQ ID NO: 19). The TEM picture shows non-aggregated VLPs assembled from spy-AP205 (SEQ ID NO: 62). The apparent average size of the antigen-coupled VLPs seem ˜25-35 nm larger compared to corresponding non-coupled spy-AP205 VLPs.

FIG. 6 : Transmission electron microscopy of SpyCatcher-AP205 virus-like particles. The TEM picture shows non-aggregated VLPs of approx. 30 nm, assembled from SpyCatcher-AP205 (SEQ ID NO: 76).

FIG. 7 : SpyCatcher-VLP/SpyTag-antigen coupling efficiency. The figure (SDS-PAGE gels) shows collected fraction after density gradient ultracentrifugation of a mixture of SpyCatcher-AP205 VLPs (SEQ ID NO: 76) and a SpyTag-antigen fusion (SEQ ID NO: 82). The molar relationship between conjugated SpyCatcher-AP205 capsid protein and SpyTag-antigen as compared to the amount of unconjugated SpyCatcher-AP205 capsid protein can be used to estimate the antigen-VLP coupling efficiency. From this experiment it is estimated that 80-100% of the SpyCatcher-AP205 capsid protein is conjugated to a SpyTag-antigen.

FIG. 8 : Transmission electron microscopy of SpyCatcher-AP205 virus-like particles coupled with SpyTag-ID1ID2a (SEQ ID NO: 82). The TEM picture shows non-aggregated VLPs assembled from SpyCatcher-AP205 (SEQ ID NO: 76). The apparent average size of the antigen-coupled VLPs seem 35 nm larger compared to corresponding non-coupled SpyCatcher-AP205 VLPs.

FIG. 9 : Dynamic light scattering of SpyTag-AP205 virus-like particles alone or coupled with SpyCatcher-antigen. The graph shows VLPs assembled from SpyTag-AP205 (SEQ ID NO: 62). The SpyTag-AP205 particles are monodisperse and have a size of 34 nm. SpyTag-AP205 virus-like particles coupled with SpyCatcher-antigen (SEQ ID NO: 19) are also monodisperse and have a size of 73 nm, which is 39 nm larger compared to corresponding non-coupled SpyTag-AP205 VLPs.

FIG. 10 : Dynamic light scattering of SpyCatcher-AP205 virus-like particles alone or coupled with SpyTag-antigen. The graph shows VLPs assembled from SpyCatcher-AP205 (SEQ ID NO: 76). The SpyCatcher-AP205 particles are monodisperse and have a size of 42 nm. SpyCatcher-AP205 virus-like particles coupled with SpyTag-antigen (SEQ ID NO: 82) are also monodisperse and have a size of 75 nm, which is 33 nm larger compared to corresponding non-coupled SpyCatcher-AP205 VLPs.

FIG. 11 : Expression of AP205 VLP. Left panel: SDS-PAGE of AP205 VLPs (SEQ ID NO: 58). The SDS-PAGE shows that the coat proteins are between 12 and 22 kDa (the theoretical size is 14 kDa). Right panel: Transmission electron microscopy of AP205 virus-like particles. The TEM picture shows non-aggregated VLPs of approx. 30 nm, assembled from AP205 (SEQ ID NO: 58).

FIG. 12 : Binding capacity of AP205-SpyTag vs. SpyTag-AP205. SDS-PAGE of the AP205-SpyTag (SEQ ID NO: 64(65)) and SpyTag-AP205 (SEQ ID NO: 62(63)) coupled to SpyC-Antigen (SEQ ID NO: 19). Both VLPs are coupled in a molar ratio of 1:2 (VLP to Ag) and the SDS-PAGE gel shows that SpyTag-AP205 has a better binding capacity compared to AP205-SpyTag.

FIG. 13 : Binding capacity of SpyTag-AP205-SpyTag (SAS) vs. SpyTag-AP205 (SA): (Left) SDS-PAGE of the SpyTag-AP205-SpyTag (SAS) VLPs (SEQ ID NO: 71(72)), lane 1, SpyTag-AP205 (SA) VLPs (SEQ ID NO: 62), lane 2 and AP205 VLPs, lane 3 (SEQ ID NO: 58) coupled in a molar ratio of 1:1 with SpyCatcher-Antigen (SEQ ID NO: 19).

FIG. 14 : Coupling of Pfs25 to SpyTag-AP205-SpyTag Virus-like particles. Reduced SDS-PAGE of SpyTag-AP205-SpyTag VLPs (SEQ ID NO: 71(72)), lane 1; SpyTag-AP205-SpyTag and Pfs25 (SEQ ID NO: 27) in a molar ratio of 1:1, lane 2; AP205 VLPs (SEQ ID NO: 58) and Pfs25 (SEQ ID NO: 27) in a molar ratio of 1:1, lane 3.

FIG. 15 : Induction of higher titres of antibodies as a result of VLP display. The figure shows the Ig response against an antigen (SEQ ID NO: 7) two weeks after a prime-boost-boost immunization regimen. The dashed lines represent individual mice immunized with SpyCatcher-AP205 (SEQ ID NO: 76) coupled with SpyTag-antigen (SEQ ID NO: 7). The gray line represents individual mice immunized with soluble SpyTag-Antigen (SEQ ID NO: 7) and AP205 (SEQ ID NO: 58), which is unable to bind the antigen. Both vaccines were formulated without aluminum hydroxide gel. X-axis: serum dilution; Y-axis: OD490 nm.

FIG. 16 : The figure shows the Ig response against an antigen (SEQ ID NO: 27) three months after a prime-boost-boost immunization regimen. The dashed line represents individual mice immunized with SpyTag-AP205-SpyT (SEQ ID NO: 71) coupled with SpyCatcher-antigen (SEQ ID NO: 27). The gray line represents individual mice immunized with soluble SpyCatcher-antigen (SEQ ID NO: 27) and AP205 (SEQ ID NO: 58), which is unable to bind the antigen. Both vaccines were formulated with aluminum hydroxide gel. X-axis: serum dilution; Y-axis: OD490 nm.

FIG. 17 : The figure shows the avidity of antibodies induced in mice following a prime-boost-boost immunization regimen. Mouse anti-sera were obtained four month after last immunization. The black bar represents a pool of sera from mice immunized with SpyTag-AP205-SpyTag (SEQ ID NO: 71) coupled to the spyCatcher-antigen (SEQ ID NO: 27). The gray bar represents a pool of sera from mice immunized with soluble SpyCatcher-antigen (SEQ ID NO: 27) and AP205 (SEQ ID NO: 58), which is unable to bind the antigen. Both vaccines were formulated with aluminum hydroxide gel.

FIG. 18 : The figure shows the avidity of antibodies induced in mice following a prime-boost-boost immunization regimen. Mouse anti-sera were obtained three month after last immunization. The black bar represents a pool of sera from mice immunized with SpyCatcher-AP205 (SEQ ID NO: 76) coupled to SpyTag-antigen (SEQ ID NO: 7). The gray bar represents a pool of sera from mice immunized with soluble SpyTag-Ag (SEQ ID NO: 7) and AP205 (SEQ ID NO: 58), which is unable to bind the antigen. Both vaccines were formulated without aluminum hydroxide gel.

FIG. 19 : Ig response against an antigen (SEQ ID NO: 52) following a single immunization. The dashed lines represent individual mice immunized with SpyTag-AP205 (SEQ ID NO: 62) coupled to SpyCatcher-antigen (SEQ ID NO: 52). The gray lines represent individual mice immunized with soluble SpyCatcher-antigen (SEQ ID NO: 52) and AP205 (SEQ ID NO: 58), which is unable to bind the antigen. Both vaccines were formulated with aluminum hydroxide gel. X-axis: serum dilution; Y-axis: OD490 nm.

FIG. 20 : The figure shows the Ig response against an antigen (SEQ ID NO: 27) following a single immunization. The dashed lines represent individual mice immunized with SpyTag-AP205-SpyTag (SEQ ID NO: 71) coupled to SpyCatcher-antigen (SEQ ID NO: 27). The gray lines represent individual mice immunized with soluble SpyCatcher-antigen (SEQ ID NO: 27) and AP205 (SEQ ID NO: 58), which is unable to bind the antigen. Both vaccines were formulated with aluminum hydroxide gel. X-axis: serum dilution; Y-axis: OD490 nm.

FIG. 21 : Breakage of self-tolerance as a result of VLP display. The figure shows the Ig response against the self-antigen IL-5 (SEQ ID NO: 19) five months after a prime-boost-boost immunization regimen. The dashed line represents individual mice immunized with SpyTag-AP205-SpyTag (SEQ ID NO: 71) coupled to the IL-5 SpyCatcher-(self)-antigen (SEQ ID NO: 19). The gray line represents individual mice immunized with soluble SpyCatcher-antigen (SEQ ID NO: 19) and AP205 (SEQ ID NO: 58), which is unable to bind the spycatcher-antigne. Both vaccines were formulated in aluminum hydroxide gel. X-axis: serum dilution; Y-axis: OD490 nm.

FIG. 22 : The figure shows the Ig response against the self-antigen CTLA-4 (SEQ ID NO: 11) two weeks after a prime-boost immunization regimen. The dashed line represents individual mice immunized with SpyTag-AP205-SpyTag (SEQ ID NO: 71) coupled to the CTLA-4 self-antigen (SEQ ID NO: 11). The gray line represents individual mice immunized with soluble SpyCatcher-antigen (SEQ ID NO: 11) and AP205 (SEQ ID NO: 58), which is unable to bind the spycatcher-antigne. Both vaccines were formulated in aluminum hydroxide gel. X-axis: serum dilution; Y-axis: OD490 nm.

FIG. 23 : Breakage of self-tolerance as a result of VLP display. The figure shows the Ig response against the self-antigen PD-L1 (SEQ ID NO: 9) two weeks after a prime-boost immunization regimen. The dashed line represents individual mice immunized with SpyTag-AP205-SpyTag (SEQ ID NO: 71) coupled to the PD-L1 self-antigen (SEQ ID NO: 9). The gray line represents individual mice immunized with soluble self-antigen (SEQ ID NO: 9) and AP205 (SEQ ID NO: 58), which is unable to bind the SpyCatcher-antigen. Both vaccines were formulated in aluminum hydroxide gel. X-axis: serum dilution; Y-axis: OD490 nm.

FIG. 24 : Immunization with a Pfs25 VLP vaccine resulted in induction of functional antibodies which were able to block the transmission of Plasmodium falciparum parasites in vitro. Mice were immunized two times with 2.5 ug of either A) spycatcher-Pfs25 antigen (SEQ ID NO: 27) displayed on the SpyTag-AP205-SpyT (SEQ ID NO: 71) VLP or B) soluble spycatcher-Pfs25 antigen (SEQ ID NO: 27) mixed with the AP205 VLP (SEQ ID NO: 58), which is unable to bind/display the antigen. Both vaccines were formulated with aluminum hydroxide gel. Transmission-blocking efficacy of antibodies was evaluated by standard mosquito membrane feeding assay (SMFA) using purified IgG from immune sera.

FIG. 25 : Antigen-specific qualitative testing of induced immune responses: Testing of the SpyCatcher-VLP platform to induce VAR2CSA specific antibodies The parasite binding-inhibition assay show normalized parasite binding after incubation with pooled anti-sera (3-fold dilutions starting from 1:20) from mice (n=5) vaccinated with SpyTag-ID1ID2a (SEQ ID NO: 82) conjugated to SpyCatcher-VLPs (SEQ ID NO: 76) or soluble SpyTag-ID1ID2a (SEQ ID NO: 82) mixed with unmodified AP205 VLPs (SEQ ID NO: 58). Parasite binding results are shown after first (▴), second (▪) and third (•) immunization. The assay show that anti-sera from mice immunized with VLP-conjugated SpyTag-ID1ID2a (SEQ ID NO: 82) has a greater binding-inhibition capacity compared to anti-sera from mice immunized with soluble SpyTag-ID1ID2a (SEQ ID NO: 82).

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 FIG. 1 .

Definitions

The term “virus-like particle” or “VLP” refers to one or several recombinantly expressed viral capsid 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 an AP205 capsid protein and/or a phage fr capsid protein to self-assemble into virus-like particles in the absence of other viral proteins, when subjected to specific conditions. “Self-assembly” does not preclude the possibility that cellular proteins, e.g. chaperons, participate in the process of intracellular VLP assembly. 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. Virus capsid proteins may be able to form VLPs on their own, or in combination with several virus capsid proteins, these optionally all being identical. Examples of virus capsid proteins include but are not limited to: AP205 capsid protein and/or a phage fr capsid protein.
The term “consistent orientation”, as used herein, refers to the orientation of the target antigen constructs of the present invention and their spatial orientation to an AP205 capsid protein and/or a phage fr capsid protein of the present invention. When linking an antigen fused to a SpyCatcher to an AP205 VLP and/or a phage fr VLP displaying a SpyTag, a molecule of the SpyCatcher tagged vaccine antigen can only be linked to a single AP205 capsid protein and/or a phage fr capsid protein at unique sites in both the vaccine antigen and the recombinant AP205 capsid protein and/or a recombinant phage fr capsid protein, thus creating a uniform and/or consistent presentation of said antigen with a consistent orientation. In contrast, for example, a streptavidin homo-tetramer may crosslink several AP205 capsid proteins and/or recombinant phage fr capsid proteins on the surface of a biotinylated VLP, thus creating an irregular and non-consistent orientation of said antigen (Chackerian, B. et al. 2008). Besides, it is highly challenging to use 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, circle-like, 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
The term “SpyTag” refers to a part of the CnaB2 domain from the FbaB protein from Streptococcus pyogenes optimized to bind SpyCatcher consisting of another part of the CnaB2 domain. The interaction occurs when the unprotonated amine of Lys31 nucleophilically attacks the carbonyl carbon of Asp117, catalyzed by the neighboring Glu77. The minimal peptide to mediate this binding is AHIVMVDA whereas a c-terminal extension giving the sequence: AHIVMVDAYKPTK provides the most optimal region, designated “SpyTag” (Zakeri, B. et al. PNAS. 2012).
The term “SpyCatcher” refers to a part of the CnaB2 domain from the FbaB protein from Streptococcus pyogenes optimized to bind SpyTag consisting of another part of the CnaB2 domain. SpyCatcher can be residue number 1-113 of CnaB2 and binding can be optimized by the following two mutations: I34E and M69Y (Zakeri, B. et al. PNAS, 2012). Truncated and homologous versions of SpyCatcher are also objects of the present invention and thus the term SpyCatcher herein denotes any variant of SpyCatcher that is still capable of interacting with SpyTag. Variants of SpyCatcher may include, but is not limited to truncated SpyCatcher variants. Truncated SpyCatcher variants may include, but is not limited to SEQ ID NO: 60 and SEQ ID NO: 61.
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.
The term “peptide tag” as used herein refers to a peptide sequence which is genetically grafted onto a recombinant protein. A first peptide tag may facilitate interactions with a second peptide tag e.g. by forming one or more covalent bonds such as isopeptide bonds. In an embodiment the first peptide tag described in the present invention comprises a SpyTag as described herein. In an embodiment the first peptide tag described in the present invention comprises a KTag as described herein. In an embodiment the second peptide tag described in the present invention comprises a KTag as described herein. In an embodiment the second peptide tag described in the present invention comprises a SpyCatcher as described herein. In an embodiment the second peptide tag described in the present invention comprises a SpyTag as described herein.
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 potentially 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 virus capsid protein such as an AP205 capsid protein and/or a phage fr capsid protein VLP using a SpyTag and SpyCatcher fusion without disrupting the self-assembly of the VLP. Thus in a main aspect, as illustrated in FIG. 1 , the present invention concerns a vaccine for use in the prophylaxis and/or treatment of a disease wherein the vaccine comprises:

- i. a virus capsid protein comprising at least one first peptide tag, and
- ii. an antigen fused to a second peptide tag,

wherein the antigen and virus capsid protein are linked via an isopeptide bond between the first and second peptide tag, and wherein i-ii form a virus-like particle displaying said antigen.
In an embodiment the first peptide tag comprises at least one SpyCatcher, and the second peptide tag comprises a SpyTag, wherein the antigen and the virus capsid protein are linked via the interaction between the SpyCatcher and the SpyTag interaction, and wherein i-ii form a virus-like particle displaying said antigen.
In an embodiment, the first peptide tag comprises two SpyCatchers. Thus in one embodiment, two SpyCatchers are fused to the AP205 capsid protein, one at each terminus.
In some embodiments the SpyCatcher is fused to the capsid protein via a spacer. In one embodiment the SpyCatcher is fused to the AP205 capsid protein via a spacer. Suitable spacers are known in the art and include spacers such as Gly-Gly-Ser-Gly-Ser (SEQ ID NO: 83).
In an embodiment the virus capsid protein is an AP205 capsid protein and the first peptide tag is a SpyCatcher, wherein the SpyCatcher is linked to the N-terminal of the AP205 capsid protein.
In an embodiment the first peptide tag comprises at least one Spytag, and the second peptide tag comprises a SpyCatcher, wherein the antigen and virus capsid protein are linked via the interaction between the SpyCatcher and SpyTag interaction, and wherein i-ii form a virus-like particle displaying said antigen.
In an embodiment, the first peptide tag comprises two SpyTags. Thus in one embodiment, two SpyTags are fused to the AP205 capsid protein, one at each terminus.
In another embodiment the first peptide tag comprises a SpyTag, and the second peptide tag comprises a KTag, and wherein the vaccine optionally comprises a SpyLigase, and wherein the antigen and virus capsid protein are linked via the interaction between the SpyTag and KTag, and wherein i-ii form a virus-like particle displaying said antigen. In a further embodiment the first peptide tag is a SpyTag and the second peptide tag is a KTag.
In another embodiment the first peptide tag comprises a KTag, and the second peptide tag comprises a SpyTag, and wherein the vaccine optionally comprises a SpyLigase wherein the antigen and virus capsid protein are linked via the interaction between the KTag and SpyTag, and wherein i-ii form a virus-like particle displaying said antigen.
In another embodiment the virus capsid protein comprises or is an AP205 capsid protein and/or a phage fr capsid protein.
In an embodiment the first peptide tag as described herein is fused to the N- and/or C-terminus of AP205 capsid protein and/or fr protein capsid.
In an embodiment the at least one first peptide tag as described herein is fused to the N- and/or C-terminus of AP205 capsid protein and/or fr protein capsid via a spacer.
In an embodiment the first peptide tag as described herein is fused to the N-terminus of AP205 capsid protein.
In an embodiment the first peptide tag as described herein is fused to the C-terminus of AP205 capsid protein. In an embodiment the peptide tag fused to the C-terminus of AP205 is not a SpyCatcher.
In an embodiment the at least one first peptide tag as described herein is two first peptide tags, wherein the two peptide tags are identical, and wherein one of the two peptide tags is fused to the N-terminus of AP205 capsid protein and the other one is fused to the C-terminus of AP205 capsid protein. In one embodiment, the two first peptide tags are two SpyTags.
In an embodiment the first peptide tag as described herein is fused to the N-terminus of fr capsid protein.
In an embodiment the first peptide tag as described herein is fused to the C-terminus of fr capsid protein.
In an embodiment the first peptide tag comprises a SpyTag, SpyCatcher and/or KTag as described herein. In a further embodiment the first peptide tag is a SpyTag, SpyCatcher and/or KTag as described herein.
In one embodiment where the first peptide tag is a SpyCatcher, the fusion to the capsid protein is to said capsid protein's N-terminus.
In another embodiment said interaction comprises an isopeptide bond based interaction. In another embodiment the SpyTag and KTag according to any one of the preceding claims are linked by means of a SpyLigase.
In a main aspect the present invention concerns a vaccine for use in the prophylaxis and/or treatment of a disease wherein the vaccine comprises:

- i. an AP205 capsid protein and/or a phage fr capsid protein comprising a SpyCatcher, and
- ii. an antigen fused to a SpyTag,

wherein the antigen and AP205 and/or phage fr are irreversibly linked through a spontaneous isopeptide bond formation between Spytag and SpyCatcher, and wherein i-ii form a virus-like particle displaying said antigen. In a further aspect the SpyCatcher is fused to the N-terminus of the AP205 capsid 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. an AP205 capsid protein and/or a phage fr capsid protein comprising a SpyCatcher having a polypeptide 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 the polypeptide sequence of SEQ ID NO: 76, and
- ii. an antigen fused to a SpyTag,

wherein the antigen and AP205 and/or phage fr are irreversibly linked through a spontaneous isopeptide bond formation between Spytag and SpyCatcher, and wherein i-ii form a virus-like particle displaying said antigen.
In another main aspect, as illustrated in FIG. 1 , the present invention concerns a vaccine for use in the prophylaxis and/or treatment of a disease wherein the vaccine comprises:

- i. an AP205 capsid protein and/or a phage fr capsid protein comprising a SpyTag, and
- ii. an antigen fused to SpyCatcher,

wherein the antigen and AP205 capsid protein and/or a phage fr capsid protein are irreversibly linked through a spontaneous isopeptide bond formation between Spytag and SpyCatcher, and wherein i-ii form a virus-like particle displaying said antigen.
In an embodiment, the AP205 capsid protein and/or a phage fr capsid protein comprising a SpyTag is able to form a virus-like particle.
The inventors of the present invention have demonstrated formation of AP205 VLP's by recombinant expression of the AP205 capsid protein, preferably in Escherichia coli cells, such as BL21 cells. Other conditions and expression hosts (such as Saccharomyces cerevisiae or 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 a 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. an AP205 capsid protein and/or a phage fr capsid protein comprising a SpyCatcher, and
- ii. an antigen fused to SpyTag,

wherein the antigen and the AP205 capsid protein and/or a phage fr capsid protein are linked through a spontaneous isopeptide bond formation between Spytag and SpyCatcher, and wherein i-ii form an antigen display scaffold. In a further aspect the SpyCatcher is fused to the N-terminus of the AP205 capsid protein.
Thus another aspect of the present invention relates to an antigen display scaffold, comprising an assembled virus-like particle comprising:

wherein the antigen and the AP205 capsid protein and/or a phage fr capsid protein are linked through a spontaneous isopeptide bond formation between Spytag and SpyCatcher, and wherein i-ii 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

wherein the antigen and the AP205 capsid protein and/or a phage fr capsid protein are linked through a spontaneous isopeptide bond formation between Spytag and SpyCatcher, and wherein i-ii form a non-naturally occurring, ordered and repetitive antigen array. In a further aspect SpyCatcher is fused to the N-terminus of the capsid protein.
Another aspect of the present invention relates to a method of producing a non-naturally occurring, ordered and repetitive antigen array comprising

- i. an AP205 capsid protein and/or a phage fr capsid protein comprising at least one SpyTag, and
- ii. an antigen fused to SpyCatcher, wherein the antigen and the AP205 capsid protein and/or a phage fr capsid protein are linked through a spontaneous isopeptide bond formation between Spytag and SpyCatcher, and wherein i-ii 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, chronic diseases, neurologic diseases, and/or infectious diseases.
In an embodiment an antigen which is associated with at least one cancer disease is linked to the virus capsid protein, such as the AP205 capsid protein and/or a phage fr capsid protein via the interaction between SpyCatcher and SpyTag. 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 virus capsid protein, such as the AP205 capsid protein and/or a phage fr capsid protein via the interaction between SpyCatcher and SpyTag. 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 virus capsid protein, such as the AP205 capsid protein and/or a phage fr capsid protein via the interaction between SpyCatcher and SpyTag. 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 virus capsid protein, such as the AP205 capsid protein and/or a phage fr capsid protein via the interaction between SpyCatcher and SpyTag. 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.
In an embodiment, an antigen which is associated with at least one chronic disease is linked to the virus capsid protein, such as the AP205 capsid protein and/or a phage fr capsid protein via the interaction between SpyCatcher and SpyTag. In a further embodiment the present VLP vaccine can be used for prophylaxis and/or treatment of the chronic disease and/or chronic diseases which the antigen is associated with.
In an embodiment, an antigen which is associated with at least one neurologic disease is linked to the virus capsid protein, such as the AP205 capsid protein and/or a phage fr capsid protein via the interaction between SpyCatcher and SpyTag. In a further embodiment the present VLP vaccine can be used for prophylaxis and/or treatment of the neurologic disease and/or neurologic 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	Breast cancer	Females overexpressing Her2
(ERBB2)
Her2/Neu	Gastric cancer	Males and Females overexpressing Her2
(ERBB2)
Her2/Neu	Ovarian cancer	Females overexpressing Her2
(ERBB2)
Her2/Neu	Uterine serous	Postmenopausal Females overexpressing
(ERBB2)	carcinoma	Her2
Survivin	Cancer types	Males and non-pregnant Females
	overexpressing Survivin	overexpressing Survivin
PCSK9	cardiovascular disease	Males and Females with dyslipidemia
PCSK9	cardiovascular disease	Males and Females with atherosclerosis
PCSK9	cardiovascular disease	Males and Females with
		hypercholesterolemia
Interleukin-5	Asthma	Males and Females with eosinophilia
Interleukin-5	nasal polyposis	Males and Females with eosinophilia
Interleukin-5	atopic dermatitis	Males and Females with eosinophilia
Interleukin-5	eosinophilic esophagitis	Males and Females with eosinophilia
Interleukin-5	Hypereosinophilic	Males and Females with eosinophilia
	syndrome
Interleukin-5	Churg-Strauss 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
PfEMP1,	Malaria	Males and Females with malaria
CIDR1a
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
Hemagglutinin	Influenza	Males and Females with influenza
HA
Interleukin-17	Psoriasis	Males and Females with Psoriasis
Interleukin-17	Multiple sclerosis	Males and Females with multiple
		sclerosis
Interleukin-17	Rheumatoid arthritis	Males and Females with rheumatoid
		arthritis
Interleukin-17	Inflammatory bowel	Males and Females with inflammatory
	diseases	bowel diseases
Interleukin-17	Asthma	Males and Females with Asthma
IL-33	Asthma	Males and Females with Asthma
IgE	Asthma	Males and Females with Asthma
Gp160	HIV	Males and Females with HIV
Gp120	HIV	Males and Females with HIV
Gp40	HIV	Males and Females with HIV
GD2	Cancer cell types	Males and Females with GD2 expressing
	expressing GD2 (e.g.	tumors.
	melanomas,
	osteosarcoma and soft-
	tissue sarcomas)
EGF-R	Cancer cell types	Males and Females with EGF-R
	expressing EGF-R (e.g.	expressing tumors.
	metastatic colorectal and
	head and neck cancer)
CEA	Cancer cell types	Males and Females with CEA expressing
	expressing CEA (e.g.	tumors.
	colon and rectal cancer
	or pancreatic, breast,
	ovary, or lung cancer.
CD52	Chronic lymphocytic	Males and Females with chronic
	leukemia	lymphocytic leukemia (CLL), cutaneous
	(CLL), cutaneous T-cell	T-cell lymphoma (CTCL) and T-cell
	lymphoma (CTCL) and	lymphoma or multiple sclerosis.
	T-cell lymphoma or
	multiple sclerosis
CD21	B-cell cancers	Males and Females with B-cell cancers
human melanoma	Cancer cell types	Males and Females with melanoma.
protein gp100	expressing human
	melanoma protein gp
	100 (e.g. Melanoma).
human melanoma	Cancer cell types	Males and Females with melanoma.
protein melan-	expressing human
A/MART-1	melanoma protein
	melan-A/MART-1 (e.g.
	Melanoma)
tyrosinase	Melanoma	Males and Females with melanoma
NA17-A nt	Melanoma	Males and Females with melanoma
protein
MAGE-3 protein	melanoma, non-small	Males and Females with melanoma, non-
	cell lung cancer,	small cell lung cancer or hematologic
	hematologic	malignancies
	malignancies.
p53 protein	Cancer cell types	Males and Females with tumors
	expressing p53	expressing p53
HPV 16 E7	Cancers of the cervix,	HPV infected males and females
protein	vulva, vagina, penis,
	oropharynx and anus.
HPV L2	Cancers of the cervix,	HPV infected males and females
	vulva, vagina, penis,
	oropharynx and anus.
PD-L1	Cancer types PD-L1	Males and females with tumors
		expressing PD-L1
PD-L1	Cancer types PD1	Males and females with tumors
		expressing PD1
CTLA-4	Cancer types CTLA-4	Males and females with tumors
		expressing CTLA-4
hCG	Cancer cell types	Males and Females with tumors
	expressing hCG	expressing hCG
Fel d1	Cat allergy	Males and females allergic to cats
(IHNV) G-	Infectious	Salmon and trout infected with IHNV
protein	haematopoietic necrosis
	(IHN)

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.
In one embodiment, the AP205 capsid protein is fused at its N-terminus and/or at its C-terminus to a SpyCatcher and the antigen is fused to a SpyTag and is selected from the group consisting of interleukin-17, hemagglutinin, GD2, EGF-R, CEA, CD52, CD21, human melanoma protein gp100, human melanoma protein melan-A/MART1, tyrosinase, NA17-A nt, MAGE-3, HPV 16 E7, HPV L2, PD1, PD-L1, CTLA-4, p53, hCG, Fel d1 and (IHNV) G-protein. In one embodiment, the AP205 capsid protein is fused to a SpyCatcher at its N-terminus and the antigen is fused to a SpyTag and is selected from the group consisting of interleukin-17, hemagglutinin, GD2, EGF-R, CEA, CD52, CD21, human melanoma protein gp100, human melanoma protein melan-A/MART1, tyrosinase, NA17-A nt, MAGE-3, HPV 16 E7, HPV L2, PD1, PD-L1, CTLA-4, p53, hCG, Fel d1 and (IHNV) G-protein. In another embodiment, the AP205 capsid protein is fused to a SpyCatcher at its C-terminus and the antigen is fused to a SpyTag and is selected from the group consisting of interleukin-17, hemagglutinin, GD2, EGF-R, CEA, CD52, CD21, human melanoma protein gp100, human melanoma protein melan-A/MART1, tyrosinase, NA17-A nt, MAGE-3, HPV 16 E7, HPV L2, PD1, PD-L1, CTLA-4, p53, hCG, Fel d1 and (IHNV) G-protein. In another embodiment, the AP205 capsid protein is fused to a SpyCatcher at its N-terminus and at its C-terminus and the antigen is fused to a SpyTag and is selected from the group consisting of interleukin-17, hemagglutinin, GD2, EGF-R, CEA, CD52, CD21, human melanoma protein gp100, human melanoma protein melan-A/MART1, tyrosinase, NA17-A nt, MAGE-3, HPV 16 E7, HPV L2, PD1, PD-L1, CTLA-4, p53, hCG, Fel d1 and (IHNV) G-protein.
In other embodiments, the AP205 capsid protein is fused at its N-terminus and/or at its C-terminus to a SpyTag and the antigen is fused to a SpyCatcher and is selected from the group consisting of interleukin-17, hemagglutinin, GD2, EGF-R, CEA, CD52, CD21, human melanoma protein gp100, human melanoma protein melan-A/MART1, tyrosinase, NA17-A nt, MAGE-3, HPV 16 E7, HPV L2, PD1, PD-L1, CTLA-4, p53, hCG, Fel d1 and (IHNV) G-protein. In one embodiment, the AP205 capsid protein is fused to a SpyTag at its N-terminus and the antigen is fused to a SpyCatcher and is selected from the group consisting of interleukin-17, hemagglutinin, GD2, EGF-R, CEA, CD52, CD21, human melanoma protein gp100, human melanoma protein melan-A/MART1, tyrosinase, NA17-A nt, MAGE-3, HPV 16 E7, HPV L2, PD1, PD-L1, CTLA-4, p53, hCG, Fel d1 and (IHNV) G-protein. In another embodiment, the AP205 capsid protein is fused to a SpyTag at its C-terminus and the antigen is fused to a Spy SpyCatcher Tag and is selected from the group consisting of interleukin-17, hemagglutinin, GD2, EGF-R, CEA, CD52, CD21, human melanoma protein gp100, human melanoma protein melan-A/MART1, tyrosinase, NA17-A nt, MAGE-3, HPV 16 E7, HPV L2, PD1, PD-L1, CTLA-4, p53, hCG, Fel d1 and (IHNV) G-protein. AP205 capsid protein is fused to a SpyTag at its N-terminus and at its C-terminus and the antigen is fused to a SpyCatcher and is selected from the group consisting of interleukin-17, hemagglutinin, GD2, EGF-R, CEA, CD52, CD21, human melanoma protein gp100, human melanoma protein melan-A/MART1, tyrosinase, NA17-A nt, MAGE-3, HPV 16 E7, HPV L2, PD1, PD-L1, CTLA-4, p53, hCG, Fel d1 and (IHNV) G-protein.

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
	GD2/Homo Sapiens.
	EGF-R/Homo Sapiens.
	CEA/Homo Sapiens.
	CD52/Homo Sapiens.
	human melanoma protein gp100/Homo Sapiens.
	human melanoma protein melan-A/MART-1/Homo Sapiens,
	tyrosinase/Homo Sapiens.
	NA17-A nt protein/Homo Sapiens.
	MAGE-3 protein/Homo Sapiens.
	p53 protein/Homo Sapiens.
	HPV 16 E7 protein/Human papillomavirus
	HPV L2 protein/Human papillomavirus
	PD1/Homo Sapiens
	PD-L1/Homo Sapiens
	CTLA-4/Homo Sapiens
	hCG/Homo Sapiens.
	(IHNV) G-protein/Infectious haematopoietic necrosis virus
Cardiovascular disease:	PCSK9 (Proprotein convertase subtilisin/kexin type 9)/
	Homo Sapiens
Asthma/Allergies:	IL-5 (Interleukin-5)/Homo Sapiens
	Fel d1
Tuberculosis:	Ag85A (Diacylglycerol cyltransferase/mycolyltransferase)/
	Mycobacterium tuberculosis
Malaria:	Reticulocyte-binding protein homologue 5 (PfRH5)/
	Plasmodium falciparum
	VAR2CSA (domain, ID1-ID2a)/Plasmodium falciparum
	CIDR1a domain of PfEMPl, 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
Multiple sclerosis	CD52/Homo Sapiens
Contraception	hCG
Influenza	HA

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 cardiovascular disease, an immune-inflammatory disease, a chronic disease, a neurologic disease and an infectious disease and cancer. In a particular embodiment the medical indication is an immune-inflammatory disease. In another particular embodiment the medical indication is a cardiovascular disease. In another embodiment the medical indication is a chronic disease. In another embodiment the medical indication is a neurologic disease. In another embodiment the medical indication is a cardiovascular disease or an immune-inflammatory disease.
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 virus capsid protein of the present invention. By way of example the antigen is linked to the AP205 capsid protein and/or a phage fr capsid protein of the present invention via the interaction between SpyCatcher and SpyTag (see FIG. 1 for a general concept of the present invention). In one embodiment, the antigen is linked to AP205 capsid protein fused to one or more SpyTag via the interaction between SpyTag and SpyCatcher. In one embodiment, the antigen is linked to AP205 capsid protein fused to two SpyTags, one at each terminus.
In one embodiment the antigen is linked to AP205 capsid protein fused to one or more SpyCatcher via the interaction between SpyTag and SpyCatcher. In one embodiment, the antigen is linked to AP205 capsid protein fused to two SpyCatchers, one at each terminus.
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 AP205 capsid protein and/or a phage fr capsid protein via the interaction between SpyCatcher and SpyTag. 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 oncoviruses can cause cancer. Therefore in an embodiment the vaccine of the present invention comprises an oncovirus associated antigen linked to the AP205 capsid protein and/or phage fr capsid protein via the interaction between the SpyCatcher, KTag and/or SpyTag.
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, Myelodysplastic 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. Such antigens may be chosen from the group consisting of interleukin-17, hemagglutinin, GD2, EGF-R, CEA, CD52, CD21, human melanoma protein gp100, human melanoma protein melan-A/MART1, tyrosinase, NA17-A nt, MAGE-3, HPV 16 E7, HPV L2, PD1, PD-L1, CTLA-4, p53, hCG, Fel d1 and (IHNV) G-protein.
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 an embodiment the antigen of the present disclosure is interleukin-17, hemagglutinin, GD2, EGF-R, CEA, CD52, CD21, human melanoma protein gp100, human melanoma protein melan-A/MART1, tyrosinase, NA17-A nt, MAGE-3, HPV 16 E7, HPV L2, PD1, PD-L1, CTLA-4, HPV L2, PD1, PD-L1, CTLA-4, p53, hCG, Fel d1 and (IHNV) G-protein or an antigenic fragment thereof, wherein the antigen is associated with and directed against at least one of the herein disclosed types of cancers.
In an 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. virus capsid protein, such as AP205 capsid protein and/or phage fr capsid protein comprising SpyCatcher, and
- ii. 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 SpyTag,

wherein the antigen and the virus capsid protein, such as AP205 capsid protein and/or phage fr capsid protein are linked via the interaction between the Spytag and the SpyCatcher insert of the AP205 capsid protein and/or phage fr capsid protein, and wherein i-ii form a virus-like particle displaying said antigen.
In an 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. virus capsid protein, such as AP205 capsid protein and/or phage fr capsid protein comprising SpyTag and/or KTag, and
- ii. 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 SpyCatcher,

wherein the antigen and the virus capsid protein, such as AP205 capsid protein and/or phage fr capsid protein are linked via the interaction between the SpyCatcher and the Spytag and/or KTag insert of the AP205 capsid protein and/or phage fr capsid protein, and wherein i-ii form a virus-like particle displaying said antigen.
In another 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. virus capsid protein, such as AP205 capsid protein and/or phage fr capsid protein comprising SpyTag and/or KTag, and
- ii. a cancer associated antigen consisting of interleukin-17, hemagglutinin, GD2, EGF-R, CEA, CD52, CD21, human melanoma protein gp100, human melanoma protein melan-A/MART1, tyrosinase, NA17-A nt, MAGE-3, HPV 16 E7, HPV L2, PD1, PD-L1, CTLA-4, p53, hCG, Fel d1 and (IHNV) G-protein or an antigenic fragment hereof fused to SpyCatcher,

wherein the antigen and the virus capsid protein, such as AP205 capsid protein and/or phage fr capsid protein are linked via the interaction between the SpyCatcher and the Spytag and/or KTag insert of the AP205 capsid protein and/or phage fr capsid protein, and wherein i-ii form a virus-like particle displaying said antigen.
In an embodiment the antigen fused to SpyCatcher at its N or C-termini and is selected from the group consisting of interleukin-17, hemagglutinin, GD2, EGF-R, CEA, CD52, CD21, human melanoma protein gp100, human melanoma protein melan-A/MART1, tyrosinase, NA17-A nt, MAGE-3, HPV 16 E7, HPV L2, PD1, PD-L1, CTLA-4, p53, hCG, Fel d1 and (IHNV) G-protein or an antigenic fragment hereof.
In an 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. virus capsid protein, such as AP205 capsid protein and/or phage fr capsid protein comprising a SpyCatcher, and
- ii. a cancer associated antigen such as GD2, EGF-R, CEA, CD52, CD21, human melanoma protein gp100, human melanoma protein melan-A/MART1, tyrosinase, NA17-A nt, MAGE-3, HPV 16 E7, HPV L2, PD1, PD-L1, CTLA-4, p53 and hCG or an antigenic fragment thereof fused to a SpyTag,

wherein the antigen and the virus capsid protein are linked via the interaction between the SpyCatcher and the Spytag, and wherein i-ii form a virus-like particle displaying said antigen. In a further embodiment SpyCatcher is fused to the N-terminus of the AP205 capsid protein. In a further embodiment, SpyCatcher is fused to the N-terminus of the AP205 capsid protein via a spacer. In a further embodiment SpyCatcher is fused to the C-terminus of the AP205 capsid protein. In a further embodiment, SpyCatcher is fused to the C-terminus of the AP205 capsid protein via a spacer. In a further embodiment SpyCatcher is fused to the C-terminus and to the N-terminus of the AP205 capsid protein. In a further embodiment, SpyCatcher is fused to the C-terminus and to the N-terminus of the AP205 capsid protein via a spacer.
In another 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. virus capsid protein, such as AP205 capsid protein and/or phage fr capsid protein comprising one or more SpyTags, and
- ii. a cancer associated antigen such as GD2, EGF-R, CEA, CD52, CD21, human melanoma protein gp100, human melanoma protein melan-A/MART1, tyrosinase, NA17-A nt, MAGE-3, HPV 16 E7, HPV L2, PD1, PD-L1, CTLA-4, p53 and hCG or an antigenic fragment thereof fused to a SpyCatcher,

wherein the antigen and the virus capsid protein are linked via the interaction between the SpyCatcher and the Spytag, and wherein i-ii form a virus-like particle displaying said antigen. In a further embodiment SpyTag is fused to the N-terminus of the AP205 capsid protein. In a further embodiment SpyTag is fused to the C-terminus of the AP205 protein. In another embodiment two SpyTags are fused to the AP205 capsid protein, one in each terminus.
In an embodiment the antigen fused to SpyTag comprises a polypeptide sequence of SEQ ID NO: 79 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 arteriosclerotic condition (e.g., atherosclerosis), a coronary artery disease, a cardiovascular 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 produce antibodies that bind PCSK9 and either clear PCSK9 from the bloodstream or hinders binding of PCSK9 to the LDL receptor, 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 an 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 virus capsid protein, such as AP205 capsid protein and/or phage fr capsid protein comprising a SpyTag and/or KTag, and
- ii. a cardiovascular disease associated antigen such as PCSK9 or an antigenic fragment hereof fused to a SpyCatcher,

wherein the antigen and the virus capsid protein, such as the AP205 capsid protein and/or a phage fr capsid protein are linked via the interaction between the SpyCatcher and the Spytag and/or KTag of the virus capsid protein, such as the AP205 capsid protein and/or a phage fr capsid protein, and wherein i-ii form a virus-like particle displaying said antigen.
In an embodiment the antigen fused to SpyCatcher comprises SEQ ID NO: 20 and/or a sequence variant hereof. In an embodiment, the SpyTag is fused to the N-terminus of the AP205 capsid protein, optionally via a spacer.
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 virus capsid protein, such as AP205 capsid protein and/or phage fr capsid protein comprising a SpyCatcher, and
- ii. a cardiovascular disease associated antigen such as PCSK9 or an antigenic fragment hereof fused to a SpyTag,

wherein the antigen and the virus capsid protein, such as AP205 capsid protein and/or phage fr capsid protein are linked via the interaction between the SpyCatcher and the Spytag, and wherein i-ii form a virus-like particle displaying said antigen. In a further embodiment SpyCatcher is fused to the N-terminus of the AP205 capsid protein.
In an embodiment the antigen fused to SpyTag comprises SEQ ID NO: 81 and/or a sequence variant hereof. In an embodiment, the SpyCatcher is fused to the N-terminus of the AP205 capsid protein, optionally via a spacer. In another embodiment two SpyCatchers are fused to the AP205 capsid protein, one at each terminus.
In one embodiment the vaccine for use in the prophylaxis and/or treatment of at least one of the herein disclosed cardiovascular diseases comprises:

- i. a virus capsid protein, such as AP205 capsid protein and/or phage fr capsid protein comprising one or more SpyTag, and
- ii. a cardiovascular disease associated antigen such as PCSK9 or an antigenic fragment hereof fused to a SpyCatcher,

wherein the antigen and the virus capsid protein, such as AP205 capsid protein and/or phage fr capsid protein are linked via the interaction between the SpyCatcher and the Spytag, and wherein i-ii form a virus-like particle displaying said antigen. In a further embodiment SpyTag is fused to the N-terminus of the AP205 capsid protein, optionally via a spacer. In another embodiment two SpyTags are fused to the AP205 capsid protein, one at each terminus.
Immune-Inflammatory Diseases and Associated Antigens
The prevalence of immune-inflammatory 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 other immune-inflammatory diseases. Other immune-inflammatory disease associated antigens (e.g. IgE or interleukin 17 or IL-17) may be used by the present invention using a similar reasoning. Consequently an IL-17-VLP based vaccine described in the present invention may be used in the treatment/prophylaxis of eosinophilic asthma or other immune-inflammatory diseases. The type of asthma or allergy or other immune-inflammatory 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 immune-inflammatory diseases disclosed herein. In a preferred embodiment the asthma or immune-inflammatory disease 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, IL-17 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 immune-inflammatory diseases.
In an embodiment the present invention concerns a vaccine for use in the prophylaxis and/or treatment of one of the herein disclosed immune-inflammatory diseases wherein the vaccine comprises:

- i. a virus capsid protein, such as AP205 capsid protein and/or a phage fr capsid protein comprising a SpyTag and/or KTag, and
- ii. an antigen such as IL-5 or IL-17 or an antigenic fragment hereof fused to SpyCatcher,

wherein the antigen and the virus capsid protein, such as AP205 capsid protein and/or phage fr capsid protein are linked via the interaction between the SpyCatcher and the Spytag and/or KTag site of the virus capsid protein, and wherein i-ii form a virus-like particle displaying said antigen.
In an embodiment the antigen is IL-17.
In an embodiment the antigen fused to SpyCatcher is IL-5. In one embodiment the antigen comprises SEQ ID NO: 19 and/or a sequence variant hereof
In one embodiment the AP205 capsid protein is fused to one or more SpyTags. In one embodiment, the AP205 capsid protein is fused to two SpyTags, one at each terminus of the capsid protein.
In a preferred embodiment the present invention concerns a vaccine for use in the prophylaxis and/or treatment of one of the herein disclosed immune-inflammatory diseases wherein the vaccine comprises:

- i. virus capsid protein, such as AP205 capsid protein and/or phage fr capsid protein comprising a SpyCatcher, and
- ii. an antigen such as IL-5 or IL-17 or an antigenic fragment hereof fused to SpyTag,

wherein the antigen and the virus capsid protein such as AP205 capsid protein and/or phage fr capsid protein are linked via the interaction between the SpyCatcher and the Spytag, and wherein i-ii form a virus-like particle displaying said antigen. In a further embodiment SpyCatcher is fused to the N-terminus of the AP205 capsid protein. In one embodiment SpyCatcher is fused to the AP205 capsid protein via a spacer.
In an embodiment the antigen is IL-17.
In an embodiment the antigen fused to SpyTag comprises SEQ ID NO: 80 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 SpyTag or SpyCatcher 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 SpyCatcher of the present invention for use in treatment/prophylaxis of malaria.
In a further embodiment an antigen from Mycobacterium tuberculosis is fused to the SpyCatcher 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, CIDR1a domain, of PfEMP1 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 a further embodiment the antigen is a hemagglutinin (HA) antigen from the influenza virus or an antigenic fragment thereof.
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 one 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. virus capsid protein, such as AP205 capsid protein and/or phage fr capsid protein comprising a SpyTag insert, and
- ii. an antigen associated with an infectious disease such as Ag85A from Mycobacterium tuberculosis, PfRH5 from Plasmodium falciparum, VAR2CSA (domain, ID1-ID2a) from Plasmodium falciparum, CIDR1a domain, of PfEMP1 from Plasmodium falciparum, GLURP from Plasmodium falciparum, MSP3 from Plasmodium falciparum, Pfs25 from Plasmodium falciparum, CSP from Plasmodium falciparum, PfSEA-1 from Plasmodium falciparum and/or HA from influenza virus or an antigenic fragment hereof fused to SpyCatcher,

wherein the antigen and the virus capsid protein, such as AP205 capsid protein and/or phage fr capsid protein are linked via the interaction between the SpyCatcher and the Spytag, and wherein i-ii form a virus-like particle displaying said antigen.
In an embodiment the antigen fused to SpyCatcher 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, SEQ ID NO: 28 and/or SEQ ID NO: 82 and/or a sequence variant hereof. In one embodiment the antigen fused to SpyCatcher comprises SEQ ID NO: 21. In one embodiment the antigen fused to SpyCatcher comprises SEQ ID NO: 24. In one embodiment the antigen fused to SpyCatcher comprises SEQ ID NO: 28. In one embodiment, the antigen fused to SpyCatcher comprises SEQ ID NO: 82.
In one 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. virus capsid protein, such as AP205 capsid protein and/or phage fr capsid protein comprising a SpyCatcher insert, and
- ii. an antigen associated with an infectious disease such as Ag85A from Mycobacterium tuberculosis, PfRH5 from Plasmodium falciparum, VAR2CSA (domain, ID1-ID2a) from Plasmodium falciparum, CIDR1a domain, of PfEMP1 from Plasmodium falciparum, GLURP from Plasmodium falciparum, MSP3 from Plasmodium falciparum, Pfs25 from Plasmodium falciparum, CSP from Plasmodium falciparum, PfSEA-1 from Plasmodium falciparum and/or HA from influenza virus or an antigenic fragment hereof fused to SpyTag,

wherein the antigen and the virus capsid protein, such as AP205 capsid protein and/or phage fr capsid protein are linked via the interaction between the SpyCatcher and the Spytag, and wherein i-ii form a virus-like particle displaying said antigen. In a further embodiment SpyCatcher is fused to the N-terminus of the AP205 capsid protein.
In an embodiment the antigen fused to Spytag 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, SEQ ID NO: 28 and/or SEQ ID NO: 82 and/or a sequence variant hereof. In one embodiment the antigen fused to Spytag comprises SEQ ID NO: 21. In one embodiment the antigen fused to Spytag comprises SEQ ID NO: 24. In one embodiment the antigen fused to Spytag comprises SEQ ID NO: 28. In one embodiment, the antigen fused to Spytag comprises SEQ ID NO: 82.
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.
The AP205 VLP
An important element of the present VLP based vaccine is the AP205 capsid protein, which has the ability to spontaneously self-assemble into virus-like particles (VLPs). The use of AP205 VLP in the present invention is illustrated in FIG. 1 . Surprisingly the present inventors have found that amino acid residues may be fused to the AP205 capsid protein at the AP205 capsid proteins N or C terminus without preventing VLP assembly while at the same time presenting the added amino acids on the outside of the assembled VLP where they are accessible for interactions. Specifically, SpyTag, SpyCatcher, and Ktag encoding residues may be fused to the N and/or C terminus of AP205. Thus it is an object of the present invention to fuse a protein tag to the N and/or C-terminus of the AP205 capsid protein. The AP205 capsid protein sequence is disclosed in SEQ ID NO: 58. Any variant of the AP205 protein that is capable of being expressed with a protein tag and that is still able to self-assemble into a VLP while presenting the protein tag on the outer surface of the VLP is an object of the present invention.
In an embodiment the AP205 capsid protein of the present invention comprises the amino acid sequence SEQ ID NO: 58, or a biologically active sequence variant that has at least 85%, or 90% or 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: 58. By “biological active” is meant the ability to form a virus-like particle.
Direct fusion of six different peptide sequences to the N or C-terminus of the AP205 capsid protein was shown in 2010 by Tissot A C. et al. (Tissot A C. et al. PLoS ONE. 2010) to result in hybrid proteins capable of self-assembling into virus-like particles. However, the ability of fusing peptides to the N- or C-terminus of the AP205 coat protein without preventing VLP assembly is by no means certain and depends greatly on both the length and the precise amino acid composition of the fused peptide. Recently, Cielens I. et al. (Cielens I. et al. Mol. Biotechnol. 2014) tried to fuse a 111 amino acid sequence of the virus-neutralising domain III (DIII) of the West Nile virus glycoprotein E to the C-terminus of the AP205 coat protein. In this study recombinant expression of the AP205-DIII fusion protein failed to assemble into VLPs. Moreover, it has also been investigated if the coat protein of the distantly related bacteriophage fr can tolerate the insertion of peptide sequences at different amino acid positions near the N- and C-terminus. This study show that several N-terminal insertion mutants of the fr coat protein failed to assemble into VLPs but instead formed dimers (P. Pushko. et al. Protein Eng. 1993). Also, in this study the C-terminus of the fr coat protein could only tolerate insertion of three amino acids whereas insertion of a longer peptide prevented VLP assembly. The mentioned peptide insertion was specifically at position 2/3, and 128/129 of the fr coat protein and hence only internal insertions of amino acids into the coat protein fr have been described to date. The present inventors also observed that fusion of a monovalent streptavidin domain (mSA) in the N-terminus of AP205 prevented formation of VLPs; mSA has a size comparable to the size of the SpyCatcher.
In a preferred embodiment the virus capsid protein comprises an AP205 capsid protein fused to a SpyCatcher, wherein the capsid protein—SpyCatcher fusion protein is capable of forming a virus-like particle. In a further embodiment SpyCatcher is fused to the N-terminus of the AP205 capsid protein optionally via a linker or spacer. In another embodiment SpyCatcher is fused to the C-terminus of the AP205 capsid protein optionally via a linker or spacer. In one embodiment, one SpyCatcher is fused to the C-terminus of the Ap205 capsid protein and one SpyCatcher is fused to the N-terminus of the AP205 capsid protein.
The inventors of the present invention have surprisingly shown that a SpyCatcher comprising more than 100 amino acids can be fused to a capsid protein such as an AP205 capsid protein and/or phage fr capsid protein, without disrupting the sensitive self-assembly process. In an embodiment the SpyCatcher is fused to the N-terminal of the AP205 capsid protein using a short flexible Gly-Gly-Ser-Gly-Ser linker (SEQ ID NO: 83 or any other appropriate linker sequence). Attempts to fuse SpyCatcher to the C-terminal of AP205 capsid protein resulted in no assembly of VLPs. In another embodiment a SpyCacther is fused to the N- and/or C-terminal of phage fr capsid protein using a short flexible Gly-Gly-Ser-Gly-Ser linker (SEQ ID NO: 83 or any other appropriate linker sequence).
In a most preferred embodiment the AP205-SpyCatcher fusion protein comprises the amino acid sequence of SEQ ID NO: 76 or a biologically active sequence variant that 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 amino acid sequence of SEQ ID NO:76 and/or any of the herein disclosed AP205 capsid proteins comprising the SpyCatcher polypeptide in the N terminal. By “biologically active” is meant the ability to form a virus-like particle.
A preferred embodiment of the present invention relates to an AP205 capsid protein comprising an N-terminal SpyCatcher which is capable of forming/self-assembling into a virus-like particle. In an embodiment SpyCatcher is fused to the N-terminus of the AP205 capsid protein optionally via a linker. In an embodiment the SpyCatcher is fused to the first 100 amino acids in the N terminus of the AP205. In an embodiment the SpyCatcher is fused to the AP205 using a peptide linker, such as SEQ ID NO: 83. In an embodiment the AP205 capsid protein comprising a SpyCatcher has a polypeptide 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 the polypeptide sequence of SEQ ID NO: 76. Another aspect of the present invention relates to an AP205 capsid protein comprising a N-terminal SpyCatcher which spontaneously can for a virus-like particle. In an embodiment SpyCatcher is fused to the N-terminus of the AP205 capsid protein optionally via a linker. In an embodiment the SpyCatcher is fused to the first 100 amino acids in the N terminal of the AP205. In an embodiment the SpyCatcher is fused to the AP205 N terminal using a peptide linker, such as SEQ ID NO: 83. In an embodiment the AP205 capsid protein comprising a SpyCatcher is having a polypeptide 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 the polypeptide sequence of SEQ ID NO: 76.
In another embodiment the AP205 capsid protein is fused to SpyTag.
The Phage Fr VLP
Phage fr, or more precisely the phage fr capsid protein, is also an important element of the present invention. The phage fr capsid protein has the ability to spontaneously self-assemble into virus-like particles. Furthermore the phage fr capsid protein is capable of self-assembly even when amino acid residues are be fused to the phage fr capsid protein at the fr capsid proteins N terminus. Importantly, the fused amino acids are presented on the outer surface of the assembled fr VLP. Specifically, SpyTag, SpyCatcher, and/or Ktag encoding residues may be fused to the N terminus and/or the C-terminus of phage fr capsid protein. Thus it is an object of the present invention to fuse a protein tag to the N-terminus of the phage fr capsid protein. The phage fr capsid protein sequence is disclosed in SEQ ID NO: 59 and/or 78. Any variant of the phage fr capsid protein that is still capable of being expressed with a protein tag and still self-assemble is an object of the present invention.
In an embodiment the Phage fr capsid protein of the present invention comprises the amino acid sequence SEQ ID NO: 59 and/or 78, or a biologically active sequence variant that has at least 70%, such as 75%, such as 80%, such as 85%, such as 90%, such as95%, 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: 59 and/or 78. By “biological active” is meant the ability to form a virus-like particle.
A further aspect of the present invention relates to a phage fr capsid protein comprising a SpyCatcher which is capable of forming/self-assembling into a virus-like particle. In an embodiment the SpyCatcher is fused to any one of the first 50 amino acids in the N terminal end and/or any one of the last 50 in the C terminal end of the phage fr capsid protein. In an embodiment the SpyCatcher is fused to the phage fr capsid protein using a peptide linker, such as SEQ ID NO: 83. In an embodiment the phage fr capsid protein comprising a SpyCatcher is having a polypeptide 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 the polypeptide sequence of SEQ ID NO: 78.
Another aspect of the present invention relates to a phage fr capsid protein comprising a SpyCatcher which spontaneously can form a virus-like particle. In an embodiment the SpyCatcher is fused to any one of the first 50 amino acids in the N terminal and/or any one of the last 50 in the C terminal of the phage fr capsid protein. In an embodiment the SpyCatcher is fused to the phage fr capsid protein using a peptide linker, such as SEQ ID NO: 83. In an embodiment the phage fr capsid protein comprising a SpyCatcher is having a polypeptide 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 the polypeptide sequence of SEQ ID NO: 78.
SpyTag and/or KTag and its Position in AP205 and/or Phage Fr
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. AP205 capsid protein comprising one or more SpyTag and/or KTag, and
- ii. an antigen fused to SpyCatcher,

wherein the antigen and the AP205 capsid protein are linked via the interaction between the SpyCatcher and the Spytag and/or KTag, and wherein i-ii form a virus-like particle displaying said antigen.
In an embodiment the SpyTag and/or KTag polypeptide is fused to the N or C terminal of a virus capsid protein, such as an AP205 capsid protein and/or phage fr capsid protein. In an embodiment, two tags, for example two SpyTags or two SpyCatcher tags, are fused to the capsid protein such as the AP205 capsid protein, one at each terminus. Without being bound by theory, increasing the number of accessible SpyTags or SpyCatchers on the surface of a VLP should maximize the antigen binding capacity and result in a higher density of displayed antigen. This is the case for fusion of two SpyTags to the AP205 capsid protein, as shown in the below examples and FIG. 13 . Whether fusion of two tags instead of one improves antigen binding can easily be tested by the skilled person.
In an embodiment the AP205 capsid protein comprising the SpyTag and/or KTag polypeptide comprises the amino acid sequences selected from the group consisting of SEQ ID NO: (62, 64, 68, 69, 71, 74, and 76) or a biologically active sequence variant that 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 any of the herein disclosed AP205 capsid proteins comprising the SpyTag and/or KTag polypeptide. By “biological active” is meant the ability to form a virus-like particle.
In another embodiment the present invention concerns a vaccine for use in the prophylaxis and/or treatment of a disease wherein the vaccine comprises:

- i. a phage fr capsid protein comprising one or more SpyTag and/or KTag, and
- ii. an antigen fused to SpyCatcher,
- wherein the antigen and phage fr capsid protein are linked via the interaction between the SpyCatcher and the Spytag insert site of the phage fr capsid protein, and wherein i-ii form a virus-like particle displaying said antigen.

In an embodiment the phage fr capsid protein comprising the SpyTag and/or KTag polypeptide comprises the amino acid sequences selected from the group consisting of SEQ ID NO: 66, SEQ ID NO: 70, and SEQ ID NO: 78 or a biologically active sequence variant that has at least 70%, such as 75%, such as 80%, such as 85%, such as 90%, 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 phage fr capsid proteins comprising the SpyTag and/or KTag polypeptide. By “biological active” is meant the ability to form a virus-like particle.
SpyCatcher and its Position in AP205 and/or Phage Fr.
In an embodiment the SpyCatcher polypeptide is fused to the N or C terminal and/or fused to the first 1-15 amino acids (N-terminal) or the last 1-15 amino acids (C-terminal) of a phage fr capsid protein, optionally using a linker as described herein. In an embodiment the SpyCatcher polypeptide is fused to the N terminal and/or fused to the first 1-15 amino acids (N-terminal) of a AP205 capsid protein, optionally using a linker as described herein. In an embodiment the SpyCatcher fused to the virus capsid protein comprises the amino acid sequence selected from the group comprising SEQ ID NO. 76, and SEQ ID NO. 78, or a biologically active sequence variant that 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. 76, and SEQ ID NO. 78. By “biologically active” is meant the ability to form a virus-like particle.
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. the AP205 capsid protein and/or phage fr capsid protein comprising SpyCatcher, and
- ii. an antigen fused to SpyTag and/or KTag,

wherein the antigen and AP205 protein are linked via the interaction between the Spytag and/or KTag and the SpyCatcher, and wherein i-ii form a virus-like particle displaying said antigen. In an embodiment SpyCatcher is fused to the N-terminus of the AP205 capsid protein optionally via a linker. In one embodiment SpyCatcher is fused to the N-terminus of the AP205 capsid protein via a GGSGS linker (SEQ ID NO: 83).
In an embodiment the SpyCatcher polypeptide is fused to the N terminal of a virus capsid protein, such as the AP205 capsid protein or phage fr capsid protein. In one embodiment the SpyCatcher is fused to the N-terminal and to the C-terminal ends of a virus capsid protein such as the AP205 capsid protein.
In an embodiment the AP205 capsid protein comprising the SpyCatcher polypeptide comprises the amino acid sequences selected from the group consisting of SEQ ID NO: 76 and/or 78 or a biologically active sequence variant that 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 any of the amino acid sequence of SEQ ID NO:76 and/or any of the herein disclosed AP205 capsid proteins comprising the SpyCatcher polypeptide in the N terminal. By “biologically active” is meant the ability to form a virus-like particle.
In a preferred embodiment the virus capsid protein comprises an AP205 capsid protein fused to a SpyCatcher, wherein the capsid protein—SpyCatcher fusion protein is capable of forming a virus-like particle. In an embodiment SpyCatcher is fused to the N-terminus of the AP205 capsid protein optionally via a linker.
The inventors of the present invention have surprisingly shown that a SpyCatcher comprising more than 100 amino acids can be fused to a capsid protein such as an AP205 capsid protein and/or phage fr capsid protein, without disrupting the sensitive self-assembly process. In an embodiment the SpyCatcher is fused to the N-terminal of the AP205 capsid protein and/or phage fr capsid protein using a short flexible Gly-Gly-Ser-Gly-Ser linker such SEQ ID NO: 83 or a sequence variant that 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: 83. Other peptide linkers may be used by the present invention.
In a most preferred embodiment the AP205-SpyCatcher fusion protein comprises the amino acid sequence of SEQ ID NO: 76 or a biologically active sequence variant that 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 amino acid sequence of SEQ ID NO: 76 and/or any of the herein disclosed AP205 capsid proteins comprising the SpyCatcher polypeptide in the N-terminal. By “biologically active” is meant the ability to form a virus-like particle.
In another embodiment the present invention concerns a vaccine for use in the prophylaxis and/or treatment of a disease wherein the vaccine comprises:

- i. a phage fr capsid protein comprising SpyCatcher, and
- ii. an antigen fused to SpyTag and/or KTag,

wherein the antigen and phage fr capsid protein are linked via the interaction between the Spytag and/or KTag and the SpyCatcher insert site of the phage fr capsid protein, and wherein i-ii form a virus-like particle displaying said antigen.
In an embodiment the phage fr capsid protein comprising the SpyCatcher polypeptide comprises the amino acid sequences selected from the group consisting of SEQ ID NO: 78 or a biologically active sequence variant that has at least 70%, such as 75%, such as 80%, such as 85%, such as 90%, 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 phage fr capsid proteins comprising the SpyCatcher polypeptide. By “biological active” is meant the ability to form a virus-like particle.
Antigen Fused to SpyCatcher, SpyTag, and/or KTag.
SpyTag refers to a part of the CnaB2 domain from the FbaB protein from Streptococcus pyogenes optimized to bind SpyCatcher consisting of another part of the CnaB2 domain. The interaction occurs when the unprotonated amine of Lys31 nucleophilically attacks the carbonyl carbon of Asp117, catalyzed by the neighboring Glu77. The minimal peptide to mediate this binding is AHIVMVDA whereas a C-terminal extension giving the sequence: AHIVMVDAYKPTK provides the most optimal region, designated “SpyTag” (Zakeri et al PNAS 2012).
SpyCatcher is a part of the CnaB2 domain from the FbaB protein from Streptococcus pyogenes and binds SpyTag consisting of another part of the CnaB2 domain. When these two polypeptides of the CnaB2 domain are mixed, they will spontaneously form an irreversible isopeptide bond thereby completing the formation of the CnaB2 domain.
Thus when fusing an antigen to SpyCatcher and mixing e.g. with a VLP comprising a genetically engineered SpyTag we obtain a uniform presentation of said antigens. The 1:1 interaction between the SpyTag and SpyCatcher enables 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 an embodiment the antigen as described by the present invention is fused to SpyCatcher or truncated versions hereof, SpyTag, and/or KTag. Examples of antigens fused to Spytag is illustrated, but not limited to SEQ ID NO: 79, 80, 81 and/or 82. The SpyTag may be fused any of the herein disclosed antigens. In addition, KTag and/or SpyCacther can be used instead of the SpyTag for example in, but not limited to SEQ ID NO: 79, 80, 81 and/or 82.
Surprisingly the inventors have found that the SpyCatcher-antigen fusion proteins of the present invention express very well under expression conditions described herein. Previous attempts of expressing antigen-monovalent streptavidin fusion proteins have almost exclusively resulted in poor expression levels and/or insoluble protein.
In an embodiment the SpyCatcher used by the present invention comprises the amino acid sequence of SEQ ID NO: 37.
Truncated and homologous versions of SpyCatcher are also objects of the present invention and thus the term SpyCatcher herein denotes any variant of SpyCatcher that is still capable of interacting with SpyTag and/or KTag. Variants of SpyCatcher may include, but is not limited to truncated SpyCatcher variants. Truncated SpyCatcher variants may include, but are not limited to SEQ NO 60 and SEQ ID NO: 61. SpyCatcher variants such as truncated SpyCatcher variants may exhibit lower immunogenicity than wildtype SpyCatcher does, without influencing the ability to bind to SpyTag and/or KTag.
In an embodiment the SpyCatcher used by the present invention comprises the amino acid sequence of SEQ ID NO: 60.
In an embodiment the SpyCatcher used by the present invention comprises the amino acid sequence of SEQ ID NO: 61.
In another embodiment the ratio of AP205 capsid protein: SpyTag and/or KTag:SpyCatcher/antigen fusion is 1:1:1.
In another embodiment the ratio of Phage fr capsid protein: SpyTag and/or KTag: SpyCatcher/antigen fusion is 1:1:1.
In an embodiment the antigen as described by the present invention is fused to SpyCatcher or truncated versions hereof, SpyTag, and/or KTag.
Changing the position where the SpyCatcher is fused to the antigen (primarily at the N or C-termini) 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 SpyCatcher further comprises or includes an additional tag such as a purification 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 4×His, 5×His, 6×His, 7×His, 8×His, 9×His, or 10×His tag.
In an embodiment SpyCatcher is fused to the antigen in any position. In another embodiment the SpyCatcher 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 SpyCatcher, without introducing stop-codons or causing frame shift or any other mutations.
In another embodiment SpyCatcher 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 SpyCatcher 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 SpyCatcher 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.

Peptide—Peptide Ligation by SpyLigase
A KTag/SpyTag/SpyLigase system may also be used in the present invention. The CnaB2 domain from Streptococcus pyogenes can be used to generate a covalent peptide-peptide ligation system (Fierer J O. et al. 2014). This is done by splitting the CnaB2 into three parts a) the 13 amino acid SpyTag (SEQ ID NO: 36), b) the β-strand of CnaB2 (SEQ ID NO: 38)) named KTag, and c) the SpyLigase (SEQ ID 55) constructed from the remaining SpyCatcher polypeptide. By expressing the vaccine antigen with the small KTag fused at the C- or N-terminus and mixing that fusion protein with SpyTag-displaying VLPs together with the SpyLigase, the KTag-fusion antigen will be attached to the SpyTag-VLPs by covalent ligation of the SpyTag with the KTag facilitated by the SpyLigase. Conversely, the KTag may also be inserted genetically into the AP205 capsid protein and/or a phage fr capsid protein whereby the vaccine antigen should then be fused to the SpyTag at the C- or N-terminus. A general aspect of the KTag/SpyTag/SpyLigase system is illustrated in FIG. 2 . The SpyTag may also be fused to the antigen and the KTag may be inserted into to the VLP (Fierer J O. et al. 2014).
Thus, an aspect of the present invention relates to a vaccine for use in the prophylaxis and/or treatment of a disease wherein the vaccine comprises:

- i. a virus capsid protein, such as the AP205 capsid protein and/or a phage fr capsid protein comprising a SpyTag described herein, and
- ii. an antigen fused to a KTag described herein, and
- iii. optionally a SpyLigase

wherein the antigen and virus capsid protein, such as the AP205 capsid protein and/or a phage fr capsid protein are linked via the interaction between the SpyTag and KTag, and wherein i-ii form a virus-like particle displaying said antigen.
In another aspect the present invention relates to a vaccine for use in the prophylaxis and/or treatment of a disease wherein the vaccine comprises:

- i. a virus capsid protein, such as the AP205 capsid protein and/or a phage fr capsid protein comprising a KTag described herein, and
- ii. an antigen fused to a SpyTag described herein, and
- iii. optionally a SpyLigase

wherein the antigen and virus capsid protein, such as the AP205 capsid protein and/or a phage fr capsid protein are linked via the interaction between the KTag and SpyTag, and wherein i-ii form a virus-like particle displaying said antigen.
In an embodiment the SpyTag and KTag described herein are linked by means of a SpyLigase as described herein.
In an aspect of the present invention relates to a vector comprising at least one polynucleotide encoding

- i. a virus capsid protein, such as the AP205 capsid protein and/or a phage fr capsid protein comprising a SpyTag as described herein, and/or
- ii. an antigen fused to a KTag as described herein, and
- iii. optionally a SpyLigase.

Another aspect of the present invention relates to a vector comprising at least one polynucleotide encoding

- i. a virus capsid protein, such as the AP205 capsid protein and/or a phage fr capsid protein comprising a KTag as described herein, and/or
- ii. an antigen fused to a SpyTag as described herein, and
- iii. optionally a SpyLigase

Another aspect of the present invention relates to a host cell, wherein the host cell expresses:

- i. a first polypeptide; a virus capsid protein, such as the AP205 capsid protein and/or phage fr capsid protein comprising a SpyTag as described herein, and
- ii. a second polypeptide; an antigen fused to KTag as described herein, and
- iii. optionally a SpyLigase

wherein the cell is selected from the group comprising bacteria, yeast, fungi, plant, mammalian and/or insect cells.
A further aspect of the present invention relates to a host cell, wherein the host cell expresses:

- i. a first polypeptide; a virus capsid protein, such as the AP205 capsid protein and/or phage fr capsid protein comprising a KTag described herein, and
- ii. a second polypeptide; an antigen fused to SpyTag described herein, and
- iii. optionally a SpyLigase

wherein the cell is selected from the group comprising bacteria, yeast, fungi, plant, mammalian and/or insect cells.
In another aspect the present invention relates to a vaccine and/or a vector and/or a host cell and/or a method of manufacturing a pharmaceutical composition, as described in the present invention, wherein the SpyTag is replaced by a KTag and/or SpyTag, and/or wherein the SpyCatcher is replaced by SpyTag and/or KTag.
Isopeptid/C-Pilin System:
As part of a similar strategy for covalent coupling of vaccine-antigens at the surface of VLPs, another pair of split-protein binding partners may be used in the present invention. The major pilin protein, Spy0128, from Streptococcus pyogenes can be split into two fragments (split-Spy0128 (residues 18-299 of Spy0128) (SEQ ID NO: 57) and isopeptide (residues 293-308 of Spy0128 (TDKDMTITFTNKKDAE))) (SEQ ID NO: 56) which together are capable of forming an intermolecular covalent complex (Zakeri, B. et al. 2010). In line with the described SpyTag-SpyCatcher strategy, the Spy0128 isopeptide is genetically inserted into a surface exposed loop of the VLP and enables the stable attachment of vaccine antigens fused at the N or C-terminus with the split-Spy0128 binding partner.
Thus in a further aspect the present invention relates to a vaccine for use in the prophylaxis and/or treatment of a disease wherein the vaccine comprises:

- i. a virus capsid protein, such as AP205 capsid protein and/or phage fr capsid protein comprising a Spy0128 isopeptide insertion described herein, and
- ii. an antigen fused to a split-Spy0128 described herein,

wherein the antigen and virus capsid protein, such as AP205 capsid protein and/or phage fr capsid protein are linked via the interaction between the Spy0128 isopeptide and split-Spy0128, and wherein i-ii form a virus-like particle displaying said antigen.
In one aspect the present invention concerns a vector comprising at least one polynucleotide encoding a) a virus capsid protein, such as AP205 capsid protein and/or phage fr capsid protein comprising a Spy0128 isopeptide insertion, and b) an antigen fused to split-Spy0128.
In another aspect the present invention relates to a vaccine and/or a vector and/or a host cell and/or a method of manufacturing a pharmaceutical composition, as described in the present invention, wherein the SpyTag is replaced by a Spy0128 isopeptide, and wherein the SpyCatcher is replaced by split-Spy0128 as described herein.
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 two peptide components 1) a virus capsid protein, such as AP205 capsid protein and/or phage fr capsid protein comprising one or two SpyTags or SpyCatchers and 2) an antigen fused to the other one of a SpyCatcher and a SpyTag. 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 or two, 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 virus capsid protein, such as AP205 capsid protein and/or phage fr capsid protein comprising a SpyTag, and/or
- ii. an antigen fused to SpyCatcher.

In one embodiment the vaccine is a vector comprising at least one polynucleotide encoding

- i. a virus capsid protein, such as AP205 capsid protein and/or phage fr capsid protein comprising a SpyCatcher, and/or
- ii. an antigen fused to a SpyTag.

In another embodiment the vector comprises at least two polynucleotides of the following polypeptides:

- i. a virus capsid protein, such as AP205 capsid protein and/or phage fr capsid protein comprising one or more SpyTag, and/or
- ii. an antigen fused to a SpyCatcher.

- i. a virus capsid protein, such as AP205 capsid protein and/or phage fr capsid protein comprising one or more SpyCatcher, and/or
- ii. an antigen fused to a SpyTag.

In one embodiment the vector comprises sequences encoding at least

- i. a virus capsid protein such as the AP205 capsid protein fused to two SpyTags, wherein one of the two SpyTags is fused to the C-terminal end of the capsid protein and the other of the two SpyTags is fused to the N-terminal end of the capsid protein,
- ii. an antigen fused to SpyCatcher.

In one embodiment the vector comprises sequences encoding at least

- i. a virus capsid protein such as the AP205 capsid protein fused to two SpyCatchers, wherein one of the two SpyCatchers is fused to the C-terminal end of the capsid protein and the other of the two SpyCatchers is fused to the N-terminal end of the capsid protein,
- ii. an antigen fused to a SpyTag.

In a further embodiment the antigen fused to the SpyCatcher 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 11, 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 SpyTag polypeptide, comprises the nucleotide sequence SEQ ID NO: 39.
In an embodiment the present invention relates to a vector comprising at least one polynucleotide encoding

- i. an AP205 capsid protein comprising a SpyCatcher, and/or
- ii. an antigen fused to a SpyTag and/or KTag.

In a further embodiment SpyCatcher is fused to the N-terminus of the AP205 capsid protein optionally via a linker.
In an embodiment the AP205 capsid protein comprising a SpyCatcher comprises the polynucleotide sequence encoding the polypeptide sequence of Seq ID No: 76 or a biologically active sequence variant that 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 polypeptide sequence of Seq ID No: 76 and/or any of the herein disclosed AP205 capsid proteins comprising the SpyCatcher polypeptide in the N terminal. By “biological active” is meant the ability to form a virus-like particle.
In an embodiment the phage fr capsid protein comprising a SpyCatcher comprises the polynucleotide sequence encoding the polypeptide sequence of Seq ID No: 78 or a biologically active sequence variant that 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 any of the herein disclosed phage fr capsid proteins comprising the SpyCatcher polypeptide. By “biologically active” is meant the ability to form a virus-like particle.
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 virus capsid protein, such as AP205 capsid protein and/or phage fr capsid protein comprising SpyTag, and/or a second polypeptide: an antigen fused to SpyCatcher in a host cell are known in the art. The first or second 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 polynucleotide coding for the first and/or second polypeptide is cloned into the genome, and a first and/or second polynucleotide coding for the first and/or second polypeptide is comprised within a vector transformed or transfected into the host cell. Alternatively, the first and/or second polynucleotide is comprised within a first vector and the first and/or second polynucleotide is comprised within a second vector and the first and/or second is comprised within a third vector.
Expression of the first and second, 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 Solanum 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 is Trichoplusia 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 a preferred embodiment the expression of an AP205 capsid protein and/or phage fr capsid protein, comprising a SpyCatcher, wherein the capsid protein—SpyCatcher fusion protein is capable of forming a virus-like particle. In a further embodiment SpyCatcher is fused to the N-terminus of the AP205 capsid protein optionally via a linker.
The inventors of the present invention have surprisingly shown that a SpyCatcher comprising more than 100 amino acids can be fused to a capsid protein such as to the N terminal of an AP205 capsid protein and/or phage fr capsid protein, without disrupting the sensitive self-assembly process.
In an embodiment the host cell expresses:

- i. a first polypeptide; a virus capsid protein, such as AP205 capsid protein and/or phage fr capsid protein comprising a SpyCatcher, and/or
- ii. a second polypeptide; an antigen fused to a SpyTag,

wherein the cell is selected from the group comprising bacteria, yeast, fungi, plant, mammalian and/or insect cells.
In a most preferred embodiment the host cell expresses an AP205 capsid protein comprising a SpyCatcher comprising the amino acid sequence of SEQ ID NO: 76 or a biologically active sequence variant that 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: 76 and/or any of the herein disclosed AP205 capsid proteins comprising the SpyCatcher polypeptide in the N terminal. By “biological active” is meant the ability to form a virus-like particle.
In an embodiment the host cell expresses:

- i. a first polypeptide; a virus capsid protein, such as AP205 capsid protein and/or phage fr capsid protein comprising a SpyTag, and/or
- ii. a second polypeptide; an antigen fused to a SpyCatcher,

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: 62 [Spy-AP205]; SEQ ID NO: 64 [AP205-Spy], SEQ ID NO: 66 [Spy-Phage fr], SEQ ID NO: 68 [Ktag-AP205], SEQ ID NO: 69 [AP205-Ktag], SEQ ID NO: 70 [Ktag-Phage fr], SEQ ID NO: 71 [Spy-AP205-Spy], SEQ ID NO: 76, SEQ ID NO: 78,
- 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: 82, SEQ ID NO: 79, SEQ ID NO: 80 and SEQ ID NO: 81, 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 another embodiment the host cell expresses:

- i. a first polypeptide; an Phage fr capsid protein comprising a SpyTag such as 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 to SEQ ID NO: 66, and/or
- ii. a second polypeptide; an antigen fused SpyCatcher such as 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 to SEQ ID NO:18,

- i. a first polypeptide; an AP205 capsid protein comprising two SpyTags such as 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 to SEQ ID NO: 71, and/or
- ii. a second polypeptide; an antigen fused SpyCatcher such as 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 to SEQ ID NO:18,

- i. a first polypeptide; an AP205 capsid protein comprising a SpyTag such as 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 to SEQ ID NO: 64, and/or
- ii. a second polypeptide; an antigen fused SpyCatcher such as 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 to SEQ ID NO:18,

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; an AP205 capsid protein comprising a SpyTag such as 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 to SEQ ID NO: 62, and/or
- ii. a second polypeptide; an antigen fused SpyCatcher such as 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 to SEQ ID NO:18,

- i. a first polypeptide; an AP205 capsid protein comprising a KTag such as 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 to SEQ ID NO: 68, and/or
- ii. a second polypeptide; an antigen fused SpyTag such as 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 to SEQ ID NO:79, SEQ ID NO: 80, SEQ ID NO: 81, and/or SEQ ID NO: 82

- i. a first polypeptide; an AP205 capsid protein comprising a KTag such as 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 to SEQ ID NO: 69, and/or
- ii. a second polypeptide; an antigen fused SpyTag such as 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 to SEQ ID NO:79, SEQ ID NO: 80, SEQ ID NO: 81, and/or SEQ ID NO: 82

- i. a first polypeptide; an Phage fr capsid protein containing a KTag such as 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 to SEQ ID NO: 70, and/or
- ii. a second polypeptide; an antigen fused SpyTag such as 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 to SEQ ID NO:79, SEQ ID NO: 80, SEQ ID NO: 81, and/or SEQ ID NO: 82

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 AP205 VLP's using E. coli cells, such as BL21 cells, incubated at 16° C. for 18 hours. Other conditions and expression hosts may yield VLP's.
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. Aluminum 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% NaCl.
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 virus capsid protein, such as AP205 capsid protein and/or phage fr capsid protein comprising a SpyTag, and/or
- ii. obtaining a second polypeptide: an antigen fused to SpyCatcher, and
- iii. subjecting the first polypeptide to conditions which enable formation of virus-like particles, and/or
- iv. obtaining a vaccine by linkage of the second polypeptide and said virus-like particles via the interaction between the SpyCatcher and the SpyTag of said virus-like particles, and/or
- v. generating a composition comprising said vaccine.

thereby obtaining a pharmaceutical composition.
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 virus capsid protein, such as AP205 capsid protein and/or phage fr capsid protein comprising a SpyCatcher, and/or
- ii. obtaining a second polypeptide: an antigen fused to SpyTag, and
- iii. subjecting the first polypeptide to conditions which enable formation of virus-like particles, and/or
- iv. obtaining a vaccine by linkage of the second polypeptide and said virus-like particles via the interaction between the SpyCatcher and the SpyTag of said virus-like particles, and/or
- v. 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 Administrating 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.

In 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.

In 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 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 show several examples of successful introduction of SpyTag into various VLP loops without disrupting the self-assembly process. The examples below are non-limiting to the scope of the invention.
Gene Design of SpyTag-AP205
The synthetic Spytag-AP205 sequence was constructed by fusion of the SpyTag sequence (AHIVMVDAYKPTK) SEQ ID NO: 36 at either the N- and/or C-terminus of the AP205 (SEQ ID NO: 58) using a spacer sequence (GSGTAGGGSGS; for N-terminal fusion or GGSG; for C-terminal fusion of SpyTag) in between the AP205 and SpyTag sequences. The gene sequence was is further modified to contain an NcoI restriction site at the N-terminal and a C-terminal stop-codon followed by a NotI restriction site. The gene sequence may be codon-optimized for expression in Escherichia coli cells or other expression systems and synthesized by Geneart, Life Technologies. Other AP205/Phage fr SpyTag constructs of the present invention is made using a similar approach.
Gene Design of SpyCatcher-AP205
The synthetic Spycatcher-AP205 sequence was constructed by fusion of the SpyCatcher sequence SEQ ID NO: 37 at the N- of the AP205 (SEQ ID NO: 58) using a spacer sequence (GGSGS) in between the AP205 and the SpyCatcher sequences. The gene sequence is further modified to contain an NcoI restriction site at the N-terminal. The gene sequence may be codon-optimized for expression in Escherichia coli cells or other expression systems and synthesized by Geneart, Life Technologies.
Gene Design of SpyCatcher-Phage Fr
The synthetic Spycatcher-Phage fr sequence was constructed by fusion of the SpyCatcher sequence SEQ ID NO: 37 at the N-terminus of the Phage fr (SEQ ID NO: 59) using a spacer sequence (GGSGS) in between the Phage fr and the SpyCatcher sequences. The gene sequence is further modified to contain an NcoI restriction site at the N-terminal and a C-terminal stop-codon followed by a NotI restriction site. The gene sequence may be codon-optimized for expression in Escherichia coli cells or other expression systems and synthesized by Geneart, Life Technologies.
Expression and Purification of AP205 and/or Phage Fr VLPs
Plasmids were transformed into E. coli BL21 or JM109. A seed culture was prepared by inoculating a single colony into 2×YT medium containing 100 mg/l Ampicillin and the culture was grown overnight at 28° C. with shaking. For expression, the overnight culture was diluted in 2×YT medium containing 100 mg/l Ampicillin, and grown to and OD600 0.5-0.8 at 37° C. with shaking. The culture was then induced with IPTG (final concentration of 0.4 mM) and grown 4 hours 28° C. or 20 hours at 18-20° C. with vigorous aeration. Cells were resuspended in 20 mM Sodium phosphate buffer pH 7.2, 20 mM NaCl containing protease inhibitors and lysed by sonication at 80% Power with 5 pulsations for 2×5 min on ice (25 W effective). The lysates was clarified using centrifugation 40000G 30 min. and purified using a Hitrap™ SP HP column using increasing concentration of NaCl at pH 7.2. Some VLPs were additionally purified by ultracentrifugation over an iodixanol (Optiprep™) density gradient. Briefly, the lysate (containing VLPs) is first clarified by centrifugation at 5000×g and the supernatant is then layered onto an Optiprep™ density gradient (27/33/39%). VLPs are purified by density gradient ultracentrifugation in a SW60i rotor at 47,800 rpm for 3.5 hours (16° C.). Optiprep™ is subsequently removed by dialysis O/N against a PBS buffer pH 7.2, 0.02% PS80 using dialysis tubing with MWCO 300,000 kDa. Concentrations of the purified proteins are determined by the BCA assay.
Gene Design and Recombinant Expression of SpyTag-Binding Vaccine Antigens
Heterologous vaccine antigens were genetically fused with a GGS linker at either their C- or N-terminus to a previously described (WO 2011098772 A1) engineered SpyCatcher (SEQ ID NO: 37 (or 60 or 61)), thereby introducing SpyTag binding capability to the expressed antigen fusion proteins. SpyCatcher-antigen fusion genes expressed in E. coli are designed with a 6×Histidine tag and NcoI/BamHI restriction sites for subcloning into pET-15b vector. SpyCatcher-antigen fusion genes are expressed in either S2 cells, Human Embryonic Kidney 293 (HEK293) cells or in Baculovirus infected insect cells; designed with flanking EcoRI/BamHI (N-terminal) and NotI (C-terminal) sites and a 6×Histidine tag and subcloned into the pHP34s, pcDNA™4/HisMax or pAcGP67A (BD Biosciences) vector, respectively.
Engineered coat proteins were all expressed in E. coli and were purified by ultracentrifugation through an Optiprep™ step gradient (23%/29%/35%). Expression yield was determined by BCA assay. VLP assembly was confirmed by transmission electron microscopy and/or dynamic light-scattering analysis. For the estimation of the antigen coupling capacity individual VLP coat proteins were first incubated at 4° C. for 24 hours with corresponding SpyTag- or SpyCatcher-fused antigen (mixed at a 1:1 molar ratio) and each sample was subsequently analyzed by SDS-PAGE/densiometric analysis to assess the amount of antigen which was bound via the SpyTag—SpyCatcher interaction to the VLP coat protein. Results are summarized in table 4:

TABLE 4

Comparison of different engineered AP205 and Phage fr coat proteins
with regard to recombinant expression yield, ability to assemble into
a virus-like particle (VLP) and their capacity for antigen display

		Recombinant
	SEQ ID	expression		Antigen coupling
VLP name	NO.	yield	VLP assembly	capacity

	Protein	No/low/high	Yes/no	−, +, ++, +++
	(DNA)
SpyTag-AP205	62 (63)	High	Yes	++
AP205-SpyTag	64 (65)	High	Yes	+
SpyTag-Ap205-	71 (72)	High	Yes	+++
SpyTag
AP205-ggsg-	74 (73)	Low	No	N/A
SpyCatcher
SpyCatcher-	76 (75)	High	Yes	++
ggsgs-AP205
Spytag-Phage FR	66 (67)	Low	Yes	+
SpyCatcher	78 (77)	Low	No	−
Phage FR

The influence of the position of the SpyTag on the AP205 capsid protein is shown in FIG. 12 . As can be seen, fusion in the N-terminal end of AP205 results in a slightly better binding capacity compared to fusion in the C-terminal end.
Quality Assessment of SpyTag-VLPs and SpyCatcher-VLPs by Electron Microscopy
To verify the integrity of chimeric SpyTag-VLPs and SpyCatcher-VLPs, an aliquot of diluted particles 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 (FIG. 3 and FIG. 6 ). As can be seen when comparing FIGS. 3 and 6 to FIG. 11 , the AP205 VLPs obtained here have the same general structure as unmodified AP205 VLPs.
Quality Assessment of SpyTag-VLPs and SpyCatcher-VLPs by Dynamic Light Scattering
To verify particle size and polydispersity of chimeric SpyTag-VLPs and SpyCatcher-VLPs, an aliquot of particles was first clarified by centrifugation at 16000G for 10 min. The supernatant was transferred to a disposable MicroCuvette and examined by dynamic light scattering (DLS) using a DynaPro® NanoStar® (FIG. 9 and FIG. 10 ).
Verification of the SpyCatcher-Antigen Coupling onto Spytagged VLPs:
The overall amounts of antigen coupled onto the VLPs was estimated by density gradient ultracentrifugation of the VLP:spytag—Antigen:SpyCatcher mixture followed by SDS-PAGE of the VLP fraction (FIG. 4 ). The stoichiometry between the unconjugated SpyTag-AP205 capsid protein and the conjugated SpyCatcher-Antigen-AP205 capsid protein band shows the coupling efficacy. The stoichiometry can be modified by adding varying amounts of SpyCacher fused antigen.
The VLPs were also examined by transmission electron microscopy to assess their integrity after coupling of different SpyCatcher-antigens (FIG. 5 ). Specifically, an aliquot of diluted particles (post SpyCatcher-coupling) 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. The size and polydispersity of the VLPs after coupling of different SpyCatcher-antigens was examined (FIG. 9 ). Specifically, an aliquot was clarified by ultracentrifugation and transferred to a cuvette and examined by dynamic light scattering (DLS) using a DynaPro® NanoStar®.
FIG. 13 shows the binding capacity of AP205 VLPs to bind antigen when AP205 is fused to: no SpyTag, one SpyTag at the N-terminus, or two SpyTags at both the N- and C-terminus, respectively. The SDS-PAGE shows that SpyTag-AP205-SpyTag can bind either one or two SpyCatcher-Antigens per coat protein, SpyTag-AP205 can bind one SpyCatcher-Antigen per coat protein and AP205 cannot bind any SpyCatcher-Antigens. Comparing the intensities of individual protein bands shows that SpyTag-AP205-SpyTag (SAS) (SEQ ID NO: 71(72)) can bind more antigen compared to SpyTag-AP205 (SA) (SEQ ID NO: 62).
FIG. 14 shows the binding capacity of SpyTag-AP205-SpyTag VLPs to SpyCatcher-Pfs25. The SDS-PAGE shows that SpyTag-AP205-SpyTag can bind either one or two Pfs25 antigens per coat protein, whereas the AP205 VLPs do not bind to the Pfs25 antigen.
Verification of the SpyTag-Antigen Coupling onto SpyCatcher-VLPs:
The overall amounts of antigen coupled onto the VLPs was estimated by density gradient ultracentrifugation of the VLP:SpyCatcher—Antigen: SpyTag mixture followed by SDS-PAGE of the VLP fraction (FIG. 7 ). The stoichiometry between the unconjugated SpyCatcher-AP205 capsid protein and the conjugated SpyCatcher-AP205—SpyTag-antigen band shows the coupling efficacy. The stoichiometry can be modified by adding varying amounts of SpyTag fused antigen.
The VLPs were also examined by transmission electron microscopy to assess their integrity after coupling of different SpyTag-antigens (FIG. 8 ). Specifically, an aliquot of diluted particles (post SpyTag-antigen coupling) 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. The size and polydispersity of the VLPs after coupling of different SpyTag-antigens was examined (FIG. 10 ). Specifically, an aliquot was clarified by ultracentrifugation and transferred to a cuvette and examined by dynamic light scattering (DLS) using a DynaPro® NanoStar®.
Versatility of the VLP-Based Antigen Presentation Platform
The inventors have successfully engineered VLPs able to display a variety of antigens, as summarized in Table 5.

TABLE 5

Summary

			Confirmed	Confirmed	Confirmed
		Confirmed	binding to	binding to	binding to
		binding to	SpyTag-	LongSpy-	Spy-	Confirmed
SEQ		SpyTag-	AP205-	Tag-AP205-	Catcher-	binding to
ID NO:		AP205	SpyTag	LongSpy-Tag	AP205	SpyTag-fr
protein		(SEQ ID	(SEQ ID	(SEQ ID	(SEQ ID	(SEQ ID
(DNA)	Antigen	NO: 62)	NO: 71)	NO: 92)	NO: 74)	NO: 66)

18	SpyCatcher-Her2-	YES	YES
	ECD\|23-686
19	SpyCatcher-IL-	YES	YES			YES
	5(C63T/C105T)
20 (29)	PCSK9\|31-	YES	YES
	692\|:Spy-
	Catcher:HIS
21 (30)	SpyCatcher-				YES
	ID1ID2a-HIS
24 (33)	GMZ2:SpyC	YES	YES	YES
27	SpyCatcher-Pfs25-	YES	YES
	HIS
28	HIS-PfCSP(aa92-	YES	YES
	397)-SpyCatcher
84 (85)	AG85A	YES	YES
	(SpyCatcher)
86 (87)	SpyC:Survivin	YES	YES
	(MP1804)
52 (53)	Spycatcher-ggs-	YES	YES
	CIDR1a-HIS
1 (2)	L2(aa11-88x5)-	YES	YES
	ggs-spycatcher
3 (4)	SpyCatcher-R0.Pf6C	YES	YES
5 (6)	SpyCatcher Pf6C	YES	YES
7 (8)	SpyTag-DBL1-ID2a				YES
9 (10)	PDL1-SpyTag				YES
11 (12)	CTLA-4-SpyTag				YES
14 (15)	SpyTag-L-DER P2				YES
16 (17)	mini-HA-Stem-HIS-				YES
	SpyT

Immunological Testing of the VLP-Based Antigen Presentation Platform:
To assess the immunological effect of the described VLP antigen-presentation platform, we immunized groups of mice (n=5) with either VLP-coupled or non-coupled soluble antigen (control group) formulated with or without extrinsic adjuvant. To take into account the possibility that AP205 VLPs themselves may have an adjuvant effect we further included a similar amount of unmodified AP205 VLPs (i.e. with no SpyTag or SpyCatcher fused) in the control group vaccine formulations. Each mouse was administered three intra muscular immunizations (50 microliter volume injected into each Tibialis anterior muscle) on day 1, 21 and 42, and sera were collected two weeks or three months after each immunization for subsequent analysis.
To study the kinetics of antibody responses, total antigen-specific immunoglobulins in mouse sera collected after 1^st, 2^ndand 3^rdimmunization, respectively, was measured in an ELISA assay using the naked vaccine antigen (i.e. with no spyTag or SpyCatcher) as the solid phase capturing antigen (plates were coated with 1 ug/ml antigen). The antibody titer of sera from mice immunized with the VLP-coupled-antigen was subsequently compared against that of the control group to evaluate the effect of the VLP antigen-display in terms of both antibody peak titers and kinetics i.e. how quickly the antibody response develops and to what magnitude, during the vaccination regime. Specifically, a three-fold dilution of the serum starting from 1:100 down to 1:5.904.900 was performed to compare antibody responses between the two groups of animals immunized with either antigen conjugated VLPs or non-coupled antigen.
To compare the longevity of the induced humoral responses between VLP vaccinated mice and mice immunized with soluble SpyCatcher-antigen or SpyTag antigen (control group), sera are collected every third week after the last given immunization for a full year (or until a significant difference is observed between test and control groups) and are tested in the above described ELISA assay.
FIG. 15 shows the Ig response against a SpyTag coupled antigen (SEQ ID NO: 7) two weeks after prime boost-boost immunization regimen (vaccines formulated without aluminum hydroxide gel). Soluble SpyTag-antigen and AP205 are unable to bind the antigen. Immunization of mice with VLP-displayed SpyTag-antigen (SEQ ID NO: 7) induces a higher Ig response compared to mice immunized with soluble SpyTag-Ag (SEQ ID NO: 7) and AP205 (SEQ ID NO: 58).
FIG. 16 shows the Ig response against a SpyCatcher coupled antigen (SEQ ID NO: 27) three months after a prime-boost-boost immunization regimen. Immunization of mice with VLP-displayed SpyCatcher-antigen (SEQ ID NO: 27) induces a higher Ig response compared to mice immunized with soluble SpyCatcher-antigen (SEQ ID NO: 27) and AP205 (SEQ ID NO: 58).
FIGS. 15 and 16 show that the VLP vaccines disclosed herein can induce increased antibody titres.
The avidity of antibodies induced in mice following a prime-boost-boost immunization regimen was analysed (FIGS. 17 and 18 ). Mouse anti-sera were obtained four months after last immunization. Immunization of mice with VLP-displayed antigen (SEQ ID NO: 27) gives rise to antibodies with a significantly higher avidity compared to antibodies from mice immunized with soluble SpyCatcher-antigen (SEQ ID NO: 27) and AP205 (SEQ ID NO: 58) (FIG. 17 ). Both vaccines were formulated with aluminum hydroxide gel. This difference was statistically tested using non-parametric Two-sample Wilcoxon rank-sum (Mann-Whitney) test, which resulted in a probability score of P>|z|=0.00002.
FIG. 18 shows the avidity of antibodies obtained from a pool of sera from mice immunized with SpyCatcher-AP205 (SEQ ID NO: 76) coupled to SpyTag-antigen (SEQ ID NO: 7). The gray bar represents a pool of sera from mice immunized with soluble SpyTag-Ag (SEQ ID NO: 7) and AP205 (SEQ ID NO: 58), which is unable to bind the antigen. Both vaccines were formulated without aluminum hydroxide gel. Immunization of mice with SpyCatcher-AP205 (SEQ ID NO: 76) coupled to SpyTag-antigen (SEQ ID NO: 7) resulted in induction of antibodies with higher avidity compared to antibodies from mice immunized with soluble SpyTag-Ag (SEQ ID NO: 7) and AP205 (SEQ ID NO: 58).
Taken together, the results of FIGS. 17 and 18 show that the present VLP vaccines can be used to induce antibodies with increased avidity compared to corresponding soluble protein vaccines.
We also analysed how fast antibodies were induced upon vaccination with VLP-displayed antigens (FIG. 19 ). The Ig response against an antigen (SEQ ID NO: 52) following a single immunization was analysed (FIG. 18 ) in individual mice immunized with SpyTag-AP205 (SEQ ID NO: 62) coupled to SpyCatcher-antigen (SEQ ID NO: 52) or with soluble SpyCatcher-antigen (SEQ ID NO: 52) and AP205 (SEQ ID NO: 58), which is unable to bind the antigen. Both vaccines were formulated with aluminum hydroxide gel. A single immunization of mice with SpyTag-AP205 (SEQ ID NO: 62) coupled to spyCatcher-antigen (SEQ ID NO: 52) induced a faster Ig response compared to mice immunized with soluble SpyCatcher-antigen (SEQ ID NO: 52) and AP205 (SEQ ID NO: 58).
The same experiment was performed in mice immunized with SpyTag-AP205-SpyTag (SEQ ID NO: 71) coupled to SpyCatcher-antigen (SEQ ID NO: 27) or with soluble SpyCatcher-antigen (SEQ ID NO: 27) and AP205 (SEQ ID NO: 58), which is unable to bind the antigen (FIG. 20 ). Both vaccines were formulated with aluminum hydroxide gel. A single immunization of mice with SpyTag-AP205-SpyTag (SEQ ID NO: 71) coupled to SpyCatcher-antigen (SEQ ID NO: 27) induced a faster Ig response compared to mice immunized with soluble SpyCatcher-antigen (SEQ ID NO: 27) and AP205 (SEQ ID NO: 58).
The data presented in FIGS. 19 and 20 show that a single immunization with VLP-presented antigens results in a faster induction of antibodies.
Human memory B cells have been proposed to play a role in maintaining serum antibody levels over time and thus to evaluate the potential of the VLP-based antigen presentation platform to induce long-term immunological memory we also compare the ability of the VLP-based vaccine to generate memory B-cells against that of the soluble antigen vaccine (control). The memory B cell ELISPOT is the accepted standard for measuring the relative frequency of memory B cells and relies on the detection of memory B cells that have differentiated into plasma cells after stimulation with three polyclonal stimuli (CpG, Staphylococcus aureus Cowan (SAC, Sigma), and Pokeweed Mitogen (PWM, Sigma)). Following the stimulation, the number of antigen-specific memory B cells and total memory B cells are enumerated and the ratio between the number of antigen-specific spots and the total number of memory B cell spots is estimated and reported as a percentage.
Antigen-Specific Qualitative Testing of Induced Immune Responses: Testing of the VLP:SpyTag and SpyCatcher:VLP Platform, Respectively, to Induce VAR2CSA Specific Antibodies
To qualitatively assess the induced antibody responses, we performed an optimized parasite binding-inhibition assay that test the capacity of the collected sera to inhibit binding between the human receptor, Chondroitin Sulfate A (CSA), and parasitized erythrocytes expressing the VAR2CSA ligand. This was done by coating 96 well plates with the purified CSA receptor and adding radio-labeled malaria parasites expressing VAR2CSA in the presence or absence of VAR2CSA specific antibodies in sera from animals immunizing animals with VAR2CSA (SpyCatcher-ID1ID2a/SpyTag-ID1ID2a) conjugated VLPs or soluble VAR2CSA antigen alone (SpyCatcher-ID1ID2a/SpyTag-ID1ID2a). Another qualitative measure of the functional IgG response is to estimate the total amount of opsonizing IgG in a serum sample. This is done by incubating VAR2CSA expressing malaria parasites with serum in a 5 fold dilution series starting from 1:100 followed by washing of the infected erythrocytes and detection of bound VAR2CSA specific IgG using an Alexa488 conjugated secondary antibody specific to mice/rat or rabbit IgG followed by flow cytometry analysis.
Specifically, 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 (3 fold dilution series, starting from 1:20) and then allowed to incubate on decorin coated plates for 90 min. Unbound IE were washed away and the remaining IEs were quantified. Normalized parasite binding after incubation with pooled anti-sera from mice (n=5) vaccinated with SpyTag-ID1ID2a (SEQ ID NO: 82) conjugated to SpyCatcher-VLPs (SEQ ID NO: 76) or soluble SpyTag-ID1ID2a (SEQ ID NO: 82) mixed with unmodified AP205 VLPs (SEQ ID NO: 58) are shown after first (▴), second (▪) and third (•) immunization (FIG. 25 ). The assay show that anti-sera from mice immunized with VLP-conjugated SpyTag-ID1ID2a (SEQ ID NO: 82) has a greater binding-inhibition capacity compared to anti-sera from mice immunized with soluble SpyTag-ID1ID2a (SEQ ID NO: 82).
Testing of the VLP:SpyTag and SpyCatcher:VLP Platform, Respectively, to Induce Humoral Immunity Against Self-Antigens.
To demonstrate the capacity of the VLP:SpyTag and the SpyCatcher:VLP platform, respectively, to break immune tolerance to self-antigens, associated with both cardiovascular disease (PCSK9), immune-inflammatory disease (IL-5) and cancer (Her2/Survivin), we genetically fuse the self-antigens to a SpyCatcher or SpyTag and couple them onto SpyTag or Spycatcher VLPs, respectively, as previously described. In some cases (IL-5, Survivin, CTLA-4 and PD-L1) we at first use the mouse gene homologues for the immunization of mice. Specifically, our working procedure is to firstly couple HER2 (SpyCatcher:Her2-ECD|23-686|), Survivin, IL-5 (SpyCatcher:IL-5 (C63T/C105T)) and PCSK9 (PCSK9|31-692|:SpyCatcher-HIS) to the SpyTaggedVLPs or, similarly, couple the (SpyTag:Her2-ECD|23-686|), Survivin, IL-5 (SpyTag:IL-5 (C63T/C105T) to the SpyCatcher:VLPs. Then we use the antigen coupled VLPs to immunize mice, and measure seroconversion of the animals in group a) mice immunized with conjugated VLPs and b) mice immunized with the non-coupled soluble antigen and unmodified (i.e. with no SpyTag/SpyCatcher) VLPs. The antigen-specific immunoglobulin titer will be estimated in a 3 fold dilution series of the sera. A positive seroconversion is defined as ELISA OD measurements above 2× standard deviation of a mock immunized animal. Serum conversion and induction of specific antibodies to HER2 and Survivin 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).
This experiment was performed with IL-5, CTLA-4 and PD-L1.
The Ig response against the self-antigen IL-5 (SEQ ID NO: 19) was analysed five months after a prime-boost-boost immunization regimen. Individual mice were immunized with SpyTag-AP205-SpyTag (SEQ ID NO: 71) coupled to the IL-5 SpyCatcher-(self)-antigen (SEQ ID NO: 19) or with soluble SpyCatcher-antigen (SEQ ID NO: 19) and AP205 (SEQ ID NO: 58), which is unable to bind the SpyCatcher-antigen. Both vaccines were formulated in aluminum hydroxide gel. Immunization of mice with VLP-displayed self-antigen (SEQ ID NO: 19) resulted in breakage of immune tolerance and induction of antigen specific antibodies, whereas immunization with the (non-displayed) soluble self-antigen (SEQ ID NO: 19) did not induce antigen specific antibodies (FIG. 21 ).
The Ig response against the self-antigen CTLA-4 (SEQ ID NO: 11) two weeks after a prime-boost immunization regimen was analysed in individual mice immunized with SpyTag-AP205-SpyTag (SEQ ID NO: 71) coupled to the CTLA-4 self-antigen (SEQ ID NO: 11) or with soluble SpyCatcher-antigen (SEQ ID NO: 11) and AP205 (SEQ ID NO: 58), which is unable to bind the SpyCatcher-antigen. Both vaccines were formulated in aluminum hydroxide gel. Immunization of mice with VLP-displayed self-antigen (SEQ ID NO: 11) resulted in breakage of immune tolerance and induction of antigen specific antibodies, whereas immunization with the (non-displayed) soluble self-antigen (SEQ ID NO: 11) does not induce antigen specific antibodies (FIG. 22 ).
The Ig response against the self-antigen PD-L1 (SEQ ID NO: 9) two weeks after a prime-boost immunization regimen in individual mice immunized with SpyTag-AP205-SpyTag (SEQ ID NO: 71) coupled to the PD-L1 self-antigen (SEQ ID NO: 9) or with soluble self-antigen (SEQ ID NO: 9) and AP205 (SEQ ID NO: 58), which is unable to bind the SpyCatcher-antigen. Both vaccines were formulated in aluminum hydroxide gel. Immunization of mice with VLP-displayed self-antigen (SEQ ID NO: 9) resulted in breakage of immune tolerance and induction of antigen specific antibodies, whereas immunization with the (non-displayed) soluble self-antigen (SEQ ID NO: 9) does not induce antigen specific antibodies (FIG. 23 ).
Testing of the Functionality of the VLP-Presented Antigen Vaccine
Immunization with a Pfs25 VLP vaccine resulted in induction of functional antibodies which were able to block the transmission of Plasmodium falciparum parasites in vitro. Mice were immunized two times with 2.5 ug of either A) spycatcher-Pfs25 antigen (SEQ ID NO: 27) displayed on the SpyTag-AP205-SpyT (SEQ ID NO: 71) VLP or B) soluble spycatcher-Pfs25 antigen (SEQ ID NO: 27) mixed with the AP205 VLP (SEQ ID NO: 58), which is unable to bind/display the antigen. Both vaccines were formulated with aluminum hydroxide gel. Transmission-blocking efficacy of antibodies was evaluated by standard mosquito membrane feeding assay (SMFA) using purified IgG from immune sera. Results show that antibodies induced in mice immunized with VLP-displayed Pfs25 (VLP vaccine) had ˜100% percent transmission-blocking activity when tested in the SMFA in vitro assay (FIG. 24 ).
Testing of the VLP:SpyTag and the SpyCatcher:VLP Platform to Induce Cancer Inhibitory Antibodies.
Standard animal models are established 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 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 tumor cell lines. Alternatively, the therapeutic effect of the vaccine is determined by immunizing animals with established tumors and monitoring tumor regression/progression by size measurements and/or by fluorescent scannings.
Testing of the VLP:SpyTag and the SpyCatcher:VLP 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:SpyTag platform or the SpyCatcher:VLP 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 in the present invention. 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 (4 μI volume) are added to wells of a 96-well plate and 1960 of prepared cholesterol reagent added. The plate is incubated for 5 minutes at 37° C. and the absorbance of the developed color read at 600 nm within 30 minutes.
Sequences

TABLE 6

Overview of the sequences disclosed in the present invention

SEQ ID NO:
protein (DNA)		Antigens:

18	A1	>SpyCatcher- Her2-ECD\|23-686
		(Homo Sapiens)
19	A2	>SpyCatcher-IL-5(C63T/C105T)
		(Mus musculus)
20 (29)	A3	>PCSK9 31-692\|:SpyCatcher:HIS
		(Homo Sapiens)
21 (30)	A4	>SpyCatcher-ID1ID2a-HIS
		(Plasmodium falciparum)
22 (31)	A5	>SpyCatcher-RO-HIS
		(Plasmodium falciparum)
23 (32)	A6	>HIS-RO-SpyCatcher
		(Plasmodium falciparum)
24 (33)	A7	>HIS-GMZ2ggsSpyCatcher
		(Plasmodium falciparum)
25 (34)	A8	>HIS-GMZ2T:ggsSpyCatcher
		(Plasmodium falciparum)
26 (35)	A9	>SpyCatcher-PfRH5-HIS
		(Plasmodium falciparum)
27	A10	>SpyCatcher-Pfs25-HIS
		(Plasmodium falciparum)
28	A11	>HIS-PfCSP(aa92-397)- SpyCatcher
		(Plasmodium falciparum)
40	A12	>Survivin: SpyCatcher
		(Homo Sapiens)
41	A13	> SpyCatcher:Survivin
		(Homo Sapiens)
42	A14	>Survivin(F101A/L102A):
		SpyCatcher (Homo Sapiens)
43	A15	>
		SpyCatcher:Survivin(F101A/L102A)
		(Homo Sapiens)
44 (48)	A16	>
		SpyCatcher: Surviving 101 A/L 102 A)
		(Mus Musculus)
45 (49)	A17	>Survivin (F101A/L102A):
		SpyCatcher (Mus Musculus)
46 (50)	A18	> SpyCatcher:Survivin
		(Mus Musculus)
47 (51)	A19	>Survivin: SpyCatcher
		(Mus Musculus)
52 (53)	A20	>SpyCatcher:CIDR1a-HIS
84 (85)	A21	SpyCatcher-Ag85A
		(Mycobacterium tuberculosis)
86 (87)	A22	SpyCatcher-ggs-survivin
		(Homo Sapiens)
1 (2)		L2(aa11-88 x5)-ggs-spycatcher
		(Human papillomavirus)
3 (4)		SpyCatcher-R0.Pf6C
		(Plasmodium falciparum)
5 (6)		SpyCatcher Pf6C
		(Plasmodium falciparum)
7 (8)		SpyTag-DBL1-ID2a
		(Plasmodium falciparum)
9 (10)		PDL1-SpyTag (Mus musculus)
11 (12)		CTLA-4-SpyTag (Mus musculus)
14 (15)		SpyTag-L-DER P2
		(Dermatophagoides pteronyssinus)
13		AMA1-SpyTag
88 (89)		Mini-HA-stem-Spytag
90		Infectious hematopoietic necrosis
		virus (IHNV) G-protein-SpyTag
91		SpyTag-IHNV G-protein
		Misc.
36 (39)		SpyTag amino acid sequence
37 (54)		SpyCatcher
38		The β-strand of CnaB2 (KTag)
55		SpyLigase
56		isopeptide Spy0128
57		Split- Spy0128
58		AP205
59		PhageFr
60		SpyCatcherΔN
61		SpyCatcherΔNC
62 (63)		Spy-AP205
64 (65)		AP205-spy
66 (67)		Spy-Phage ft
68		Ktag-AP205
69		AP205-Ktag
70		Ktag-Phage fr
71 (72)		Spy-AP205-Spy
74 (73)		AP205-ggsg-Spycatcher
76 (75)		SpyCatcher-ggsgs-AP205
78 (77)		SpyCatcher-ggsgs-Phage ft
79		SpyTag-Her2-ECD\|23-686
80		SpyTag-IL-5(C63T/C105T)
81		PCSK9\|31-692\|:Spytag
82		SpyTag-ID1ID2a-HIS
83		Short flexible linker
92 (93)		SpyTag-IHNV G-protein
94 (95)		mSA-AP205 DNA

>L2(aa11-88 x5)-ggs-spycatcher
SEQ ID NO: 1
MKRASATQLYKTCKQAGTCPPDIIPKVEGKTIADQILQYGSMGVFFGGLGIGTGSGTG

GRTGYIPLGTRPPTATDTLAKRASVTDLYKTCKQSGTCPPDVVPKVEGTTLADKILQ

WSSLGIFLGGLGIGTGSGTGGRTGYIPLGGRSNTVVDVGPKRASATQLYQTCKLTGTC

PPDVIPKVEHNTIADQILKWGSLGVFFGGLGIGTGSGTGGRTGYVPLGTSAKPSITSGP

KRAAPKDIYPSCKISNTCPPDIQNKIEHTTIADKILQYGSLGVFLGGLGIGTARGSGGRI

GYTPLGEGGGVRVATRPKRDSVTHIYQTCKQAGTCPPDVINKVEQTTVADNILKYGS

AGVFFGGLGISTGRGTGGATGYVPLGEGPGVRVGGTPGGSGAMVDTLSGLSSEQGQS

GDMTIEEDSATHIKFSKRDEDGKELAGATMELRDSSGKTISTWISDGQVKDFYLYPG

KYTFVETAAPDGYEVATAITFTVNEQGQVTVNGKATKGDAHI

>L2(aa11-88 x5)-ggs-spycatcher DNA
SEQ ID NO: 2
ATGAAACGTGCAAGCGCAACCCAGCTGTATAAAACCTGTAAACAGGCAGGCACC

TGTCCGCCTGATATCATTCCGAAAGTTGAAGGTAAAACCATTGCCGATCAGATTC

TGCAGTATGGTAGCATGGGCGTGTTTTTTGGTGGTCTGGGTATTGGCACCGGTAG

CGGCACAGGTGGACGTACCGGTTACATTCCGCTGGGCACCCGTCCGCCTACCGCA

ACCGATACCCTGGCAAAACGTGCCAGCGTTACCGATCTGTACAAAACATGCAAAC

AGAGCGGAACATGTCCTCCGGATGTTGTTCCTAAAGTGGAAGGCACCACCCTGGC

AGATAAAATCCTGCAGTGGTCAAGCCTGGGTATTTTCCTGGGTGGCTTAGGCATA

GGTACAGGTAGTGGTACAGGCGGTCGCACAGGCTATATCCCGCTGGGTGGTCGTA

GCAATACCGTTGTTGATGTTGGTCCGAAACGTGCATCAGCCACACAGCTGTATCA

GACCTGCAAACTGACCGGTACGTGCCCACCTGATGTTATCCCGAAAGTGGAACAT

AATACAATTGCAGACCAGATTCTGAAATGGGGTTCACTGGGCGTATTCTTCGGAG

GCCTGGGCATCGGAACCGGTTCAGGTACGGGTGGCCGTACCGGCTATGTGCCTCT

GGGTACAAGCGCAAAACCGAGCATTACCAGCGGTCCTAAACGCGCAGCACCGAA

AGATATTTATCCGAGCTGTAAAATTAGCAATACCTGCCCTCCGGATATCCAGAAC

AAAATTGAACATACCACCATTGCCGACAAAATCTTACAGTACGGTTCTCTGGGTG

TGTTTCTGGGAGGTTTAGGTATCGGTACGGCACGTGGTAGCGGTGGTCGCATTGG

TTATACACCGCTGGGTGAAGGTGGTGGTGTTCGTGTTGCAACCCGTCCTAAACGT

GATAGCGTTACCCATATTTATCAGACGTGTAAACAAGCAGGTACTTGTCCACCAG

ATGTGATTAACAAAGTGGAACAGACAACCGTTGCGGATAACATTCTGAAATATG

GTAGTGCCGGTGTGTTTTTTGGCGGACTGGGCATTTCAACCGGTCGTGGTACGGG

TGGTGCAACCGGTTACGTGCCTCTGGGCGAAGGTCCGGGTGTGCGTGTGGGTGGT

ACACCGGGTGGTAGCGGTGCAATGGTTGATACCCTGAGCGGTCTGAGCAGCGAA

CAGGGTCAGAGCGGTGATATGACCATTGAAGAAGATAGCGCAACCCACATCAAA

TTCAGCAAACGTGATGAAGATGGTAAAGAACTGGCAGGCGCAACAATGGAACTG

CGTGATAGCAGCGGTAAAACCATTAGCACCTGGATTAGTGATGGTCAGGTGAAA

GATTTTTATCTGTACCCTGGCAAATACACCTTTGTTGAAACCGCAGCACCGGATG

GTTATGAAGTTGCAACCGCAATTACCTTTACCGTTAATGAACAGGGCCAGGTTAC

CGTGAATGGTAAAGCAACCAAAGGTGATGCACATATTtaa

>Spycatcher-RO.PfC
SEQ ID NO: 3
GAMVDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDEDGKELAGATMELRDSSGKTIS

TWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGKATKGDA

HIGGSRSTSENRNKRIGGPKLRGNVTSNIKFPSDNKGKIIRGSNDKLNKNSEDVLEQSE

KSLVSENVPSGLDIDDIPKESIFIQEDQEGQTHSELNPETSEHSKDLNNNGSKNESSDIIS

ENNKSNKVQNHFESLSDLELLENSSQDNLDKDTISTEPFPNQKHKDLQQDLNDEPLEP

FPTQIHKDYKEKNLINEEDSEPFPRQKHKKVDNHNEEKNVFHENGSANGNQGSLKLK

SFDEHLKDEKIENEPLVHENLSIPNDPIEQILNQPEQETNIQEQLYNEKQNVEEKQNSQI

PSLDLKEPTNEDILPNHNPLENIKQSESEINHVQDHALPKENIIDKLDNQKEHIDQSQH

NINVLQENNINNHQLEPQEKPNIESFEPKNIDSEIILPENVETEEIIDDVPSPKHSNHETFE

EETSESEHEEAVSEKNAHETVEHEETVSQESNPEKADNDGNVSQNSNNELNENEFVE

SEKSEHEARSEKKVIHGCNFSSNVSSKHTFTDSLDISLVDDSAHISCNVHLSEPKYNHL

VGLNCPGDIIPDCFFQVYQPESEELEPSNIVYLDSQINIGDIEYYEDAEGDDKIKLFGIV

GSIPKTTSFTCICKKDKKSAYMTVTIDSA

>Spycatcher-RO.PfC DNA
SEQ ID NO: 4
GGTGCAATGGTTGATACCCTGAGCGGTCTGAGCAGCGAACAGGGTCAGAGCGGT

GATATGACCATTGAAGAAGATAGCGCAACCCACATCAAATTCAGCAAACGTGAT

GAAGATGGTAAAGAACTGGCAGGCGCAACAATGGAACTGCGTGATAGCAGCGGT

AAAACCATTAGCACCTGGATTAGTGATGGTCAGGTGAAAGATTTTTATCTGTACC

CTGGCAAATACACCTTTGTTGAAACCGCAGCACCGGATGGTTATGAAGTTGCAAC

CGCAATTACCTTTACCGTTAATGAACAGGGCCAGGTTACCGTGAATGGTAAAGCA

ACCAAAGGTGATGCACATATTGGTGGTAGCAGATCCACAAGTGAGAATAGAAAT

AAACGAATCGGGGGTCCTAAATTAAGGGGTAATGTTACAAGTAATATAAAGTTCC

CATCAGATAACAAAGGTAAAATTATAAGAGGTTCGAATGATAAACTTAATAAAA

ACTCTGAAGATGTTTTAGAACAAAGCGAAAAATCGCTTGTTTCAGAAAATGTTCC

TAGTGGATTAGATATAGATGATATCCCTAAAGAATCTATTTTTATTCAAGAAGAT

CAAGAAGGTCAAACTCATTCTGAATTAAATCCTGAAACATCAGAACATAGTAAA

GATTTAAATAATAATGGTTCAAAAAATGAATCTAGTGATATTATTTCAGAAAATA

ATAAATCAAATAAAGTACAAAATCATTTTGAATCATTATCAGATTTAGAATTACT

TGAAAATTCCTCACAAGATAATTTAGACAAAGATACAATTTCAACAGAACCTTTT

CCTAATCAAAAACATAAAGACTTACAACAAGATTTAAATGATGAACCTTTAGAAC

CCTTTCCTACACAAATACATAAAGATTATAAAGAAAAAAATTTAATAAATGAAGA

AGATTCAGAACCATTTCCCAGACAAAAGCATAAAAAGGTAGACAATCATAATGA

AGAAAAAAACGTATTTCATGAAAATGGTTCTGCAAATGGTAATCAAGGAAGTTTG

AAACTTAAATCATTCGATGAACATTTAAAAGATGAAAAAATAGAAAATGAACCA

CTTGTTCATGAAAATTTATCCATACCAAATGATCCAATAGAACAAATATTAAATC

AACCTGAACAAGAAACAAATATCCAGGAACAATTGTATAATGAAAAACAAAATG

TTGAAGAAAAACAAAATTCTCAAATACCTTCGTTAGATTTAAAAGAACCAACAAA

TGAAGATATTTTACCAAATCATAATCCATTAGAAAATATAAAACAAAGTGAATCA

GAAATAAATCATGTACAAGATCATGCGCTACCAAAAGAGAATATAATAGACAAA

CTTGATAATCAAAAAGAACACATCGATCAATCACAACATAATATAAATGTATTAC

AAGAAAATAACATAAACAATCACCAATTAGAACCTCAAGAGAAACCTAATATTG

AATCGTTTGAACCTAAAAATATAGATTCAGAAATTATTCTTCCTGAAAATGTTGA

AACAGAAGAAATAATAGATGATGTGCCTTCCCCTAAACATTCTAACCATGAAACA

TTTGAAGAAGAAACAAGTGAATCTGAACATGAAGAAGCCGTATCTGAAAAAAAT

GCCCACGAAACTGTCGAACATGAAGAAACTGTGTCTCAAGAAAGCAATCCTGAA

AAAGCTGATAATGATGGAAATGTATCTCAAAACAGCAACAACGAATTAAATGAA

AATGAATTCGTTGAATCGGAAAAAAGCGAGCATGAAGCAAGATCCGAAAAAAAA

GTCATACACGGATGTAACTTCTCTTCAAATGTTAGTTCTAAACATACTTTTACAGA

TAGTTTAGATATTTCTTTAGTTGATGATAGTGCACATATTTCATGTAACGTACATT

TGTCTGAACCAAAATATAATCATTTGGTAGGTTTAAATTGTCCTGGTGATATTATA

CCAGATTGCTTTTTTCAAGTATATCAACCTGAATCAGAAGAACTTGAACCATCCA

ACATTGTTTATTTAGATTCACAAATAAATATAGGAGATATTGAATATTATGAAGA

TGCTGAAGGAGATGATAAAATTAAATTATTTGGTATAGTTGGAAGTATACCAAAA

ACGACATCTTTTACTTGTATATGTAAGAAGGATAAAAAAAGTGCTTATATGACAG

TTACTATAGATTCAGCA

>SpyCatcher-Pf6C
SEQ ID NO: 5
GAMVDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDEDGKELAGATMELRDSSGKTIS

TWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGKATKGDA

HIGGSRSEKKVIHGCNFSSNVSSKHTFTDSLDISLVDDSAHISCNVHLSEPKYNHLVGL

NCPGDIIPDCFFQVYQPESEELEPSNIVYLDSQINIGDIEYYEDAEGDDKIKLFGIVGSIP

KTTSFTCICKKDKKSAYMTVTIDSARS

>SpyCatcher-Pf6C DNA
SEQ ID NO: 6
GGTGCAATGGTTGATACCCTGAGCGGTCTGAGCAGCGAACAGGGTCAGAGCGGT

GATATGACCATTGAAGAAGATAGCGCAACCCACATCAAATTCAGCAAACGTGAT

GAAGATGGTAAAGAACTGGCAGGCGCAACAATGGAACTGCGTGATAGCAGCGGT

AAAACCATTAGCACCTGGATTAGTGATGGTCAGGTGAAAGATTTTTATCTGTACC

CTGGCAAATACACCTTTGTTGAAACCGCAGCACCGGATGGTTATGAAGTTGCAAC

CGCAATTACCTTTACCGTTAATGAACAGGGCCAGGTTACCGTGAATGGTAAAGCA

ACCAAAGGTGATGCACATATTGGTGGTAGCAGATCCGAAAAAAAAGTCATACAC

GGATGTAACTTCTCTTCAAATGTTAGTTCTAAACATACTTTTACAGATAGTTTAGA

TATTTCTTTAGTTGATGATAGTGCACATATTTCATGTAACGTACATTTGTCTGAAC

CAAAATATAATCATTTGGTAGGTTTAAATTGTCCTGGTGATATTATACCAGATTGC

TTTTTTCAAGTATATCAACCTGAATCAGAAGAACTTGAACCATCCAACATTGTTTA

TTTAGATTCACAAATAAATATAGGAGATATTGAATATTATGAAGATGCTGAAGGA

GATGATAAAATTAAATTATTTGGTATAGTTGGAAGTATACCAAAAACGACATCTT

TTACTTGTATATGTAAGAAGGATAAAAAAAGTGCTTATATGACAGTTACTATAGA

TTCAGCAAGATCTtaa

>SpyTag-DBL1-ID2a
SEQ ID NO: 7
MAHIVMVDAYKPTKNKIEEYLGAKSDDSKIDELLKADPSEVEYYRSGGDGDYLKNNI

CKITVNHSDSGKYDPCEKKLPPYDDNDQWKCQQNSSDGSGKPENICVPPRRERLCTY

NLENLKFDKIRDNNAFLADVLLTARNEGEKIVQNHPDTNSSNVCNALERSFADLADII

RGTDQWKGTNSNLEKNLKQMFAKIRENDKVLQDKYPKDQKYTKLREAWWNANRQ

KVWEVITCGARSNDLLIKRGWRTSGKSDRKKNFELCRKCGHYEKEVPTKLDYVPQF

LRWLTEWIEDFYREKQNLIDDMERHREECTREDHKSKEGTSYCSTCKDKCKKYCEC

VKKWKTEWENQENKYKDLYEQNKNKTSQKNTSRYDDYVKDFFEKLEANYSSLENY

IKGDPYFAEYATKLSFILNPSDANNPSGETANHNDEACNCNESGISSVGQAQTSGPSS

NKTCITHSSIKTNKKKECKDVKLGVRENDKDLKICVIEDTSLSGVDNCCCQDLLGILQ

ENCSDNKRGSSSNDSCDNKNQDECQKKLEKVFASLTNGYKCDKCKSGTSRSKKKWI

WKKSSGNEEGLQEEYANTIGLPPRTQSLYLGNLPKLENVCEDVKDINFDTKEKFLAG

CLIVSFHEGKNLKKRYPQNKNSGNKENLCKALEYSFADYGDLIKGTSIWDNEYTKDL

ELNLQNNFGKLFGKYIKKNNTAEQDTSYSSLDELRESWWNTNKKYIWTAMKHGAE

MNITTCNADGSVTGSGSSCDDIPTIDLIPQYLRFLQEWVENFCEQRQAKVKDVITNCK

SCKESGNKCKTECKTKCKDECEKYKKFIEACGTAGGGIGTAGSPWSKRWDQIYKRY

SKHIEDAKRNRKAGTKNCGTSSTTNAAASTDENKCVQSDIDSFFKHLIDIGLTTPSSYL

SNVLDDNICGADKAPWTTYTTYTTTEKCNKERDKSKSQSSDTLVVVNVPSPLGNTPY

RYKYACQCKIPTNEETCDDRKEYMNQWSCGSARTMKRGYKNDNYELCKYNGVDV

KPTTVRSNSSKLD

>SpyTag-DBL1-ID2a DNA
SEQ ID NO: 8
atgGCTCACATCGTGATGGTGGACGCTTACAAGCCCACCAAGAACAAGATCGAGG

AATATCTGGGAGCTAAGTCCGATGACAGCAAGATCGACGAACTGCTGAAGGCCG

ATCCTAGCGAAGTGGAGTACTACAGAAGCGGAGGCGACGGCGACTACCTGAAGA

ACAACATCTGCAAGATCACCGTGAACCACAGCGATAGCGGCAAGTATGACCCCT

GCGAGAAGAAGCTGCCCCCCTACGACGACAACGACCAGTGGAAGTGCCAGCAGA

ACAGCAGCGACGGCAGCGGCAAGCCCGAGAACATCTGCGTGCCCCCCAGACGGG

AGCGGCTGTGCACCTACAACCTGGAAAACCTGAAGTTCGACAAGATCCGGGACA

ACAACGCCTTCCTGGCCGACGTGCTGCTGACCGCCCGGAACGAGGGCGAGAAGA

TCGTGCAGAACCACCCCGACACCAACAGCAGCAACGTGTGCAACGCCCTGGAAC

GGTCCTTCGCTGACCTGGCTGACATCATCCGGGGCACCGATCAGTGGAAGGGCAC

CAACTCCAATCTGGAAAAGAACCTGAAGCAGATGTTCGCCAAGATCAGAGAAAA

CGACAAGGTGCTGCAGGACAAGTACCCCAAGGACCAGAAGTACACCAAGCTGCG

GGAGGCCTGGTGGAACGCCAACCGGCAGAAAGTGTGGGAAGTGATCACCTGTGG

CGCCAGAAGCAACGATCTGCTGATCAAGCGGGGCTGGCGGACCAGCGGCAAGAG

CGACCGGAAGAAAAACTTCGAGCTGTGCCGGAAGTGCGGCCACTACGAGAAAGA

GGTGCCCACCAAGCTGGACTACGTGCCCCAGTTCCTGCGGTGGCTGACCGAGTGG

ATCGAGGACTTCTACCGGGAGAAGCAGAACCTGATCGACGACATGGAACGGCAC

CGGGAGGAATGCACCAGAGAGGACCACAAGAGCAAAGAGGGCACCAGCTACTG

CAGCACATGCAAGGACAAGTGCAAGAAATACTGCGAGTGCGTGAAGAAATGGAA

AACCGAGTGGGAGAACCAGGAAAACAAGTACAAGGACCTGTACGAGCAGAACA

AGAACAAGACCAGCCAGAAGAACACCAGCAGATACGACGACTACGTGAAGGACT

TCTTCGAGAAGCTGGAAGCCAACTACAGCAGCCTGGAAAACTACATCAAGGGCG

ACCCCTATTTCGCTGAGTACGCTACAAAACTGAGCTTCATCCTGAACCCCAGCGA

CGCCAACAACCCCAGCGGCGAGACAGCCAACCACAACGACGAGGCCTGCAACTG

CAACGAGAGCGGCATCAGCAGCGTGGGCCAGGCTCAGACATCCGGCCCTAGCAG

CAACAAGACCTGTATCACCCACAGCTCCATCAAGACCAACAAGAAAAAAGAATG

CAAGGACGTGAAGCTGGGCGTGCGGGAGAACGACAAGGATCTGAAGATCTGCGT

GATCGAGGACACCAGCCTGAGCGGCGTGGACAACTGCTGCTGCCAGGATCTGCT

GGGCATCCTGCAGGAAAACTGCAGCGACAACAAGCGGGGCAGCAGCTCCAACGA

CAGCTGCGACAATAAGAACCAGGACGAGTGCCAGAAAAAGCTGGAAAAGGTGTT

CGCCAGCCTGACCAACGGCTACAAGTGCGATAAGTGCAAGAGCGGCACCTCCCG

GTCCAAGAAGAAGTGGATCTGGAAGAAGTCCAGCGGCAACGAGGAAGGCCTGCA

GGAAGAGTACGCCAACACCATCGGCCTGCCCCCCAGGACCCAGAGCCTGTACCT

GGGCAATCTGCCCAAACTGGAAAACGTGTGCGAGGATGTGAAGGACATCAACTT

CGACACCAAAGAGAAGTTTCTGGCCGGCTGCCTGATCGTGTCCTTCCACGAGGGC

AAGAATCTGAAGAAGCGCTACCCCCAGAATAAGAACAGCGGCAACAAAGAAAA

CCTGTGCAAGGCTCTGGAATACAGCTTCGCCGACTACGGCGACCTGATCAAGGGC

ACCTCCATCTGGGACAACGAGTACACAAAGGACCTGGAACTGAATCTGCAGAAC

AACTTCGGCAAGCTGTTCGGCAAGTACATCAAGAAGAACAATACCGCCGAGCAG

GACACCTCCTACAGCTCCCTGGACGAGCTGCGCGAGTCTTGGTGGAATACCAATA

AGAAGTACATCTGGACCGCCATGAAGCACGGCGCCGAGATGAACATCACCACCT

GTAACGCCGACGGCTCCGTGACCGGCAGCGGCTCCAGCTGCGACGACATCCCCAC

CATCGACCTGATCCCCCAGTACCTGAGATTTCTGCAGGAATGGGTCGAGAACTTC

TGCGAGCAGCGGCAGGCCAAAGTGAAGGACGTGATCACCAACTGCAAGAGCTGC

AAAGAATCCGGCAACAAATGCAAGACCGAGTGCAAAACCAAGTGCAAGGATGA

GTGCGAGAAGTACAAGAAGTTCATCGAGGCCTGCGGCACAGCCGGCGGAGGCAT

CGGAACAGCCGGCAGCCCCTGGTCCAAGAGATGGGACCAGATCTACAAGCGGTA

CAGCAAGCACATCGAGGACGCCAAGCGGAACCGGAAGGCCGGCACCAAGAACT

GCGGCACCAGCTCCACCACCAACGCCGCTGCCAGCACCGACGAGAATAAGTGCG

TGCAGAGCGACATCGACAGCTTTTTCAAGCACCTGATCGATATCGGCCTGACCAC

CCCCAGCAGCTACCTGAGCAACGTGCTGGACGACAACATCTGTGGCGCCGACAA

GGCCCCCTGGACAACCTATACAACATACACTACAACCGAGAAGTGCAACAAAGA

GCGGGACAAGAGCAAGAGCCAGAGCAGCGACACCCTGGTGGTGGTGAACGTGCC

CAGCCCCCTGGGCAACACACCCTACCGGTACAAGTACGCCTGCCAGTGCAAGATC

CCCACCAACGAGGAAACATGCGACGACCGGAAAGAATACATGAACCAGTGGTCC

TGCGGGAGCGCTCGGACCATGAAGAGAGGGTATAAGAACGATAACTACGAACTG

TGCAAGTACAACGGCGTGGATGTGAAGCCCACCACCGTGCGGAGCAACTCCAGC

AAGCTGGAC

>PD-L1-SpyTag
SEQ ID NO: 9
FTITAPKDLYVVEYGSNVTMECRFPVERELDLLALVVYWEKEDEQVIQFVAGEEDLK

PQHSNFRGRASLPKDQLLKGNAALQITDVKLQDAGVYCCIISYGGADYKRITLKVNA

PYRKINQRISVDPATSEHELICQAEGYPEAEVIWTNSDHQPVSGKRSVTTSRTEGMLL

NVTSSLRVNATANDVFYCTFWRSQPGQNHTAELIIPELPATHPPQNRTHGGSAHIVM

VDAYKPTK

>PD-L1-SpyTag DNA
SEQ ID NO: 10
TTCACCATCACCGCTCCCAAGGACCTGTACGTGGTCGAGTACGGTTCCAACGTGA

CAATGGAATGCCGTTTCCCCGTCGAGCGCGAGCTGGACCTGTTGGCTTTGGTGGT

GTACTGGGAGAAGGAAGATGAGCAAGTCATCCAGTTCGTGGCTGGCGAAGAGGA

CCTGAAGCCCCAGCACTCCAACTTCCGTGGTCGTGCTTCCCTGCCTAAGGACCAG

CTGCTGAAGGGCAACGCTGCTCTGCAGATCACCGACGTGAAGCTGCAGGACGCT

GGTGTCTACTGCTGCATCATCTCCTACGGTGGTGCTGACTACAAGCGTATCACCCT

CAAAGTGAACGCTCCCTACCGCAAGATCAACCAGCGCATCTCCGTGGACCCCGCT

ACCTCTGAGCACGAGCTGATCTGCCAGGCTGAGGGTTACCCCGAGGCTGAAGTGA

TCTGGACCAACTCCGACCACCAGCCCGTGTCCGGAAAGCGTTCCGTGACCACCTC

TCGTACCGAGGGCATGCTGCTGAACGTGACCTCCTCCCTGCGTGTGAACGCTACC

GCTAACGACGTGTTCTACTGCACCTTCTGGCGTTCCCAGCCCGGCCAGAACCACA

CCGCTGAGCTGATCATCCCCGAGCTGCCTGCTACCCACCCCCCTCAAAACCGTAC

CCACGGTGGTTCCGCTCACATCGTGATGGTGGACGCTTACAAGCCCACTAAATAA

>CTLA-4-spyTag
SEQ ID NO: 11
EAIQVTQPSVVLASSHGVASFPCEYSPSHNTDEVRVTVLRQTNDQMTEVCATTFTEK

NTVGFLDYPFCSGTFNESRVNLTIQGLRAVDTGLYLCKVELMYPPPYFVGMGNGTQI

YVIDPEPSPDSDGGSAHIVMVDAYKPTK

>CTLA-4-spyTag DNA
SEQ ID NO: 12
GAGGCTATCCAAGTGACCCAGCCCTCCGTGGTGCTGGCTTCCTCTCACGGTGTTG

CCAGCTTCCCTTGCGAGTACTCCCCCTCCCACAACACCGACGAAGTGCGTGTGAC

CGTGCTGCGTCAGACCAACGACCAGATGACCGAAGTGTGCGCTACCACCTTCACC

GAGAAGAACACCGTCGGTTTCTTGGACTACCCCTTCTGCTCCGGCACCTTCAACG

AGTCCCGTGTGAACCTGACCATCCAGGGCCTGCGTGCTGTGGACACCGGACTGTA

CCTGTGCAAGGTCGAGCTGATGTACCCTCCCCCCTACTTCGTGGGCATGGGCAAC

GGCACCCAGATCTACGTGATCGACCCCGAGCCTTCCCCCGACTCTGACGGTGGTT

CTGCTCACATCGTGATGGTGGACGCTTACAAGCCCACTAAATAA

>AMA1-SpyTag
SEQ ID NO: 13
QNYWEHPYQNSDVYRPINEHREHPKEYEYPLHQEHTYQQEDSGEDENTLQHAYPID

HEGAEPAPQEQNLFSSIEIVERSNYMGNPWTEYMAKYDIEEVHGSGIRVDLGEDAEV

AGTQYRLPSGKCPVFGKGIIIENSNTTFLTPVATGNQYLKDGGFAFPPTEPLMSPMTLD

EMRHFYKDNKYVKNLDELTLCSRHAGNMIPDNDKNSNYKYPAVYDDKDKKCHILYI

AAQENNGPRYCNKDESKRNSMFCFRPAKDISFQNYTYLSKNVVDNWEKVCPRKNLQ

NAKFGLWVDGNCEDIPHVNEFPAIDLFECNKLVFELSASDQPKQYEQHLTDYEKIKE

GFKNKNASMIKSAFLPTGAFKADRYKSHGKGYNWGNYNTETQKCEIFNVKPTCLIN

NSSYIATTALSHPIEVENNFPCSLYKDEIMKEIERESKRIKLNDNDDEGNKKIIAPRIFIS

DDKDSLKCPCDPEMVSNSTCRFFVCKCVERRAEVTSNNEVVVKEEYKDEYADIPEHK

PTYDKMKGGSGAHIVMVDAYKPTK

>SpyTag-L-Der p2
SEQ ID NO: 14
AHIVMVDAYKPTKGGSDQVDVKDCANHEIKKVLVPGCHGSEPCIIHRGKPFQLEAVF

EANQNSKTAKIEIKASIDGLEVDVPGIDPNACHYMKCPLVKGQQYDIKYTWNVPKIA

PKSENVVVTVKVMGDDGVLACAIATHAKIRDAS

>SpyTag-L-Der p2 DNA
SEQ ID NO: 15
GCTCACATCGTGATGGTGGACGCTTACAAGCCCACCAAGGGTggatccGATCAAGTC

GATGTCAAAGATTGTGCCAATCATGAAATCAAAAAAGTTTTGGTACCAGGATGCC

ATGGTTCAGAACCATGTATCATTCATCGTGGTAAACCATTCCAATTGGAAGCCGT

TTTCGAAGCCAACCAAAACTCAAAAACCGCTAAAATTGAAATCAAAGCTTCAATC

GATGGTTTAGAAGTTGATGTTCCCGGTATCGATCCAAATGCATGCCATTATATGA

AATGTCCATTGGTTAAAGGACAACAATATGATATTAAATATACATGGAATGTTCC

GAAAATTGCACCAAAATCTGAAAATGTTGTCGTCACTGTCAAAGTTATGGGTGAT

GATGGTGTTTTGGCCTGTGCTATTGCTACTCATGCTAAAATCCGCGATgctagc

>miniHAstem-HIS-SpyTag
SEQ ID NO: 16
MKVKLLVLLCTFTATYADTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENGGGG

KYVCSAKLRMVTGLRNKPSKQSQGLFGAIAGFTEGGWTGMVDGWYGYHHQNEQG

SGYAADQKSTQNAINGITNKVNSVIEKMNTQYTAIGCEYNKSERCMKQIEDKIEEIES

KIWTYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCN

DECMESVKNGTYDYPKYSEESKLNREKIDGVKLESMGVYQIEGHHHHHHHGGAHIV

MVDAYKPTK

>miniHAstem-HIS-SpyTag DNA
SEQ ID NO: 17
ATGAAAGTGAAGCTGCTGGTGCTGCTGTGCACCTTCACCGCCACCTACGCCGACA

CCATCTGCATCGGCTACCACGCCAACAACAGCACCGACACCGTGGATACCGTGCT

GGAAAAGAACGTGACCGTGACCCACAGCGTGAACCTGCTGGAAAATGGCGGCGG

AGGCAAATACGTGTGCAGCGCCAAGCTGCGGATGGTCACCGGCCTGAGAAACAA

GCCCAGCAAGCAGAGCCAGGGCCTGTTCGGAGCCATTGCCGGCTTTACAGAGGG

CGGCTGGACCGGCATGGTGGATGGGTGGTACGGCTATCACCACCAGAACGAGCA

GGGCAGCGGCTACGCCGCCGATCAGAAGTCTACCCAGAACGCCATCAACGGCAT

CACCAACAAAGTGAACAGCGTGATCGAGAAGATGAACACCCAGTACACCGCCAT

CGGCTGCGAGTACAACAAGAGCGAGCGGTGCATGAAGCAGATCGAGGACAAGAT

CGAAGAGATCGAGTCTAAGATCTGGACCTACAACGCCGAACTGCTGGTGCTGCTG

GAAAACGAGCGGACCCTGGACTTCCACGACAGCAACGTGAAGAACCTGTACGAG

AAAGTGAAAAGCCAGCTGAAGAACAACGCCAAAGAGATCGGCAACGGCTGCTTC

GAGTTCTACCACAAGTGCAACGACGAGTGCATGGAAAGCGTGAAGAATGGCACC

TACGACTACCCCAAGTACAGCGAGGAAAGCAAGCTGAACCGCGAGAAGATCGAC

GGCGTGAAGCTGGAATCTATGGGCGTGTACCAGATTGAGGGCCACCACCATCACC

ATCATCACGGCGGAGCCCACATCGTGATGGTGGACGCCTACAAGCCCACCAAAT

AA

>A1>SpyCatcher-Her2-ECD\|23-686
SEQ ID NO: 18
GAMVDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDEDGKELAGATMELRDSSGKTIS

TWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGKATKGDA

HIGGSTQVCTGTDMKLRLPASPETHLDMLRHLYQGCQVVQGNLELTYLPTNASLSFL

QDIQEVQGYVLIAHNQVRQVPLQRLRIVRGTQLFEDNYALAVLDNGDPLNNTTPVTG

ASPGGLRELQLRSLTEILKGGVLIQRNPQLCYQDTILWKDIFHKNNQLALTLIDTNRSR

ACHPCSPMCKGSRCWGESSEDCQSLTRTVCAGGCARCKGPLPTDCCHEQCAAGCTG

PKHSDCLACLHFNHSGICELHCPALVTYNTDTFESMPNPEGRYTFGASCVTACPYNY

LSTDVGSCTLVCPLHNQEVTAEDGTQRCEKCSKPCARVCYGLGMEHLREVRAVTSA

NIQEFAGCKKIFGSLAFLPESFDGDPASNTAPLQPEQLQVFETLEEITGYLYISAWPDSL

PDLSVFQNLQVIRGRILHNGAYSLTLQGLGISWLGLRSLRELGSGLALIHHNTHLCFV

HTVPWDQLFRNPHQALLHTANRPEDECVGEGLACHQLCARGHCWGPGPTQCVNCS

QFLRGQECVEECRVLQGLPREYVNARHCLPCHPECQPQNGSVTCFGPEADQCVACA

HYKDPPFCVARCPSGVKPDLSYMPIWKFPDEEGACQPCPINCTHSCVDLDDKGCPAE

QRASPLTSIISAVVGILLVVVLGVVFGILIKRRQQKIRKYTHHHHHH

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

TWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGKATKGDA

HIGGSIPTEIPTSALVKETLALLSTHRTLLIANETLRIPVPVHKNHQLTTEEIFQGIGTLES

QTVQGGTVERLFKNLSLIKKYIDGQKKKTGEERRRVNQFLDYLQEFLGVMNTEWIIE

S*SGRK

>A3>PCSK9\|31-692\|:SpyCatcher:HIS
SEQ ID NO: 20
QEDEDGDYEELVLALRSEEDGLAEAPEHGTTATFHRCAKDPWRLPGTYVVVLKEET

HLSQSERTARRLQAQAARRGYLTKILHVFHGLLPGFLVKMSGDLLELALKLPHVDYI

EEDSSVFAQSIPWNLERITPPRYRADEYQPPDGGSLVEVYLLDTSIQSDHREIEGRVMV

TDFENVPEEDGTRFHRQASKCDSHGTHLAGVVSGRDAGVAKGASMRSLRVLNCQG

KGTVSGTLIGLEFIRKSQLVQPVGPLVVLLPLAGGYSRVLNAACQRLARAGVVLVTA

AGNFRDDACLYSPASAPEVITVGATNAQDQPVTLGTLGTNFGRCVDLFAPGEDIIGAS

SDCSTCFVSQSGTSQAAAHVAGIAAMMLSAEPELTLAELRQRLIHFSAKDVINEAWFP

EDQRVLTPNLVAALPPSTHGAGWQLFCRTVWSAHSGPTRMATAVARCAPDEELLSC

SSFSRSGKRRGERMEAQGGKLVCRAHNAFGGEGVYAIARCCLLPQANCSVHTAPPA

EASMGTRVHCHQQGHVLTGCSSHWEVEDLGTHKPPVLRPRGQPNQCVGHREASIHA

SCCHAPGLECKVKEHGIPAPQEQVTVACEEGWTLTGCSALPGTSHVLGAYAVDNTC

VVRSRDVSTTGSTSEGAVTAVAICCRSRHLAQASQELQGGSGAMVDTLSGLSSEQGQ

SGDMTIEEDSATHIKFSKRDEDGKELAGATMELRDSSGKTISTWISDGQVKDFYLYPG

KYTFVETAAPDGYEVATAITFTVNEQGQVTVNGKATKGDAHIHHHHHH

>A4>SpyCatcher-IDHD2a-HIS
SEQ ID NO: 21
GAMVDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDEDGKELAGATMELRDSSGKTIS

TWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGKATKGDA

HIGGSNYIKGDPYFAEYATKLSFILNPSDANNPSGETANHNDEACNCNESGISSVGQA

QTSGPSSNKTCITHSSIKTNKKKECKDVKLGVRENDKDLKICVIEDTSLSGVDNCCCQ

DLLGILQENCSDNKRGSSSNDSCDNKNQDECQKKLEKVFASLTNGYKCDKCKSGTSR

SKKKWIWKKSSGNEEGLQEEYANTIGLPPRTQSLYLGNLPKLENVCEDVKDINFDTK

EKFLAGCLIVSFHEGKNLKKRYPQNKNSGNKENLCKALEYSFADYGDLIKGTSIWDN

EYTKDLELNLQNNFGKLFGKYIKKNNTAEQDTSYSSLDELRESWWNTNKKYIWTAM

KHGAEMNITTCNADGSVTGSGSSCDDIPTIDLIPQYLRFLQEWVENFCEQRQAKVKD

VITNCKSCKESGNKCKTECKTKCKDECEKYKKFIEACGTAGGGIGTAGSPWSKRWD

QIYKRYSKHIEDAKRNRKAGTKNCGTSSTTNAAASTDENKCVQSDIDSFFKHLIDIGL

TTPSSYLSNVLDDNICGADKAPWTTYTTYTTTEKCNKERDKSKSQSSDTLVVVNVPS

PLGNTPYRYKYACQCKIPTNEETCDDRKEYMNQWSCGSARTMKRGYKNDNYELCK

YNGVDVKPTTVRSNSSKLDHHHHHH

>A5>SpyCatcher-RO-HIS
SEQ ID NO: 22
GAMVDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDEDGKELAGATMELRDSSGKTIS

TWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGKATKGDA

HIGGSTSENRNKRIGGPKLRGNVTSNIKFPSDNKGKIIRGSNDKLNKNSEDVLEQSEKS

LVSENVPSGLDIDDIPKESIFIQEDQEGQTHSELNPETSEHSKDLNNNGSKNESSDIISEN

NKSNKVQNHFESLSDLELLENSSQDNLDKDTISTEPFPNQKHKDLQQDLNDEPLEPFP

TQIHKDYKEKNLINEEDSEPFPRQKHKKVDNHNEEKNVFHENGSANGNQGSLKLKSF

DEHLKDEKIENEPLVHENLSIPNDPIEQILNQPEQETNIQEQLYNEKQNVEEKQNSQIPS

LDLKEPTNEDILPNHNPLENIKQSESEINHVQDHALPKENIIDKLDNQKEHIDQSQHNI

NVLQENNINNHQLEPQEKPNIESFEPKNIDSEIILPENVETEEIIDDVPSPKHSNHETFEE

ETSESEHEEAVSEKNAHETVEHEETVSQESNPEKADNDGNVSQNSNNELNENEFVES

EKSEHEARSKAKEASSYDYILGWEFGGGVPEHKKEENMLSHLYVSSKDKENISKEND

DVLDEKEEEAEETEEEELEEKNEEETESEISEDEEEEEEEEEKEEENDKKKEQEKEQSN

ENNDQKKDMEAQNLISKNQNNNEKNVKEAAESIMKTLAGLIKGNNQIDSTLKDLVE

ELSKYFKNHRSHHHHHH

>A6>HIS-RO-SpyCatcher
SEQ ID NO: 23
GSTSENRNKRIGGPKLRGNVTSNIKFPSDNKGKIIRGSNDKLNKNSEDVLEQSEKSLVS

ENVPSGLDIDDIPKESIFIQEDQEGQTHSELNPETSEHSKDLNNNGSKNESSDIISENNK

SNKVQNHFESLSDLELLENSSQDNLDKDTISTEPFPNQKHKDLQQDLNDEPLEPFPTQI

HKDYKEKNLINEEDSEPFPRQKHKKVDNHNEEKNVFHENGSANGNQGSLKLKSFDE

HLKDEKIENEPLVHENLSIPNDPIEQILNQPEQETNIQEQLYNEKQNVEEKQNSQIPSLD

LKEPTNEDILPNHNPLENIKQSESEINHVQDHALPKENIIDKLDNQKEHIDQSQHNINV

LQENNINNHQLEPQEKPNIESFEPKNIDSEIILPENVETEEIIDDVPSPKHSNHETFEEETS

ESEHEEAVSEKNAHETVEHEETVSQESNPEKADNDGNVSQNSNNELNENEFVESEKS

EHEAGGSGAMVDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDEDGKELAGATMELR

DSSGKTISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNG

KATKGDAHI

>A7>HIS-GMZ2ggsSpyCatcher
SEQ ID NO: 24
GSTSENRNKRIGGPKLRGNVTSNIKFPSDNKGKIIRGSNDKLNKNSEDVLEQSEKSLVS

ENVPSGLDIDDIPKESIFIQEDQEGQTHSELNPETSEHSKDLNNNGSKNESSDIISENNK

SNKVQNHFESLSDLELLENSSQDNLDKDTISTEPFPNQKHKDLQQDLNDEPLEPFPTQI

HKDYKEKNLINEEDSEPFPRQKHKKVDNHNEEKNVFHENGSANGNQGSLKLKSFDE

HLKDEKIENEPLVHENLSIPNDPIEQILNQPEQETNIQEQLYNEKQNVEEKQNSQIPSLD

LKEPTNEDILPNHNPLENIKQSESEINHVQDHALPKENIIDKLDNQKEHIDQSQHNINV

LQENNINNHQLEPQEKPNIESFEPKNIDSEIILPENVETEEIIDDVPSPKHSNHETFEEETS

ESEHEEAVSEKNAHETVEHEETVSQESNPEKADNDGNVSQNSNNELNENEFVESEKS

EHEARSKAKEASSYDYILGWEFGGGVPEHKKEENMLSHLYVSSKDKENISKENDDVL

DEKEEEAEETEEEELEEKNEEETESEISEDEEEEEEEEEKEEENDKKKEQEKEQSNENN

DQKKDMEAQNLISKNQNNNEKNVKEAAESIMKTLAGLIKGNNQIDSTLKDLVEELSK

YFKNHGGSGAMVDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDEDGKELAGATMEL

RDSSGKTISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVN

GKATKGDAHI

>A8>HIS-GMZ2T:ggsSpyCatcher
SEQ ID NO: 25
GSTSENRNKRIGGPKLRGNVTSNIKFPSDNKGKIIRGSNDKLNKNSEDVLEQSEKSLVS

ENVPSGLDIDDIPKESIFIQEDQEGQTHSELNPETSEHSKDLNNNGSKNESSDIISENNK

SNKVQNHFESLSDLELLENSSQDNLDKDTISTEPFPNQKHKDLQQDLNDEPLEPFPTQI

HKDYKEKNLINEEDSEPFPRQKHKKVDNHNEEKNVFHENGSANGNQGSLKLKSFDE

HLKDEKIENEPLVHENLSIPNDPIEQILNQPEQETNIQEQLYNEKQNVEEKQNSQIPSLD

LKEPTNEDILPNHNPLENIKQSESEINHVQDHALPKENIIDKLDNQKEHIDQSQHNINV

LQENNINNHQLEPQEKPNIESFEPKNIDSEIILPENVETEEIIDDVPSPKHSNHETFEEETS

ESEHEEAVSEKNAHETVEHEETVSQESNPEKADNDGNVSQNSNNELNENEFVESEKS

EHEARSKTKEYAEKAKNAYEKAKNAYQKANQAVLKAKEASSYDYILGWEFGGGVP

EHKKEENMLSHLYVSSKDKENISKENDDVLDEKEEEAEETEEEELEGGSGAMVDTLS

GLSSEQGQSGDMTIEEDSATHIKFSKRDEDGKELAGATMELRDSSGKTISTWISDGQV

KDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGKATKGDAHI

>A9>SpyCatcher-PfRH5-HIS
SEQ ID NO: 26
GAMVDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDEDGKELAGATMELRDSSGKTIS

TWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGKATKGDA

HIGGSLSFENAIKKTKNQENNLTLLPIKSTEEEKDDIKNGKDIKKEIDNDKENIKTNNA

KDHSTYIKSYLNTNVNDGLKYLFIPSHNSFIKKYSVFNQINDGMLLNEKNDVKNNED

YKNVDYKNVNFLQYHFKELSNYNIANSIDILQEKEGHLDFVIIPHYTFLDYYKHLSYN

SIYHKYSTYGKYIAVDAFIKKINETYDKVKSKCNDIKNDLIATIKKLEHPYDINNKND

DSYRYDISEEIDDKSEETDDETEEVEDSIQDTDSNHTPSNKKKNDLMNRTFKKMMDE

YNTKKKKLIKCIKNHENDFNKICMDMKNYGTNLFEQLSCYNNNFCNTNGIRFHYDE

YIHKLILSVKSKNLNKDLSDMTNILQQSELLLTNLNKKMGSYIYIDTIKFIHKEMKHIF

NRIEYHTKIINDKTKIIQDKIKLNIWRTFQKDELLKRILDMSNEYSLFITSDHLRQMLYN

TFYSKEKHLNNIFHHLIYVLQMKFNDVPIKMEYFQTYKKNKPLTQHHHHHH

>A10>SpyCatcher-Pfs25-HIS
SEQ ID NO: 27
GAMVDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDEDGKELAGATMELRDSSGKTIS

TWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGKATKGDA

HIGGSNKLYSLFLFLFIQLSIKYNNAKVTVDTVCKRGFLIQMSGHLECKCENDLVLVN

EETCEEKVLKCDEKTVNKPCGDFSKCIKIDGNPVSYACKCNLGYDMVNNVCIPNECK

NVTCGNGKCILDTSNPVKTGVCSCNIGKVPNVQDQNKCSKDGETKCSLKCLKENETC

KAVDGIYKCDCKDGFIIDNESSICTAFSAYNILNLSIMFILFSVCFFIM

>A11>HIS-PfCSP(aa92-397)-SpyCatcher
SEQ ID NO: 28
KLKQPADGNPDPNANPNVDPNANPNVDPNANPNVDPNANPNANPNANPNANPNAN

PNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNVDPNA

NPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPN

ANPNANPNANPNANPNKNNQGNGQGHNMPNDPNRNVDENANANSAVKNNNNEEP

SDKHIKEYLNKIQNSLSTEWSPCSVTCGNGIQVRIKPGSANKPKDELDYANDIEKKICK

MEKCSSVFNVVNSSIGLIMVLSFLFLNGGSGAMVDTLSGLSSEQGQSGDMTIEEDSAT

HIKFSKRDEDGKELAGATMELRDSSGKTISTWISDGQVKDFYLYPGKYTFVETAAPD

GYEVATAITFTVNEQGQVTVNGKATKGDAHI

>A3>PCSK9\|31-692\|:SpyCatcher:HIS DNA
SEQ ID NO: 29
TTTCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCT

GGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTAC

GTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGC

GTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAA

TGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAAC

TCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAA

GCAGAGCTCTCTGGCTAACTAGAGAACCCACTGCTTACTGGCTTATCGAAATTAA

TACGACTCACTATAGGGAGACCCAAGCTGGCTAGCGTTTAAACTTAAGCTTAGCG

CAGAGGCTTGGGGCAGCCGAGCGGCAGCCAGGCCCCGGCCCGGGCCTCGGTTCC

AGAAGGGAGAGGAGCCCGCCAAGGCGCGCAAGAGAGCGGGCTGCCTCGCAGTCC

GAGCCGGAGAGGGAGCGCGAGCCGCGCCGGCCCCGGACGGCCTCCGAAACCATG

CAGGAAGATGAGGACGGCGACTACGAGGAACTGGTGCTGGCCCTGCGGAGCGAA

GAGGATGGACTGGCCGAGGCCCCTGAGCACGGCACCACCGCCACCTTCCACAGA

TGCGCCAAGGACCCTTGGCGGCTGCCCGGCACATACGTGGTGGTGCTGAAAGAG

GAAACCCACCTGAGCCAGAGCGAGCGGACCGCCAGAAGGCTGCAGGCCCAGGCC

GCCAGAAGAGGCTACCTGACCAAGATCCTGCACGTGTTCCACGGCCTGCTGCCCG

GCTTCCTGGTGAAAATGAGCGGCGACCTGCTGGAACTGGCCCTGAAGCTGCCCCA

CGTGGACTACATCGAAGAGGACAGCAGCGTGTTCGCCCAGAGCATCCCCTGGAA

CCTGGAACGGATCACCCCCCCCAGATACCGGGCCGACGAGTACCAGCCTCCTGAC

GGCGGCAGCCTGGTGGAAGTGTACCTGCTGGACACCAGCATCCAGAGCGACCAC

CGCGAGATCGAGGGCAGAGTGATGGTGACAGACTTCGAGAACGTGCCCGAAGAG

GACGGCACCCGGTTCCACAGACAGGCCAGCAAGTGCGACAGCCACGGCACACAT

CTGGCCGGCGTGGTGTCTGGCAGAGATGCCGGCGTGGCCAAGGGCGCCAGCATG

AGAAGCCTGCGGGTGCTGAACTGCCAGGGCAAGGGCACCGTGTCCGGCACCCTG

ATCGGCCTGGAATTCATCCGGAAGTCCCAGCTGGTGCAGCCCGTGGGCCCTCTGG

TGGTGCTGCTGCCTCTGGCTGGCGGCTACAGCAGAGTGCTGAACGCCGCCTGCCA

GAGACTGGCCAGAGCTGGCGTGGTGCTGGTGACAGCCGCCGGAAACTTCCGGGA

CGACGCCTGCCTGTACAGCCCCGCCTCTGCCCCCGAAGTGATCACCGTGGGCGCC

ACCAACGCCCAGGACCAGCCTGTGACACTGGGCACCCTGGGCACAAACTTCGGC

AGATGCGTGGACCTGTTCGCCCCTGGCGAGGACATCATCGGCGCCAGCAGCGACT

GCAGCACCTGTTTCGTGTCCCAGAGCGGCACCAGCCAGGCCGCTGCCCATGTGGC

CGGAATCGCCGCCATGATGCTGAGCGCCGAGCCTGAGCTGACCCTGGCCGAGCTG

CGGCAGCGGCTGATCCACTTCTCCGCCAAGGACGTGATCAACGAGGCCTGGTTCC

CCGAGGACCAGAGAGTGCTGACCCCCAACCTGGTGGCCGCCCTGCCTCCTTCTAC

ACACGGCGCTGGCTGGCAGCTGTTCTGCAGGACAGTGTGGTCCGCCCACAGCGGC

CCCACCAGAATGGCCACAGCCGTGGCCAGATGCGCCCCTGATGAGGAACTGCTG

AGCTGCAGCAGCTTCTCCAGAAGCGGCAAGCGGAGAGGCGAGCGGATGGAAGCC

CAGGGCGGCAAGCTCGTGTGCAGAGCCCACAATGCCTTCGGCGGCGAGGGCGTG

TACGCCATTGCCAGATGCTGCCTGCTGCCTCAGGCCAACTGCAGCGTGCACACAG

CCCCTCCAGCCGAGGCCAGCATGGGCACCAGAGTGCACTGCCACCAGCAGGGCC

ACGTGCTGACCGGCTGTAGCAGCCACTGGGAGGTGGAAGATCTGGGCACCCACA

AGCCCCCCGTGCTGAGGCCCAGAGGCCAGCCTAATCAGTGCGTGGGCCACAGAG

AGGCCTCCATCCACGCCAGCTGTTGCCACGCCCCTGGCCTGGAATGCAAAGTGAA

AGAGCACGGCATCCCTGCCCCCCAGGAACAGGTCACAGTGGCCTGCGAGGAAGG

CTGGACCCTGACAGGCTGTTCCGCCCTGCCAGGCACCTCTCACGTGCTGGGCGCC

TACGCCGTGGACAATACCTGCGTCGTGCGCAGCCGGGACGTGTCCACAACCGGCT

CTACAAGCGAGGGCGCCGTGACCGCCGTGGCCATCTGCTGCAGAAGCAGACACC

TGGCCCAGGCCTCCCAGGAACTGCAGGGCGGATCTGGTGCAATGGTTGATACCCT

GAGCGGTCTGAGCAGCGAACAGGGTCAGAGCGGTGATATGACCATTGAAGAAGA

TAGCGCAACCCACATCAAATTCAGCAAACGTGATGAAGATGGTAAAGAACTGGC

AGGCGCAACAATGGAACTGCGTGATAGCAGCGGTAAAACCATTAGCACCTGGAT

TAGTGATGGTCAGGTGAAAGATTTTTATCTGTACCCTGGCAAATACACCTTTGTTG

AAACCGCAGCACCGGATGGTTATGAAGTTGCAACCGCAATTACCTTTACCGTTAA

TGAACAGGGCCAGGTTACCGTGAATGGTAAAGCAACCAAAGGTGATGCACATAT

TCACCACCACCATCACCACTAAGCGGCCGCTTTT

>A4>SpyCatcher-ggs-ID1ID2a-HIS DNA
SEQ ID NO: 30
GGTGCAATGGTTGATACCCTGAGCGGTCTGAGCAGCGAACAGGGTCAGAGCGGT

GATATGACCATTGAAGAAGATAGCGCAACCCACATCAAATTCAGCAAACGTGAT

GAAGATGGTAAAGAACTGGCAGGCGCAACAATGGAACTGCGTGATAGCAGCGGT

AAAACCATTAGCACCTGGATTAGTGATGGTCAGGTGAAAGATTTTTATCTGTACC

CTGGCAAATACACCTTTGTTGAAACCGCAGCACCGGATGGTTATGAAGTTGCAAC

CGCAATTACCTTTACCGTTAATGAACAGGGCCAGGTTACCGTGAATGGTAAAGCA

ACCAAAGGTGATGCACATATTGGTGGTAGCGGTCCGGCAACCGAACAGGGTCAG

GATACCTTTACCAAAGTTAAAGGTGGCAGCAACTATATCAAAGGCGATCCGTATT

TTGCAGAGTATGCAACCAAACTGAGCTTTATTCTGAATCCGAGTGATGCAAATAA

TCCGAGCGGTGAAACCGCAAATCACAATGATGAAGCCTGTAATTGTAACGAAAG

CGGTATTAGCAGCGTTGGTCAGGCACAGACCAGCGGTCCGAGCAGCAATAAAAC

CTGTATTACCCATAGCAGCATTAAAACCAATAAAAAGAAAGAATGCAAAGATGT

GAAACTGGGCGTGCGCGAAAATGATAAAGATCTGAAAATTTGCGTGATCGAGGA

TACCAGCCTGAGCGGTGTTGATAATTGTTGTTGTCAGGATCTGCTGGGTATTCTGC

AAGAAAATTGCAGCGATAATAAACGTGGTAGCAGCAGCAATGATAGCTGCGATA

ACAAAAATCAGGATGAATGCCAGAAAAAACTGGAAAAAGTTTTTGCCAGCCTGA

CGAATGGTTACAAATGCGATAAATGTAAAAGCGGCACCAGCCGCAGCAAAAAGA

AATGGATTTGGAAAAAAAGCAGCGGCAATGAAGAAGGTCTGCAAGAGGAATATG

CAAATACCATTGGTCTGCCTCCGCGTACCCAGAGCCTGTATCTGGGTAATCTGCC

GAAACTGGAAAATGTGTGTGAAGATGTGAAAGATATCAATTTTGATACCAAAGA

AAAATTTCTGGCAGGCTGCCTGATTGTGAGCTTTCATGAAGGTAAAAACCTGAAA

AAACGCTATCCGCAGAATAAAAACAGCGGTAACAAAGAAAATCTGTGCAAAGCA

CTGGAATACAGCTTTGCAGATTATGGCGATCTGATTAAAGGCACCAGCATTTGGG

ATAACGAGTATACCAAAGATCTGGAACTGAATCTGCAGAACAATTTCGGTAAACT

GTTCGGCAAATATATCAAAAAAAACAATACCGCAGAGCAGGATACCAGCTATAG

CAGCCTGGATGAACTGCGTGAAAGTTGGTGGAATACCAACAAAAAATACATTTG

GACCGCCATGAAACATGGTGCCGAAATGAATATTACCACCTGTAATGCAGATGGT

AGCGTTACCGGTAGCGGTAGCAGCTGTGATGATATTCCGACCATTGATCTGATTC

CGCAGTATCTGCGTTTTCTGCAAGAATGGGTTGAAAACTTTTGTGAACAGCGTCA

GGCGAAAGTGAAAGATGTTATTACCAATTGCAAAAGCTGCAAAGAAAGCGGCAA

TAAATGCAAAACCGAGTGCAAAACCAAATGCAAAGACGAGTGCGAGAAATACAA

AAAATTCATTGAAGCATGTGGTACAGCCGGTGGTGGTATTGGCACCGCAGGTAGC

CCGTGGTCAAAACGTTGGGATCAGATCTATAAACGCTACAGCAAACACATCGAA

GATGCCAAACGTAATCGTAAAGCAGGCACCAAAAATTGTGGCACCAGCAGCACC

ACCAATGCAGCAGCAAGCACCGATGAAAACAAATGTGTTCAGAGCGATATCGAT

AGCTTCTTCAAACATCTGATTGATATTGGTCTGACCACCCCGAGCAGCTATCTGA

GCAATGTTCTGGATGATAACATTTGCGGTGCAGATAAAGCACCGTGGACCACCTA

TACCACATATACCACCACAGAAAAATGCAACAAAGAGCGCGATAAAAGCAAAAG

CCAGAGCAGCGATACCCTGGTTGTTGTTAATGTTCCGAGTCCGCTGGGTAATACC

CCGTATCGTTATAAGTATGCCTGCCAGTGTAAAATCCCGACCAATGAAGAAACCT

GTGATGATCGCAAAGAATACATGAATCAGTGGTCATGTGGTAGCGCACGTACCAT

GAAACGTGGCTATAAAAACGATAATTATGAACTGTGCAAATATAACGGCGTGGA

TGTTAAACCGACCACCGTTCGTAGCAATAGCAGCAAACTGGATCATCATCATCAC

CATCATTAAGGATCC

>A5>SpyCatcher-ggs-RO-HIS DNA
SEQ ID NO: 31
GGTGCAATGGTTGATACCCTGAGCGGTCTGAGCAGCGAACAGGGTCAGAGCGGT

GATATGACCATTGAAGAAGATAGCGCAACCCACATCAAATTCAGCAAACGTGAT

GAAGATGGTAAAGAACTGGCAGGCGCAACAATGGAACTGCGTGATAGCAGCGGT

AAAACCATTAGCACCTGGATTAGTGATGGTCAGGTGAAAGATTTTTATCTGTACC

CTGGCAAATACACCTTTGTTGAAACCGCAGCACCGGATGGTTATGAAGTTGCAAC

CGCAATTACCTTTACCGTTAATGAACAGGGCCAGGTTACCGTGAATGGTAAAGCA

ACCAAAGGTGATGCACATATTGGTGGTAGCACAAGTGAGAATAGAAATAAACGA

ATCGGGGGTCCTAAATTAAGGGGTAATGTTACAAGTAATATAAAGTTCCCATCAG

ATAACAAAGGTAAAATTATAAGAGGTTCGAATGATAAACTTAATAAAAACTCTG

AAGATGTTTTAGAACAAAGCGAAAAATCGCTTGTTTCAGAAAATGTTCCTAGTGG

ATTAGATATAGATGATATCCCTAAAGAATCTATTTTTATTCAAGAAGATCAAGAA

GGTCAAACTCATTCTGAATTAAATCCTGAAACATCAGAACATAGTAAAGATTTAA

ATAATAATGGTTCAAAAAATGAATCTAGTGATATTATTTCAGAAAATAATAAATC

AAATAAAGTACAAAATCATTTTGAATCATTATCAGATTTAGAATTACTTGAAAAT

TCCTCACAAGATAATTTAGACAAAGATACAATTTCAACAGAACCTTTTCCTAATC

AAAAACATAAAGACTTACAACAAGATTTAAATGATGAACCTTTAGAACCCTTTCC

TACACAAATACATAAAGATTATAAAGAAAAAAATTTAATAAATGAAGAAGATTC

AGAACCATTTCCCAGACAAAAGCATAAAAAGGTAGACAATCATAATGAAGAAAA

AAACGTATTTCATGAAAATGGTTCTGCAAATGGTAATCAAGGAAGTTTGAAACTT

AAATCATTCGATGAACATTTAAAAGATGAAAAAATAGAAAATGAACCACTTGTTC

ATGAAAATTTATCCATACCAAATGATCCAATAGAACAAATATTAAATCAACCTGA

ACAAGAAACAAATATCCAGGAACAATTGTATAATGAAAAACAAAATGTTGAAGA

AAAACAAAATTCTCAAATACCTTCGTTAGATTTAAAAGAACCAACAAATGAAGAT

ATTTTACCAAATCATAATCCATTAGAAAATATAAAACAAAGTGAATCAGAAATAA

ATCATGTACAAGATCATGCGCTACCAAAAGAGAATATAATAGACAAACTTGATA

ATCAAAAAGAACACATCGATCAATCACAACATAATATAAATGTATTACAAGAAA

ATAACATAAACAATCACCAATTAGAACCTCAAGAGAAACCTAATATTGAATCGTT

TGAACCTAAAAATATAGATTCAGAAATTATTCTTCCTGAAAATGTTGAAACAGAA

GAAATAATAGATGATGTGCCTTCCCCTAAACATTCTAACCATGAAACATTTGAAG

AAGAAACAAGTGAATCTGAACATGAAGAAGCCGTATCTGAAAAAAATGCCCACG

AAACTGTCGAACATGAAGAAACTGTGTCTCAAGAAAGCAATCCTGAAAAAGCTG

ATAATGATGGAAATGTATCTCAAAACAGCAACAACGAATTAAATGAAAATGAAT

TCGTTGAATCGGAAAAAAGCGAGCATGAAGCAAGATCCAAAGCAAAAGAAGCTT

CTAGTTATGATTATATTTTAGGTTGGGAATTTGGAGGAGGCGTTCCAGAACACAA

AAAAGAAGAAAATATGTTATCACATTTATATGTTTCTTCAAAGGATAAGGAAAAT

ATATCTAAGGAAAATGATGATGTATTAGATGAGAAGGAAGAAGAGGCAGAAGAA

ACAGAAGAAGAAGAACTTGAAGAAAAAAATGAAGAAGAAACAGAATCAGAAAT

AAGTGAAGATGAAGAAGAAGAAGAAGAAGAAGAAGAAAAGGAAGAAGAAAAT

GACAAAAAAAAAGAACAAGAAAAAGAACAAAGTAATGAAAATAATGATCAAAA

AAAAGATATGGAAGCACAGAATTTAATTTCTAAAAACCAGAATAATAATGAGAA

AAACGTAAAAGAAGCTGCTGAAAGCATCATGAAAACTTTAGCTGGTTTAATCAA

GGGAAATAATCAAATAGATTCTACCTTAAAAGATTTAGTAGAAGAATTATCCAAA

TATTTTAAAAATCATAGATCTCATCACCATCATCACCATTAGggatccttt

>A6>HIS-RO-ggs-Spycatcher DNA
SEQ ID NO: 32
GGATCCACAAGTGAGAATAGAAATAAACGAATCGGGGGTCCTAAATTAAGGGGT

AATGTTACAAGTAATATAAAGTTCCCATCAGATAACAAAGGTAAAATTATAAGA

GGTTCGAATGATAAACTTAATAAAAACTCTGAAGATGTTTTAGAACAAAGCGAA

AAATCGCTTGTTTCAGAAAATGTTCCTAGTGGATTAGATATAGATGATATCCCTA

AAGAATCTATTTTTATTCAAGAAGATCAAGAAGGTCAAACTCATTCTGAATTAAA

TCCTGAAACATCAGAACATAGTAAAGATTTAAATAATAATGGTTCAAAAAATGA

ATCTAGTGATATTATTTCAGAAAATAATAAATCAAATAAAGTACAAAATCATTTT

GAATCATTATCAGATTTAGAATTACTTGAAAATTCCTCACAAGATAATTTAGACA

AAGATACAATTTCAACAGAACCTTTTCCTAATCAAAAACATAAAGACTTACAACA

AGATTTAAATGATGAACCTTTAGAACCCTTTCCTACACAAATACATAAAGATTAT

AAAGAAAAAAATTTAATAAATGAAGAAGATTCAGAACCATTTCCCAGACAAAAG

CATAAAAAGGTAGACAATCATAATGAAGAAAAAAACGTATTTCATGAAAATGGT

TCTGCAAATGGTAATCAAGGAAGTTTGAAACTTAAATCATTCGATGAACATTTAA

AAGATGAAAAAATAGAAAATGAACCACTTGTTCATGAAAATTTATCCATACCAA

ATGATCCAATAGAACAAATATTAAATCAACCTGAACAAGAAACAAATATCCAGG

AACAATTGTATAATGAAAAACAAAATGTTGAAGAAAAACAAAATTCTCAAATAC

CTTCGTTAGATTTAAAAGAACCAACAAATGAAGATATTTTACCAAATCATAATCC

ATTAGAAAATATAAAACAAAGTGAATCAGAAATAAATCATGTACAAGATCATGC

GCTACCAAAAGAGAATATAATAGACAAACTTGATAATCAAAAAGAACACATCGA

TCAATCACAACATAATATAAATGTATTACAAGAAAATAACATAAACAATCACCA

ATTAGAACCTCAAGAGAAACCTAATATTGAATCGTTTGAACCTAAAAATATAGAT

TCAGAAATTATTCTTCCTGAAAATGTTGAAACAGAAGAAATAATAGATGATGTGC

CTTCCCCTAAACATTCTAACCATGAAACATTTGAAGAAGAAACAAGTGAATCTGA

ACATGAAGAAGCCGTATCTGAAAAAAATGCCCACGAAACTGTCGAACATGAAGA

AACTGTGTCTCAAGAAAGCAATCCTGAAAAAGCTGATAATGATGGAAATGTATCT

CAAAACAGCAACAACGAATTAAATGAAAATGAATTCGTTGAATCGGAAAAAAGC

GAGCATGAAGCAGGTGGTAGCGGTGCAATGGTTGATACCCTGAGCGGTCTGAGC

AGCGAACAGGGTCAGAGCGGTGATATGACCATTGAAGAAGATAGCGCAACCCAC

ATCAAATTCAGCAAACGTGATGAAGATGGTAAAGAACTGGCAGGCGCAACAATG

GAACTGCGTGATAGCAGCGGTAAAACCATTAGCACCTGGATTAGTGATGGTCAG

GTGAAAGATTTTTATCTGTACCCTGGCAAATACACCTTTGTTGAAACCGCAGCAC

CGGATGGTTATGAAGTTGCAACCGCAATTACCTTTACCGTTAATGAACAGGGCCA

GGTTACCGTGAATGGTAAAGCAACCAAAGGTGATGCACATATT

>A7>HIS-GMZ2ggs-Spycatcher
SEQ ID NO: 33
GGATCCACAAGTGAGAATAGAAATAAACGAATCGGGGGTCCTAAATTAAGGGGT

AATGTTACAAGTAATATAAAGTTCCCATCAGATAACAAAGGTAAAATTATAAGA

GGTTCGAATGATAAACTTAATAAAAACTCTGAAGATGTTTTAGAACAAAGCGAA

AAATCGCTTGTTTCAGAAAATGTTCCTAGTGGATTAGATATAGATGATATCCCTA

AAGAATCTATTTTTATTCAAGAAGATCAAGAAGGTCAAACTCATTCTGAATTAAA

TCCTGAAACATCAGAACATAGTAAAGATTTAAATAATAATGGTTCAAAAAATGA

ATCTAGTGATATTATTTCAGAAAATAATAAATCAAATAAAGTACAAAATCATTTT

GAATCATTATCAGATTTAGAATTACTTGAAAATTCCTCACAAGATAATTTAGACA

AAGATACAATTTCAACAGAACCTTTTCCTAATCAAAAACATAAAGACTTACAACA

AGATTTAAATGATGAACCTTTAGAACCCTTTCCTACACAAATACATAAAGATTAT

AAAGAAAAAAATTTAATAAATGAAGAAGATTCAGAACCATTTCCCAGACAAAAG

CATAAAAAGGTAGACAATCATAATGAAGAAAAAAACGTATTTCATGAAAATGGT

TCTGCAAATGGTAATCAAGGAAGTTTGAAACTTAAATCATTCGATGAACATTTAA

AAGATGAAAAAATAGAAAATGAACCACTTGTTCATGAAAATTTATCCATACCAA

ATGATCCAATAGAACAAATATTAAATCAACCTGAACAAGAAACAAATATCCAGG

AACAATTGTATAATGAAAAACAAAATGTTGAAGAAAAACAAAATTCTCAAATAC

CTTCGTTAGATTTAAAAGAACCAACAAATGAAGATATTTTACCAAATCATAATCC

ATTAGAAAATATAAAACAAAGTGAATCAGAAATAAATCATGTACAAGATCATGC

GCTACCAAAAGAGAATATAATAGACAAACTTGATAATCAAAAAGAACACATCGA

TCAATCACAACATAATATAAATGTATTACAAGAAAATAACATAAACAATCACCA

ATTAGAACCTCAAGAGAAACCTAATATTGAATCGTTTGAACCTAAAAATATAGAT

TCAGAAATTATTCTTCCTGAAAATGTTGAAACAGAAGAAATAATAGATGATGTGC

CTTCCCCTAAACATTCTAACCATGAAACATTTGAAGAAGAAACAAGTGAATCTGA

ACATGAAGAAGCCGTATCTGAAAAAAATGCCCACGAAACTGTCGAACATGAAGA

AACTGTGTCTCAAGAAAGCAATCCTGAAAAAGCTGATAATGATGGAAATGTATCT

CAAAACAGCAACAACGAATTAAATGAAAATGAATTCGTTGAATCGGAAAAAAGC

GAGCATGAAGCAAGATCCAAAGCAAAAGAAGCTTCTAGTTATGATTATATTTTAG

GTTGGGAATTTGGAGGAGGCGTTCCAGAACACAAAAAAGAAGAAAATATGTTAT

CACATTTATATGTTTCTTCAAAGGATAAGGAAAATATATCTAAGGAAAATGATGA

TGTATTAGATGAGAAGGAAGAAGAGGCAGAAGAAACAGAAGAAGAAGAACTTG

AAGAAAAAAATGAAGAAGAAACAGAATCAGAAATAAGTGAAGATGAAGAAGAA

GAAGAAGAAGAAGAAGAAAAGGAAGAAGAAAATGACAAAAAAAAAGAACAAG

AAAAAGAACAAAGTAATGAAAATAATGATCAAAAAAAAGATATGGAAGCACAG

AATTTAATTTCTAAAAACCAGAATAATAATGAGAAAAACGTAAAAGAAGCTGCT

GAAAGCATCATGAAAACTTTAGCTGGTTTAATCAAGGGAAATAATCAAATAGATT

CTACCTTAAAAGATTTAGTAGAAGAATTATCCAAATATTTTAAAAATCATGGTGG

TAGCGGTGCAATGGTTGATACCCTGAGCGGTCTGAGCAGCGAACAGGGTCAGAG

CGGTGATATGACCATTGAAGAAGATAGCGCAACCCACATCAAATTCAGCAAACG

TGATGAAGATGGTAAAGAACTGGCAGGCGCAACAATGGAACTGCGTGATAGCAG

CGGTAAAACCATTAGCACCTGGATTAGTGATGGTCAGGTGAAAGATTTTTATCTG

TACCCTGGCAAATACACCTTTGTTGAAACCGCAGCACCGGATGGTTATGAAGTTG

CAACCGCAATTACCTTTACCGTTAATGAACAGGGCCAGGTTACCGTGAATGGTAA

AGCAACCAAAGGTGATGCACATATT

>A8>HIS-GMZ2T:ggs-Spycatcher DNA
SEQ ID NO: 34
GGATCCACAAGTGAGAATAGAAATAAACGAATCGGGGGTCCTAAATTAAGGGGT

AATGTTACAAGTAATATAAAGTTCCCATCAGATAACAAAGGTAAAATTATAAGA

GGTTCGAATGATAAACTTAATAAAAACTCTGAAGATGTTTTAGAACAAAGCGAA

AAATCGCTTGTTTCAGAAAATGTTCCTAGTGGATTAGATATAGATGATATCCCTA

AAGAATCTATTTTTATTCAAGAAGATCAAGAAGGTCAAACTCATTCTGAATTAAA

TCCTGAAACATCAGAACATAGTAAAGATTTAAATAATAATGGTTCAAAAAATGA

ATCTAGTGATATTATTTCAGAAAATAATAAATCAAATAAAGTACAAAATCATTTT

GAATCATTATCAGATTTAGAATTACTTGAAAATTCCTCACAAGATAATTTAGACA

AAGATACAATTTCAACAGAACCTTTTCCTAATCAAAAACATAAAGACTTACAACA

AGATTTAAATGATGAACCTTTAGAACCCTTTCCTACACAAATACATAAAGATTAT

AAAGAAAAAAATTTAATAAATGAAGAAGATTCAGAACCATTTCCCAGACAAAAG

CATAAAAAGGTAGACAATCATAATGAAGAAAAAAACGTATTTCATGAAAATGGT

TCTGCAAATGGTAATCAAGGAAGTTTGAAACTTAAATCATTCGATGAACATTTAA

AAGATGAAAAAATAGAAAATGAACCACTTGTTCATGAAAATTTATCCATACCAA

ATGATCCAATAGAACAAATATTAAATCAACCTGAACAAGAAACAAATATCCAGG

AACAATTGTATAATGAAAAACAAAATGTTGAAGAAAAACAAAATTCTCAAATAC

CTTCGTTAGATTTAAAAGAACCAACAAATGAAGATATTTTACCAAATCATAATCC

ATTAGAAAATATAAAACAAAGTGAATCAGAAATAAATCATGTACAAGATCATGC

GCTACCAAAAGAGAATATAATAGACAAACTTGATAATCAAAAAGAACACATCGA

TCAATCACAACATAATATAAATGTATTACAAGAAAATAACATAAACAATCACCA

ATTAGAACCTCAAGAGAAACCTAATATTGAATCGTTTGAACCTAAAAATATAGAT

TCAGAAATTATTCTTCCTGAAAATGTTGAAACAGAAGAAATAATAGATGATGTGC

CTTCCCCTAAACATTCTAACCATGAAACATTTGAAGAAGAAACAAGTGAATCTGA

ACATGAAGAAGCCGTATCTGAAAAAAATGCCCACGAAACTGTCGAACATGAAGA

AACTGTGTCTCAAGAAAGCAATCCTGAAAAAGCTGATAATGATGGAAATGTATCT

CAAAACAGCAACAACGAATTAAATGAAAATGAATTCGTTGAATCGGAAAAAAGC

GAGCATGAAGCAAGATCCAAAACAAAAGAATATGCTGAAAAAGCAAAAAATGCT

TATGAAAAGGCAAAAAATGCTTATCAAAAAGCAAACCAAGCTGTTTTAAAAGCA

AAAGAAGCTTCTAGTTATGATTATATTTTAGGTTGGGAATTTGGAGGAGGCGTTC

CAGAACACAAAAAAGAAGAAAATATGTTATCACATTTATATGTTTCTTCAAAGGA

TAAGGAAAATATATCTAAGGAAAATGATGATGTATTAGATGAGAAGGAAGAAGA

GGCAGAAGAAACAGAAGAAGAAGAACTTGAAGGTGGTAGCGGTGCAATGGTTG

ATACCCTGAGCGGTCTGAGCAGCGAACAGGGTCAGAGCGGTGATATGACCATTG

AAGAAGATAGCGCAACCCACATCAAATTCAGCAAACGTGATGAAGATGGTAAAG

AACTGGCAGGCGCAACAATGGAACTGCGTGATAGCAGCGGTAAAACCATTAGCA

CCTGGATTAGTGATGGTCAGGTGAAAGATTTTTATCTGTACCCTGGCAAATACAC

CTTTGTTGAAACCGCAGCACCGGATGGTTATGAAGTTGCAACCGCAATTACCTTT

ACCGTTAATGAACAGGGCCAGGTTACCGTGAATGGTAAAGCAACCAAAGGTGAT

GCACATATT

>A9>Spycatcher-ggs-PfRH5-HIS DNA
SEQ ID NO: 35
GGTGCAATGGTTGATACCCTGAGCGGTCTGAGCAGCGAACAGGGTCAGAGCGGT

GATATGACCATTGAAGAAGATAGCGCAACCCACATCAAATTCAGCAAACGTGAT

GAAGATGGTAAAGAACTGGCAGGCGCAACAATGGAACTGCGTGATAGCAGCGGT

AAAACCATTAGCACCTGGATTAGTGATGGTCAGGTGAAAGATTTTTATCTGTACC

CTGGCAAATACACCTTTGTTGAAACCGCAGCACCGGATGGTTATGAAGTTGCAAC

CGCAATTACCTTTACCGTTAATGAACAGGGCCAGGTTACCGTGAATGGTAAAGCA

ACCAAAGGTGATGCACATATTGGTGGTAGCCTGTCCTTCGAGAACGCCATCAAGA

AGACCAAGAACCAGGAAAACAACCTGACCCTGCTGCCCATCAAGTCCACCGAGG

AAGAGAAGGACGACATCAAGAACGGCAAGGATATCAAGAAGGAAATCGACAAC

GACAAGGAAAACATCAAGACCAACAACGCCAAGGACCACTCCACCTACATCAAG

TCTTACCTGAACACCAACGTGAACGACGGCCTGAAGTACCTGTTCATCCCATCCC

ACAACAGCTTCATCAAGAAGTACTCCGTTTTCAACCAGATCAACGACGGCATGCT

GCTGAACGAGAAGAACGACGTGAAGAACAACGAGGACTACAAGAACGTCGACT

ACAAGAACGTGAACTTCCTGCAGTACCACTTCAAGGAACTGTCCAACTACAACAT

CGCCAACTCCATCGACATCCTGCAAGAAAAGGAAGGCCACCTGGACTTCGTGATC

ATCCCCCACTACACTTTCTTGGACTACTACAAGCACCTGTCCTACAACAGCATCTA

CCACAAGTACAGCACCTACGGCAAGTACATCGCTGTGGACGCTTTCATCAAGAAG

ATCAACGAGACTTACGACAAAGTGAAGTCCAAGTGTAACGATATCAAGAACGAC

CTGATCGCCACCATCAAGAAGCTCGAGCACCCCTACGACATCAACAACAAGAAC

GACGACAGCTACCGCTACGACATCTCCGAAGAGATCGACGACAAGTCCGAGGAA

ACCGACGACGAGACTGAGGAAGTCGAGGACTCCATCCAGGACACCGACTCCAAC

CACACCCCCTCCAACAAGAAGAAGAACGATCTGATGAACCGCACCTTCAAGAAG

ATGATGGACGAGTACAACACTAAGAAGAAGAAGCTGATCAAGTGCATCAAGAAC

CACGAGAACGACTTCAACAAGATCTGCATGGACATGAAGAACTACGGCACCAAC

CTGTTCGAGCAGCTGTCCTGCTACAACAACAACTTCTGCAACACTAACGGCATCC

GCTTCCACTACGATGAGTACATCCACAAGCTGATCCTGTCCGTCAAGAGCAAGAA

CCTGAACAAGGACCTGAGCGACATGACCAACATCCTCCAGCAGTCCGAGCTGCTG

CTGACCAACTTGAACAAGAAGATGGGCTCCTACATCTACATCGACACTATCAAGT

TCATCCACAAGGAAATGAAGCACATCTTCAACCGCATCGAGTACCACACCAAGAT

CATCAACGATAAGACTAAGATCATCCAAGACAAGATCAAGCTGAACATCTGGCG

CACTTTCCAAAAGGACGAACTGCTGAAGCGTATCCTGGACATGTCTAACGAGTAC

TCCCTCTTCATCACCTCCGACCACCTGAGGCAGATGCTGTACAACACCTTCTACTC

CAAGGAAAAGCACCTCAACAACATCTTCCACCACCTGATCTACGTGCTGCAGATG

AAGTTCAACGACGTCCCCATCAAGATGGAATACTTCCAGACCTACAAGAAGAAC

AAGCCCCTGACCCAGCATCATCACCACCACCAC

>(SpyTag sequence)
SEQ ID NO: 36
AHIVMVDAYKPTK

>SpyCatcher
SEQ ID NO: 37
GAMVDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDEDGKELAGATMELRDSSGKTIS

TWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGKATKGDA

HI

>The β-strand of CnaB2 (KTag)
SEQ ID NO: 38
ATHIKFSKRD

>DNA sequence of the SpyTag
SEQ ID NO: 39
GCTCACATCGTGATGGTGGACGCTTACAAGCCCACCAAG

>>Survivin:ggs-Spycatcher (Homo Sapiens)
SEQ ID NO: 40
MGAPTLPPAWQPFLKDHRISTFKNWPFLEGCACTPERMAEAGFIHCPTENEPDLAQCF

FCFKELEGWEPDDDPIEEHKKHSSGCAFLSVKKQFEELTLGEFLKLDRERAKNKIAKE

TNNKKKEFEETAKKVRRAIEQLAAMDggsGAMVDTLSGLSSEQGQSGDMTIEEDSAT

HIKFSKRDEDGKELAGATMELRDSSGKTISTWISDGQVKDFYLYPGKYTFVETAAPD

GYEVATAITFTVNEQGQVTVNGKATKGDAHI

>>Spycatcher-ggs-Survivin (Homo Sapiens)
SEQ ID NO: 41
GAMVDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDEDGKELAGATMELRDSSGKTIS

TWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGKATKGDA

HIggsGAPTLPPAWQPFLKDHRISTFKNWPFLEGCACTPERMAEAGFIHCPTENEPDLA

QCFFCFKELEGWEPDDDPIEEHKKHSSGCAFLSVKKQFEELTLGEFLKLDRERAKNKI

AKETNNKKKEFEETAKKVRRAIEQLAAMD

>>Survivin(F101A/L102A)-ggs-Spycatcher (Homo Sapiens)
SEQ ID NO: 42
MGAPTLPPAWQPFLKDHRISTFKNWPFLEGCACTPERMAEAGFIHCPTENEPDLAQCF

FCFKELEGWEPDDDPIEEHKKHSSGCAFLSVKKQFEELTLGEAAKLDRERAKNKIAK

ETNNKKKEFEETAKKVRRAIEQLAAMDggsGAMVDTLSGLSSEQGQSGDMTIEEDSA

THIKFSKRDEDGKELAGATMELRDSSGKTISTWISDGQVKDFYLYPGKYTFVETAAP

DGYEVATAITFTVNEQGQVTVNGKATKGDAHI

>>Spycatcher-ggs-Survivin(F101A/L102A) (Homo Sapiens)
SEQ ID NO: 43
GAMVDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDEDGKELAGATMELRDSSGKTIS

TWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGKATKGDA

HIggsGAPTLPPAWQPFLKDHRISTFKNWPFLEGCACTPERMAEAGFIHCPTENEPDLA

QCFFCFKELEGWEPDDDPIEEHKKHSSGCAFLSVKKQFEELTLGEAAKLDRERAKNKI

AKETNNKKKEFEETAKKVRRAIEQLAAMD

>>Spycatcher-ggs-Survivin (F101A/L102A) (Mus Musculus)
SEQ ID NO: 44
GAMVDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDEDGKELAGATMELRDSSGKTIS

TWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGKATKGDA

HIGGSGAPALPQIWQLYLKNYRIATFKNWPFLEDCACTPERMAEAGFIHCPTENEPDL

AQCFFCFKELEGWEPDDNPIEEHRKHSPGCAFLTVKKQMEELTVSEAAKLDRQRAKN

KIAKETNNKQKEFEETAKTTRQSIEQLAASGRF

>>Survivin (F101A/L102A)-ggs-Spycatcher (Mus Musculus)
SEQ ID NO: 45
GAPALPQIWQLYLKNYRIATFKNWPFLEDCACTPERMAEAGFIHCPTENEPDLAQCFF

CFKELEGWEPDDNPIEEHRKHSPGCAFLTVKKQMEELTVSEAAKLDRQRAKNKIAKE

TNNKQKEFEETAKTTRQSIEQLAAggsGAMVDTLSGLSSEQGQSGDMTIEEDSATHIKF

SKRDEDGKELAGATMELRDSSGKTISTWISDGQVKDFYLYPGKYTFVETAAPDGYEV

ATAITFTVNEQGQVTVNGKATKGDAHI

>>Spycatcher-ggs-Survivin (Mus Musculus)
SEQ ID NO: 46
GAMVDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDEDGKELAGATMELRDSSGKTIS

TWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGKATKGDA

HIGGSGAPALPQIWQLYLKNYRIATFKNWPFLEDCACTPERMAEAGFIHCPTENEPDL

AQCFFCFKELEGWEPDDNPIEEHRKHSPGCAFLTVKKQMEELTVSEFLKLDRQRAKN

KIAKETNNKQKEFEETAKTTRQSIEQLAASGRF

>>Survivin-ggs-Spycatcher (Mus Musculus)
SEQ ID NO: 47
GAPALPQIWQLYLKNYRIATFKNWPFLEDCACTPERMAEAGFIHCPTENEPDLAQCFF

CFKELEGWEPDDNPIEEHRKHSPGCAFLTVKKQMEELTVSEFLKLDRQRAKNKIAKE

TNNKQKEFEETAKTTRQSIEQLAAGGSGAMVDTLSGLSSEQGQSGDMTIEEDSATHIK

FSKRDEDGKELAGATMELRDSSGKTISTWISDGQVKDFYLYPGKYTFVETAAPDGYE

VATAITFTVNEQGQVTVNGKATKGDAHI

>>Spycatcher-ggs-Survivin (F101A/L102A) (Mus Musculus) DNA
SEQ ID NO: 48
GGTGCAATGGTTGATACCCTGAGCGGTCTGAGCAGCGAACAGGGTCAGAGCGGT

GATATGACCATTGAAGAAGATAGCGCAACCCACATCAAATTCAGCAAACGTGAT

GAAGATGGTAAAGAACTGGCAGGCGCAACAATGGAACTGCGTGATAGCAGCGGT

AAAACCATTAGCACCTGGATTAGTGATGGTCAGGTGAAAGATTTTTATCTGTACC

CTGGCAAATACACCTTTGTTGAAACCGCAGCACCGGATGGTTATGAAGTTGCAAC

CGCAATTACCTTTACCGTTAATGAACAGGGCCAGGTTACCGTGAATGGTAAAGCA

ACCAAAGGTGATGCACATATTGGTGGTAGCGGTGCACCGGCACTGCCGCAGATTT

GGCAGCTGTATCTGAAAAACTATCGTATCGCCACCTTTAAAAACTGGCCGTTTCT

GGAAGATTGTGCATGTACACCGGAACGTATGGCAGAAGCAGGTTTTATTCATTGT

CCGACCGAAAATGAACCGGATCTGGCACAGTGTTTTTTTTGCTTTAAAGAACTGG

AAGGTTGGGAGCCGGATGATAATCCGATTGAAGAACATCGTAAACATAGTCCGG

GTTGTGCATTTCTGACCGTGAAAAAACAAATGGAAGAACTGACCGTTAGCGAGG

CAGCAAAACTGGATCGTCAGCGTGCCAAAAACAAAATTGCAAAAGAAACCAATA

ACAAACAGAAAGAATTCGAAGAAACCGCCAAAACCACCCGTCAGAGCATTGAAC

AGCTGGCAGCAagcggccgcttt

>>Survivin (F101A/L102A)-ggs-Spycatcher (Mus Musculus) DNA
SEQ ID NO: 49
GGTGCACCGGCACTGCCGCAGATTTGGCAGCTGTATCTGAAAAACTATCGTATCG

CCACCTTTAAAAACTGGCCGTTTCTGGAAGATTGTGCATGTACACCGGAACGTAT

GGCAGAAGCAGGTTTTATTCATTGTCCGACCGAAAATGAACCGGATCTGGCACAG

TGTTTTTTTTGCTTTAAAGAACTGGAAGGTTGGGAGCCGGATGATAATCCGATTG

AAGAACATCGTAAACATAGTCCGGGTTGTGCATTTCTGACCGTGAAAAAACAAAT

GGAAGAACTGACCGTTAGCGAGGCAGCAAAACTGGATCGTCAGCGTGCCAAAAA

CAAAATTGCAAAAGAAACCAATAACAAACAGAAAGAATTCGAAGAAACCGCCA

AAACCACCCGTCAGAGCATTGAACAGCTGGCAGCAGGTGGCAGCGGTGCAATGG

TTGATACCCTGAGCGGTCTGAGCAGCGAACAGGGTCAGAGCGGTGATATGACCA

TTGAAGAAGATAGCGCAACCCACATCAAATTCAGCAAACGTGATGAAGATGGTA

AAGAACTGGCAGGCGCAACAATGGAACTGCGTGATAGCAGCGGTAAAACCATTA

GCACCTGGATTAGTGATGGTCAGGTGAAAGATTTTTATCTGTACCCTGGCAAATA

CACCTTTGTTGAAACCGCAGCACCGGATGGTTATGAAGTTGCAACCGCAATTACC

TTTACCGTTAATGAACAGGGCCAGGTTACCGTGAATGGTAAAGCAACCAAAGGT

GATGCACATATT

>>Spycatcher-ggs-Survivin (Mus Musculus) DNA
SEQ ID NO: 50
GGTGCAATGGTTGATACCCTGAGCGGTCTGAGCAGCGAACAGGGTCAGAGCGGT

GATATGACCATTGAAGAAGATAGCGCAACCCACATCAAATTCAGCAAACGTGAT

GAAGATGGTAAAGAACTGGCAGGCGCAACAATGGAACTGCGTGATAGCAGCGGT

AAAACCATTAGCACCTGGATTAGTGATGGTCAGGTGAAAGATTTTTATCTGTACC

CTGGCAAATACACCTTTGTTGAAACCGCAGCACCGGATGGTTATGAAGTTGCAAC

CGCAATTACCTTTACCGTTAATGAACAGGGCCAGGTTACCGTGAATGGTAAAGCA

ACCAAAGGTGATGCACATATTGGTGGTAGCGGTGCACCGGCACTGCCGCAGATTT

GGCAGCTGTATCTGAAAAACTATCGTATCGCCACCTTTAAAAACTGGCCGTTTCT

GGAAGATTGTGCATGTACACCGGAACGTATGGCAGAAGCAGGTTTTATTCATTGT

CCGACCGAAAATGAACCGGATCTGGCACAGTGTTTTTTTTGCTTTAAAGAACTGG

AAGGTTGGGAGCCGGATGATAATCCGATTGAAGAACATCGTAAACATAGTCCGG

GTTGTGCATTTCTGACCGTGAAAAAACAAATGGAAGAACTGACCGTTAGCGAGTT

TCTGAAACTGGATCGTCAGCGTGCCAAAAACAAAATTGCAAAAGAAACCAATAA

CAAACAGAAAGAATTCGAAGAAACCGCCAAAACCACCCGTCAGAGCATTGAACA

GCTGGCAGCAagcggccgcttt

>>Survivin-ggs-Spycatcher (Mus Musculus) DNA
SEQ ID NO: 51
GGTGCACCGGCACTGCCGCAGATTTGGCAGCTGTATCTGAAAAACTATCGTATCG

CCACCTTTAAAAACTGGCCGTTTCTGGAAGATTGTGCATGTACACCGGAACGTAT

GGCAGAAGCAGGTTTTATTCATTGTCCGACCGAAAATGAACCGGATCTGGCACAG

TGTTTTTTTTGCTTTAAAGAACTGGAAGGTTGGGAGCCGGATGATAATCCGATTG

AAGAACATCGTAAACATAGTCCGGGTTGTGCATTTCTGACCGTGAAAAAACAAAT

GGAAGAACTGACCGTTAGCGAGTTTCTGAAACTGGATCGTCAGCGTGCCAAAAA

CAAAATTGCAAAAGAAACCAATAACAAACAGAAAGAATTCGAAGAAACCGCCA

AAACCACCCGTCAGAGCATTGAACAGCTGGCAGCAGGTGGCAGCGGTGCAATGG

TTGATACCCTGAGCGGTCTGAGCAGCGAACAGGGTCAGAGCGGTGATATGACCA

TTGAAGAAGATAGCGCAACCCACATCAAATTCAGCAAACGTGATGAAGATGGTA

AAGAACTGGCAGGCGCAACAATGGAACTGCGTGATAGCAGCGGTAAAACCATTA

GCACCTGGATTAGTGATGGTCAGGTGAAAGATTTTTATCTGTACCCTGGCAAATA

CACCTTTGTTGAAACCGCAGCACCGGATGGTTATGAAGTTGCAACCGCAATTACC

TTTACCGTTAATGAACAGGGCCAGGTTACCGTGAATGGTAAAGCAACCAAAGGT

GATGCACATATT

>>Spycatcher-ggs-CIDR1a-HIS Protein
SEQ ID NO: 52
GAMVDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDEDGKELAGATMELRDSSGKTIS

TWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGKATKGDA

HIGGSKITSFDEFFDFWVRKLLIDTIKWETELTYCINNTDVTDCNKCNKNCVCFDKWV

KQKEDEWTNIMKLFTNKHDIPKKYYLNINDLFDSFFFQVIYKFNEGEAKWNELKENL

KKQIASSKANNGTKDSEAAIKVLFNHIKEIATICKDNNTN

>>Spycatcher-ggs-CIDR1a-HIS DNA
SEQ ID NO: 53
GGTGCAATGGTTGATACCCTGAGCGGTCTGAGCAGCGAACAGGGTCAGAGCGGT

GATATGACCATTGAAGAAGATAGCGCAACCCACATCAAATTCAGCAAACGTGAT

GAAGATGGTAAAGAACTGGCAGGCGCAACAATGGAACTGCGTGATAGCAGCGGT

AAAACCATTAGCACCTGGATTAGTGATGGTCAGGTGAAAGATTTTTATCTGTACC

CTGGCAAATACACCTTTGTTGAAACCGCAGCACCGGATGGTTATGAAGTTGCAAC

CGCAATTACCTTTACCGTTAATGAACAGGGCCAGGTTACCGTGAATGGTAAAGCA

ACCAAAGGTGATGCACATATTGGTGGTAGCAAAATAACGTCATTTGATGAATTTT

TTGATTTTTGGGTTAGAAAATTATTAATAGACACTATAAAGTGGGAAACCGAACT

TACGTATTGTATAAATAATACTGATGTCACGGATTGTAATAAATGTAACAAAAAT

TGCGTATGTTTTGACAAATGGGTTAAACAAAAAGAAGACGAATGGACAAATATA

ATGAAACTATTCACAAACAAACACGATATACCGAAAAAATATTATCTTAATATTA

ATGATCTTTTTGATAGTTTTTTTTTCCAAGTTATATATAAGTTTAACGAAGGAGAA

GCAAAATGGAATGAACTTAAAGAAAATTTAAAAAAGCAAATTGCGTCTTCCAAA

GCAAATAACGGAACCAAAGATTCAGAAGCTGCAATAAAAGTGTTGTTTAATCAC

ATAAAAGAAATTGCAACAATATGCAAAGATAATAATACAAAC

>>SpyCatcher
SEQ ID NO: 54
GGTGCAATGGTTGATACCCTGAGCGGTCTGAGCAGCGAACAGGGTCAGAGCGGT

GATATGACCATTGAAGAAGATAGCGCAACCCACATCAAATTCAGCAAACGTGAT

GAAGATGGTAAAGAACTGGCAGGCGCAACAATGGAACTGCGTGATAGCAGCGGT

AAAACCATTAGCACCTGGATTAGTGATGGTCAGGTGAAAGATTTTTATCTGTACC

CTGGCAAATACACCTTTGTTGAAACCGCAGCACCGGATGGTTATGAAGTTGCAAC

CGCAATTACCTTTACCGTTAATGAACAGGGCCAGGTTACCGTGAATGGTAAAGCA

ACCAAAGGTGATGCACATATT

>SpyLigase:
SEQ ID NO: 55
HHHHHHDYDGQSGDGKELAGATMELRDSSGKTISTWISDGQVKDFYLYPGKYTFVE

TAAPDGYEVATAITFTVNEQGQVTVNGKATKGGSGGSGGSGEDSATHI

>isopeptide Spy0128
SEQ ID NO: 56
TDKDMTITFTNKKDAE

>Split-Spy0128
SEQ ID NO: 57
ATTVHGETVVNGAKLTVTKNLDLVNSNALIPNTDFTFKIEPDTTVNEDGNKFKGVAL

NTPMTKVTYTNSDKGGSNTKTAEFDFSEVTFEKPGVYYYKVTEEKIDKVPGVSYDTT

SYTVQVHVLWNEEQQKPVATYIVGYKEGSKVPIQFKNSLDSTTLTVKKKVSGTGGD

RSKDFNFGLTLKANQYYKASEKVMIEKTTKGGQAPVQTEASIDQLYHFTLKDGESIK

VTNLPVGVDYVVTEDDYKSEKYTTNVEVSPQDGAVKNIAGNSTEQETSTDKDMTI

>AP205 capsid protein
SEQ ID NO: 58
ANKPMQPITSTANKIVWSDPTRLSTTFSASLLRQRVKVGIAELNNVSGQYVSVYKRPA

PKPEGCADACVIMPNENQSIRTVISGSAENLATLKAEWETHKRNVDTLFASGNAGLG

FLDPTAAIVSSDTTA

>PhageFr capsid protein
SEQ ID NO: 59
ASNFEEFVLVDNGGTGDVKVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSANNR

KYTVKVEVPKVATQVQGGVELPVAAWRSYMNMELTIPVFATNDDCALIVKALQGTF

KTGNPIATAIAANSGIY

>SpyCatcherAN
SEQ ID NO: 60
DSATHIKFSKRDEDGKELAGATMELRDSSGKTISTWISDGQVKDFYLYPGKYTFVET

AAPDGYEVATAITFTVNEQGQVTVNGKATKGDAHI

>SpyCatcherANC
SEQ ID NO: 61
DSATHIKFSKRDEDGKELAGATMELRDSSGKTISTWISDGQVKDFYLYPGKYTFVET

AAPDGYEVATAITFTVNEQGQVTVNGKATKG

>Spy-AP205
SEQ ID NO: 62
MAHIVMVDAYKPTKGSGTAGGGSGSANKPMQPITSTANKIVWSDPTRLSTTFSASLL

RQRVKVGIAELNNVSGQYVSVYKRPAPKPEGCADACVIMPNENQSIRTVISGSAENL

ATLKAEWETHKRNVDTLFASGNAGLGFLDPTAAIVSSDTTA

>Spy-AP205
SEQ ID NO: 63
ATGGCACATATTGTTATGGTGGATGCATATAAACCGACCAAAGGTAGCGGTACAG

CCGGTGGTGGTAGTGGTAGCGCAAATAAACCGATGCAGCCGATTACCAGCACCG

CAAACAAAATTGTTTGGAGCGATCCGACCCGTCTGAGCACCACCTTTAGCGCAAG

CCTGCTGCGTCAGCGTGTTAAAGTTGGTATTGCAGAACTGAATAATGTGAGCGGT

CAGTATGTTAGCGTGTATAAACGTCCGGCACCGAAACCGGAAGGTTGTGCAGATG

CATGTGTTATTATGCCGAATGAAAATCAGAGCATTCGTACCGTTATTAGCGGTAG

CGCAGAAAATCTGGCAACCCTGAAAGCAGAATGGGAAACCCATAAACGTAATGT

GGATACCCTGTTTGCAAGCGGTAATGCAGGTCTGGGTTTTCTGGACCCGACCGCA

GCAATTGTTAGCAGCGATACCACCGCATAA

>AP205-spy
SEQ ID NO: 64
MANKPMQPITSTANKIVWSDPTRLSTTFSASLLRQRVKVGIAELNNVSGQYVSVYKR

PAPKPEGCADACVIMPNENQSIRTVISGSAENLATLKAEWETHKRNVDTLFASGNAG

LGFLDPTAAIVSSDTTAGTAGGSGAHIVMVDAYKPTK

>AP205-spy
SEQ ID NO: 65
ATGGCAAATAAACCGATGCAGCCGATTACCAGCACCGCAAACAAAATTGTTTGG

AGCGATCCGACCCGTCTGAGCACCACCTTTAGCGCAAGCCTGCTGCGTCAGCGTG

TTAAAGTTGGTATTGCAGAACTGAATAATGTGAGCGGTCAGTATGTTAGCGTGTA

TAAACGTCCGGCACCGAAACCGGAAGGTTGTGCAGATGCATGTGTTATTATGCCG

AATGAAAATCAGAGCATTCGTACCGTTATTAGCGGTAGCGCAGAAAATCTGGCA

ACCCTGAAAGCAGAATGGGAAACCCATAAACGTAATGTGGATACCCTGTTTGCA

AGCGGTAATGCAGGTCTGGGTTTTCTGGACCCGACCGCAGCAATTGTTAGCAGCG

ATACCACCGCAGGTACAGCCGGTGGTAGCGGTGCACATATTGTTATGGTTGATGC

ATATAAACCGACCAAATAA

>Spy-Phage fr
SEQ ID NO: 66
MAHTVMVDAYKPTKGSGTAGGGSGSASNFEEFVLVDNGGTGDVKVAPSNFANGVA

EWISSNSRSQAYKVTCSVRQSSANNRKYTVKVEVPKVATQVQGGVELPVAAWRSY

MNMELTIPVFATNDDCALIVKALQGTFKTGNPIATAIAANSGIY

>Spy-Phage fr
SEQ ID NO: 67
ATGGCACATATTGTTATGGTGGATGCATATAAACCGACCAAAGGTAGCGGTACAG

CCGGTGGTGGTAGTGGTAGCGCAAGCAATTTTGAAGAATTTGTGCTGGTTGATAA

TGGTGGCACCGGTGATGTTAAAGTTGCACCGAGTAATTTTGCAAATGGTGTTGCA

GAATGGATTAGCAGCAATAGCCGTAGCCAGGCATATAAAGTTACCTGTAGCGTTC

GTCAGAGCAGCGCAAATAATCGTAAATATACCGTTAAAGTCGAGGTTCCGAAAG

TTGCAACCCAGGTTCAGGGTGGTGTTGAACTGCCGGTTGCAGCATGGCGTAGCTA

TATGAATATGGAACTGACCATTCCGGTTTTTGCCACCAATGATGATTGTGCCCTGA

TTGTTAAAGCACTGCAGGGCACCTTTAAAACCGGTAATCCGATTGCAACCGCAAT

TGCAGCAAATAGCGGTATCTATTAA

>Ktag-AP205
SEQ ID NO: 68
ATHIKFSKRDGSGTAGGGSGSANKPMQPITSTANKIVWSDPTRLSTTFSASLLRQRVK

VGIAELNNVSGQYVSVYKRPAPKPEGCADACVIMPNENQSIRTVISGSAENLATLKAE

WETHKRNVDTLFASGNAGLGFLDPTAAIVSSDTTA

>AP205-Ktag
SEQ ID NO: 69
MANKPMQPITSTANKIVWSDPTRLSTTFSASLLRQRVKVGIAELNNVSGQYVSVYKR

PAPKPEGCADACVIMPNENQSIRTVISGSAENLATLKAEWETHKRNVDTLFASGNAG

LGFLDPTAAIVSSDTTAGTAGGSGATHIKFSKRD

>Ktag-Phage fr
SEQ ID NO: 70
ATHIKFSKRDGSGTAGGGSGSASNFEEFVLVDNGGTGDVKVAPSNFANGVAEWISSN

SRSQAYKVTCSVRQSSANNRKYTVKVEVPKVATQVQGGVELPVAAWRSYMNMELT

IPVFATNDDCALIVKALQGTFKTGNPIATAIAANSGIY

>Spy-AP205-Spy
SEQ ID NO: 71
MAHIVMVDAYKPTKGSGTAGGGSGSANKPMQPITSTANKIVWSDPTRLSTTFSASLL

RQRVKVGIAELNNVSGQYVSVYKRPAPKPEGCADACVIMPNENQSIRTVISGSAENL

ATLKAEWETHKRNVDTLFASGNAGLGFLDPTAAIVSSDTTAGTAGGSGAHIVMVDA

YKPTK

>Spy-AP205-Spy
SEQ ID NO: 72
CACATATTGTTATGGTGGATGCATATAAACCGACCAAAGGTAGCGGTACAG

CCGGTGGTGGTAGTGGTAGCGCAAATAAACCGATGCAGCCGATTACCAGCACCG

CAAACAAAATTGTTTGGAGCGATCCGACCCGTCTGAGCACCACCTTTAGCGCAAG

CCTGCTGCGTCAGCGTGTTAAAGTTGGTATTGCAGAACTGAATAATGTGAGCGGT

CAGTATGTTAGCGTGTATAAACGTCCGGCACCGAAACCGGAAGGTTGTGCAGATG

CATGTGTTATTATGCCGAATGAAAATCAGAGCATTCGTACCGTTATTAGCGGTAG

CGCAGAAAATCTGGCAACCCTGAAAGCAGAATGGGAAACCCATAAACGTAATGT

GGATACCCTGTTTGCAAGCGGTAATGCAGGTCTGGGTTTTCTGGACCCGACCGCA

GCAATTGTTAGCAGCGATACCACCGCAGGTACAGCCGGTGGTAGCGGTGCACAT

ATTGTTATGGTTGATGCATATAAACCGACCAAATAA

>AP205-ggsg-Spycatcher
SEQ ID NO: 73
ATGGCAAATAAACCGATGCAGCCGATTACCAGCACCGCAAACAAAATTGTTTGG

AGCGATCCGACCCGTCTGAGCACCACCTTTAGCGCAAGCCTGCTGCGTCAGCGTG

TTAAAGTTGGTATTGCAGAACTGAATAATGTGAGCGGTCAGTATGTTAGCGTGTA

TAAACGTCCGGCACCGAAACCGGAAGGTTGTGCAGATGCATGTGTTATTATGCCG

AATGAAAATCAGAGCATTCGTACCGTTATTAGCGGTAGCGCAGAAAATCTGGCA

ACCCTGAAAGCAGAATGGGAAACCCATAAACGTAATGTGGATACCCTGTTTGCA

AGCGGTAATGCAGGTCTGGGTTTTCTGGACCCGACCGCAGCAATTGTTAGCAGCG

ATACCACCGCAGGTACAGCCGGTGGTAGCGGTGGTGCAATGGTTGATACCCTG

AGCGGTCTGAGCAGCGAACAGGGTCAGAGCGGTGATATGACCATTGAAGAAGAT

AGCGCAACCCACATCAAATTCAGCAAACGTGATGAAGATGGTAAAGAACTGGCA

GGCGCAACAATGGAACTGCGTGATAGCAGCGGTAAAACCATTAGCACCTGGATT

AGTGATGGTCAGGTGAAAGATTTTTATCTGTACCCTGGCAAATACACCTTTGTTG

AAACCGCAGCACCGGATGGTTATGAAGTTGCAACCGCAATTACCTTTACCGTTAA

TGAACAGGGCCAGGTTACCGTGAATGGTAAAGCAACCAAAGGTGATGCACATAT

Ttaa

>AP205-ggsg-Spycatcher
SEQ ID NO: 74
MANKPMQPITSTANKIVWSDPTRLSTTFSASLLRQRVKVGIAELNNVSGQYVSVYKR

PAPKPEGCADACVIMPNENQSIRTVISGSAENLATLKAEWETHKRNVDTLFASGNAG

LGFLDPTAAIVSSDTTAGTAGGSGGAMVDTLSGLSSEQGQSGDMTIEEDSATHIKFSK

RDEDGKELAGATMELRDSSGKTISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVAT

AITFTVNEQGQVTVNGKATKGDAHI

>SpyCatcher-ggsgs-AP205
SEQ ID NO: 75
GGTGCAATGGTTGATACCCTGAGCGGTCTGAGCAGCGAACAGGGTCAGAGCGGT

GATATGACCATTGAAGAAGATAGCGCAACCCACATCAAATTCAGCAAACGTGAT

GAAGATGGTAAAGAACTGGCAGGCGCAACAATGGAACTGCGTGATAGCAGCGGT

AAAACCATTAGCACCTGGATTAGTGATGGTCAGGTGAAAGATTTTTATCTGTACC

CTGGCAAATACACCTTTGTTGAAACCGCAGCACCGGATGGTTATGAAGTTGCAAC

CGCAATTACCTTTACCGTTAATGAACAGGGCCAGGTTACCGTGAATGGTAAAGCA

ACCAAAGGTGATGCACATATT TAAACCGATGCAGC

CGATTACCAGCACCGCAAACAAAATTGTTTGGAGCGATCCGACCCGTCTGAGCAC

CACCTTTAGCGCAAGCCTGCTGCGTCAGCGTGTTAAAGTTGGTATTGCAGAACTG

AATAATGTGAGCGGTCAGTATGTTAGCGTGTATAAACGTCCGGCACCGAAACCGG

AAGGTTGTGCAGATGCATGTGTTATTATGCCGAATGAAAATCAGAGCATTCGTAC

CGTTATTAGCGGTAGCGCAGAAAATCTGGCAACCCTGAAAGCAGAATGGGAAAC

CCATAAACGTAATGTGGATACCCTGTTTGCAAGCGGTAATGCAGGTCTGGGTTTT

CTGGACCCGACCGCAGCAATTGTTAGCAGCGATACCACCGCATAA

>SpyCatcher-ggsgs-AP205
SEQ ID NO: 76
MVDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDEDGKELAGATMELRDSSGKTIST

WISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGKATKGDAH

IGGSGSANKPMQPITSTANKIVWSDPTRLSTTFSASLLRQRVKVGIAELNNVSGQYVS

VYKRPAPKPEGCADACVIMPNENQSIRTVISGSAENLATLKAEWETHKRNVDTLFAS

GNAGLGFLDPTAAIVSSDTTA

>SpyCatcher-ggsgs-Phage fr
SEQ ID NO: 77
GGTGCAATGGTTGATACCCTGAGCGGTCTGAGCAGCGAACAGGGTCAGAGCGGT

GATATGACCATTGAAGAAGATAGCGCAACCCACATCAAATTCAGCAAACGTGAT

GAAGATGGTAAAGAACTGGCAGGCGCAACAATGGAACTGCGTGATAGCAGCGGT

AAAACCATTAGCACCTGGATTAGTGATGGTCAGGTGAAAGATTTTTATCTGTACC

CTGGCAAATACACCTTTGTTGAAACCGCAGCACCGGATGGTTATGAAGTTGCAAC

CGCAATTACCTTTACCGTTAATGAACAGGGCCAGGTTACCGTGAATGGTAAAGCA

ACCAAAGGTGATGCACATATTGGTGGTAGCGGTAGCGCAAGCAATTTTGAAGA

ATTTGTGCTGGTTGATAATGGTGGCACCGGTGATGTTAAAGTTGCACCGAGTAAT

TTTGCAAATGGTGTTGCAGAATGGATTAGCAGCAATAGCCGTAGCCAGGCATATA

AAGTTACCTGTAGCGTTCGTCAGAGCAGCGCAAATAATCGTAAATATACCGTTAA

AGTCGAGGTTCCGAAAGTTGCAACCCAGGTTCAGGGTGGTGTTGAACTGCCGGTT

GCAGCATGGCGTAGCTATATGAATATGGAACTGACCATTCCGGTTTTTGCCACCA

ATGATGATTGTGCCCTGATTGTTAAAGCACTGCAGGGCACCTTTAAAACCGGTAA

TCCGATTGCAACCGCAATTGCAGCAAATAGCGGTATCTATTAA

>SpyCatcher-ggsgs-Phage fr
SEQ ID NO: 78
MVDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDEDGKELAGATMELRDSSGKTIST

WISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGKATKGDAH

IGGSGSASNFEEFVLVDNGGTGDVKVAPSNFANGVAEWISSNSRSQAYKVTCSVRQS

SANNRKYTVKVEVPKVATQVQGGVELPVAAWRSYMNMELTIPVFATNDDCALIVK

ALQGTFKTGNPIATAIAANSGIY

>>SpyTag-Her2-ECD\|23-686-HIS
SEQ ID NO: 79
MAHIVMVDAYKPTKGGSTQVCTGTDMKLRLPASPETHLDMLRHLYQGCQVVQGNL

ELTYLPTNASLSFLQDIQEVQGYVLIAHNQVRQVPLQRLRIVRGTQLFEDNYALAVLD

NGDPLNNTTPVTGASPGGLRELQLRSLTEILKGGVLIQRNPQLCYQDTILWKDIFHKN

NQLALTLIDTNRSRACHPCSPMCKGSRCWGESSEDCQSLTRTVCAGGCARCKGPLPT

DCCHEQCAAGCTGPKHSDCLACLHFNHSGICELHCPALVTYNTDTFESMPNPEGRYT

FGASCVTACPYNYLSTDVGSCTLVCPLHNQEVTAEDGTQRCEKCSKPCARVCYGLG

MEHLREVRAVTSANIQEFAGCKKIFGSLAFLPESFDGDPASNTAPLQPEQLQVFETLEE

ITGYLYISAWPDSLPDLSVFQNLQVIRGRILHNGAYSLTLQGLGISWLGLRSLRELGSG

LALIHHNTHLCFVHTVPWDQLFRNPHQALLHTANRPEDECVGEGLACHQLCARGHC

WGPGPTQCVNCSQFLRGQECVEECRVLQGLPREYVNARHCLPCHPECQPQNGSVTC

FGPEADQCVACAHYKDPPFCVARCPSGVKPDLSYMPIWKFPDEEGACQPCPINCTHS

CVDLDDKGCPAEQRASPLTSIISAVVGILLVVVLGVVFGILIKRRQQKIRKYTHHHHH

H

>>SpyTag-IL-5(C63T/C105T)
SEQ ID NO: 80
MAHIVMVDAYKPTKGGSIPTEIPTSALVKETLALLSTHRTLLIANETLRIPVPVHKNHQ

LTTEEIFQGIGTLESQTVQGGTVERLFKNLSLIKKYIDGQKKKTGEERRRVNQFLDYL

QEFLGVMNTEWIIES*SGRK

>>PCSK9\|31-692\|:SpyTag
SEQ ID NO: 81
QEDEDGDYEELVLALRSEEDGLAEAPEHGTTATFHRCAKDPWRLPGTYVVVLKEET

HLSQSERTARRLQAQAARRGYLTKILHVFHGLLPGFLVKMSGDLLELALKLPHVDYI

EEDSSVFAQSIPWNLERITPPRYRADEYQPPDGGSLVEVYLLDTSIQSDHREIEGRVMV

TDFENVPEEDGTRFHRQASKCDSHGTHLAGVVSGRDAGVAKGASMRSLRVLNCQG

KGTVSGTLIGLEFIRKSQLVQPVGPLVVLLPLAGGYSRVLNAACQRLARAGVVLVTA

AGNFRDDACLYSPASAPEVITVGATNAQDQPVTLGTLGTNFGRCVDLFAPGEDIIGAS

SDCSTCFVSQSGTSQAAAHVAGIAAMMLSAEPELTLAELRQRLIHFSAKDVINEAWFP

EDQRVLTPNLVAALPPSTHGAGWQLFCRTVWSAHSGPTRMATAVARCAPDEELLSC

SSFSRSGKRRGERMEAQGGKLVCRAHNAFGGEGVYAIARCCLLPQANCSVHTAPPA

EASMGTRVHCHQQGHVLTGCSSHWEVEDLGTHKPPVLRPRGQPNQCVGHREASIHA

SCCHAPGLECKVKEHGIPAPQEQVTVACEEGWTLTGCSALPGTSHVLGAYAVDNTC

VVRSRDVSTTGSTSEGAVTAVAICCRSRHLAQASQELQGGSAHIVMVDAYKPTK

>SpyTag-IDHD2a-HIS
SEQ ID NO: 82
AHIVMVDAYKPTKGGSNYIKGDPYFAEYATKLSFILNPSDANNPSGETANHNDEACN

CNESGISSVGQAQTSGPSSNKTCITHSSIKTNKKKECKDVKLGVRENDKDLKICVIEDT

SLSGVDNCCCQDLLGILQENCSDNKRGSSSNDSCDNKNQDECQKKLEKVFASLTNGY

KCDKCKSGTSRSKKKWIWKKSSGNEEGLQEEYANTIGLPPRTQSLYLGNLPKLENVC

EDVKDINFDTKEKFLAGCLIVSFHEGKNLKKRYPQNKNSGNKENLCKALEYSFADYG

DLIKGTSIWDNEYTKDLELNLQNNFGKLFGKYIKKNNTAEQDTSYSSLDELRESWWN

TNKKYIWTAMKHGAEMNITTCNADGSVTGSGSSCDDIPTIDLIPQYLRFLQEWVENF

CEQRQAKVKDVITNCKSCKESGNKCKTECKTKCKDECEKYKKFIEACGTAGGGIGTA

GSPWSKRWDQIYKRYSKHIEDAKRNRKAGTKNCGTSSTTNAAASTDENKCVQSDID

SFFKHLIDIGLTTPSSYLSNVLDDNICGADKAPWTTYTTYTTTEKCNKERDKSKSQSS

DTLVVVNVPSPLGNTPYRYKYACQCKIPTNEETCDDRKEYMNQWSCGSARTMKRG

YKNDNYELCKYNGVDVKPTTVRSNSSKLDHHHHHH

>Short flexible linker
SEQ ID NO: 83
GGSGS

>SpyCatcher-Ag85A
SEQ ID NO: 84
GAMVDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDEDGKELAGATMELRDSSGKTIS

TWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGKATKGDA

HIGGSFSRPGLPVEYLQVPSPSMGRDIKVQFQSGGANSPALYLLDGLRAQDDFSGWDI

NTPAFEWYDQSGLSVVMPVGGQSSFYSDWYQPACGKAGCQTYKWETFLTSELPGW

LQANRHVKPTGSAVVGLSMAASSALTLAIYHPQQFVYAGAMSGLLDPSQAMGPTLI

GLAMGDAGGYKASDMWGPKEDPAWQRNDPLLNVGKLIANNTRVWVYCGNGKLS

DLGGNNLPAKFLEGFVRTSNIKFQDAYNAGGGHNGVFDFPDSGTHSWEYWGAQLN

AMKPDLQRALGATPNTGPAPQGA

>SpyCatcher-Ag85A DNA
SEQ ID NO: 85
GGTGCAATGGTTGATACCCTGAGCGGTCTGAGCAGCGAACAGGGTCAGAGCGGT

GATATGACCATTGAAGAAGATAGCGCAACCCACATCAAATTCAGCAAACGTGAT

GAAGATGGTAAAGAACTGGCAGGCGCAACAATGGAACTGCGTGATAGCAGCGGT

AAAACCATTAGCACCTGGATTAGTGATGGTCAGGTGAAAGATTTTTATCTGTACC

CTGGCAAATACACCTTTGTTGAAACCGCAGCACCGGATGGTTATGAAGTTGCAAC

CGCAATTACCTTTACCGTTAATGAACAGGGCCAGGTTACCGTGAATGGTAAAGCA

ACCAAAGGTGATGCACATATTGGTGGTAGCTTCTCCCGTCCCGGACTGCCTGTGG

AATACCTCCAGGTGCCCTCCCCCTCTATGGGTCGTGACATCAAGGTGCAGTTCCA

GTCCGGTGGTGCTAACTCCCCCGCTCTGTACCTGCTGGACGGACTGCGTGCTCAG

GACGACTTCTCCGGCTGGGACATCAACACTCCCGCTTTCGAGTGGTACGACCAGT

CCGGCCTGTCCGTGGTTATGCCTGTGGGTGGCCAGTCCTCCTTCTACTCCGACTGG

TACCAACCCGCTTGCGGCAAGGCTGGCTGCCAGACCTACAAGTGGGAGACTTTCC

TGACCTCCGAGCTGCCCGGATGGCTGCAGGCTAACCGTCACGTGAAGCCCACCGG

TTCCGCTGTCGTGGGCCTGTCTATGGCTGCTTCCTCCGCTCTGACCCTGGCTATCT

ACCACCCCCAGCAGTTCGTGTACGCTGGCGCTATGTCCGGACTGCTGGACCCCTC

TCAGGCTATGGGTCCTACCCTGATCGGCCTGGCTATGGGCGACGCTGGTGGTTAC

AAGGCTTCCGACATGTGGGGTCCCAAGGAAGATCCCGCTTGGCAGCGTAACGAC

CCCCTGCTGAACGTGGGCAAGCTGATCGCTAACAACACCCGTGTGTGGGTGTACT

GCGGCAACGGCAAGCTGTCCGACCTGGGTGGCAACAACCTGCCCGCTAAGTTCCT

CGAGGGTTTCGTGCGCACCTCCAACATCAAGTTCCAGGACGCTTACAACGCTGGC

GGTGGTCACAACGGCGTGTTCGACTTCCCCGACTCCGGAACCCACTCCTGGGAGT

ACTGGGGTGCTCAGCTGAACGCTATGAAGCCCGACCTGCAGCGTGCTCTGGGTGC

TACCCCTAACACCGGTCCAGCTCCTCAGGGTGCTTAA

>SEQ ID NO: 86: SpyCatcher-ggs-Survivin DNA
GAMVDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDEDGKELAGATMELRDSSGKTIS

TWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGKATKGDA

HIGGSGAPALPQIWQLYLKNYRIATFKNWPFLEDCACTPERMAEAGFIHCPTENEPDL

AQCFFCFKELEGWEPDDNPIEEHRKHSPGCAFLTVKKQMEELTVSEFLKLDRQRAKN

KIAKETNNKQKEFEETAKTTRQSIEQLAA

>SEQ ID NO: 87: SpyCatcher-ggs-Survivin DNA
GGTGCAATGGTTGATACCCTGAGCGGTCTGAGCAGCGAACAGGGTCAGAGCGGT

GATATGACCATTGAAGAAGATAGCGCAACCCACATCAAATTCAGCAAACGTGAT

GAAGATGGTAAAGAACTGGCAGGCGCAACAATGGAACTGCGTGATAGCAGCGGT

AAAACCATTAGCACCTGGATTAGTGATGGTCAGGTGAAAGATTTTTATCTGTACC

CTGGCAAATACACCTTTGTTGAAACCGCAGCACCGGATGGTTATGAAGTTGCAAC

CGCAATTACCTTTACCGTTAATGAACAGGGCCAGGTTACCGTGAATGGTAAAGCA

ACCAAAGGTGATGCACATATTGGTGGTAGCGGTGCACCGGCACTGCCGCAGATTT

GGCAGCTGTATCTGAAAAACTATCGTATCGCCACCTTTAAAAACTGGCCGTTTCT

GGAAGATTGTGCATGTACACCGGAACGTATGGCAGAAGCAGGTTTTATTCATTGT

CCGACCGAAAATGAACCGGATCTGGCACAGTGTTTTTTTTGCTTTAAAGAACTGG

AAGGTTGGGAGCCGGATGATAATCCGATTGAAGAACATCGTAAACATAGTCCGG

GTTGTGCATTTCTGACCGTGAAAAAACAAATGGAAGAACTGACCGTTAGCGAGTT

TCTGAAACTGGATCGTCAGCGTGCCAAAAACAAAATTGCAAAAGAAACCAATAA

CAAACAGAAAGAATTCGAAGAAACCGCCAAAACCACCCGTCAGAGCATTGAACA

GCTGGCAGCATAA

>SEQ ID NO: 88: Mini-HA-stem-Spytag
MKVKLLVLLCTFTATYADTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENGGGG

KYVCSAKLRMVTGLRNKPSKQSQGLFGAIAGFTEGGWTGMVDGWYGYHHQNEQG

SGYAADQKSTQNAINGITNKVNSVIEKMNTQYTAIGCEYNKSERCMKQIEDKIEEIES

KIWTYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCN

DECMESVKNGTYDYPKYSEESKLNREKIDGVKLESMGVYQIEGAHIVMVDAYKPTK

>SEQ ID NO: 89: Mini-HA-stem-Spytag DNA
ATGAAAGTGAAGCTGCTGGTGCTGCTGTGCACCTTCACCGCCACCTACGCCGACA

CCATCTGCATCGGCTACCACGCCAACAACAGCACCGACACCGTGGATACCGTGCT

GGAAAAGAACGTGACCGTGACCCACAGCGTGAACCTGCTGGAAAATGGCGGCGG

AGGCAAATACGTGTGCAGCGCCAAGCTGCGGATGGTCACCGGCCTGAGAAACAA

GCCCAGCAAGCAGAGCCAGGGCCTGTTCGGAGCCATTGCCGGCTTTACAGAGGG

CGGCTGGACCGGCATGGTGGATGGGTGGTACGGCTATCACCACCAGAACGAGCA

GGGCAGCGGCTACGCCGCCGATCAGAAGTCTACCCAGAACGCCATCAACGGCAT

CACCAACAAAGTGAACAGCGTGATCGAGAAGATGAACACCCAGTACACCGCCAT

CGGCTGCGAGTACAACAAGAGCGAGCGGTGCATGAAGCAGATCGAGGACAAGAT

CGAAGAGATCGAGTCTAAGATCTGGACCTACAACGCCGAACTGCTGGTGCTGCTG

GAAAACGAGCGGACCCTGGACTTCCACGACAGCAACGTGAAGAACCTGTACGAG

AAAGTGAAAAGCCAGCTGAAGAACAACGCCAAAGAGATCGGCAACGGCTGCTTC

GAGTTCTACCACAAGTGCAACGACGAGTGCATGGAAAGCGTGAAGAATGGCACC

TACGACTACCCCAAGTACAGCGAGGAAAGCAAGCTGAACCGCGAGAAGATCGAC

GGCGTGAAGCTGGAATCTATGGGCGTGTACCAGATTGAGGGCGCCCACATCGTG

ATGGTGGACGCCTACAAGCCTACCAAG

>SEQ ID NO: 90: Infectious hematopoietic necrosis virus (IHNV) G-
protein-SpyTag
MDTMITTPLILILITCGANSQTVQPDTASESDQPTWSNPLFTYPEGCTLDKLSKVNASQ

LRCPRIFNDENRGLIAYPTSIRSLSVGNDLGNIHTQGNYIHKVLYRTICSTGFFGGQTIE

KALVEMKLSTREAGVYDTTTAAALYFPAPRCQWYTDNVQNDLIFYYTTQKSVLRDP

YTRDFLDSDFIGGKCTKSPCQTHWSNVVWMGDAGIPACDSSQEIKGHLFVDKISNRV

VKATSYGHHPWGLHHACMIDFCGKPWIRTDLGDLISVEYNSGAKTLSFPKCEDKTVG

MRGNLDDFAYLDDLVKASESREECLEAHAEIISTNSVTPYLLSKFRSPHPGINDVYAM

HKGSIYHGMCMTVAVDEVSKDRTTYRAHRATSFTKWERPFGDEWEGFHGLHGNNT

TIIPDLEKYVAQYKTSMMEPMSIKSVPHPSILAHYNETDVSGISIRKLDSFDLQSLHWS

GSGAHIVMVDAYKPTK

>SEQ ID NO: 91: SpyTag-IHNV G-protein
AHIVMVDAYKPTKGGSDTMITTPLILILITCGANSQTVQPDTASESDQPTWSNPLFTYP

EGCTLDKLSKVNASQLRCPRIFNDENRGLIAYPTSIRSLSVGNDLGNIHTQGNYIHKVL

YRTICSTGFFGGQTIEKALVEMKLSTREAGVYDTTTAAALYFPAPRCQWYTDNVQND

LIFYYTTQKSVLRDPYTRDFLDSDFIGGKCTKSPCQTHWSNVVWMGDAGIPACDSSQ

EIKGHLFVDKISNRVVKATSYGHHPWGLHHACMIDFCGKPWIRTDLGDLISVEYNSG

AKTLSFPKCEDKTVGMRGNLDDFAYLDDLVKASESREECLEAHAEIISTNSVTPYLLS

KFRSPHPGINDVYAMHKGSIYHGMCMTVAVDEVSKDRTTYRAHRATSFTKWERPFG

DEWEGFHGLHGNNTTIIPDLEKYVAQYKTSMMEPMSIKSVPHPSILAHYNETDVSGIS

IRKLDSFDLQSLHWS

>SEQ ID NO: 92: LongSpyTag-AP205-LongSpyTag
MAHIVMVDAYKPTKGSGTAGGGSGSANKPMQPITSTANKIVWSDPTRLSTTFSASLL

RQRVKVGIAELNNVSGQYVSVYKRPAPKPEGCADACVIMPNENQSIRTVISGSAENL

ATLKAEWETHKRNVDTLFASGNAGLGFLDPTAAIVSSDTTAGTASGGSGGSGAHIVM

VDAYKPTK

>SEQ ID NO: 93: LongSpyTag-AP205-LongSpyTag DNA
ATGGCACATATTGTTATGGTGGATGCATATAAACCGACCAAAGGTAGCGGTACAG

CCGGTGGTGGTAGTGGTAGCGCAAATAAACCGATGCAGCCGATTACCAGCACCG

CAAACAAAATTGTTTGGAGCGATCCGACCCGTCTGAGCACCACCTTTAGCGCAAG

CCTGCTGCGTCAGCGTGTTAAAGTTGGTATTGCAGAACTGAATAATGTGAGCGGT

CAGTATGTTAGCGTGTATAAACGTCCGGCACCGAAACCGGAAGGTTGTGCAGATG

CATGTGTTATTATGCCGAATGAAAATCAGAGCATTCGTACCGTTATTAGCGGTAG

CGCAGAAAATCTGGCAACCCTGAAAGCAGAATGGGAAACCCATAAACGTAATGT

GGATACCCTGTTTGCAAGCGGTAATGCAGGTCTGGGTTTTCTGGACCCGACCGCA

GCAATTGTTAGCAGCGATACCACCGCAGGTACAGCCAGCGGTGGTAGCGGTGGT

AGCGGTGCACATATTGTTATGGTTGATGCATATAAACCGACCAAATAA

>SEQ ID NO: 94: mSA-AP205
MAEAGITGTWYNQHGSTFTVTAGADGNLTGQYENRAQGTGCQNSPYTLTGRYNGT

KLEWRVEWNNSTENCHSRTEWRGQYQGGAEARINTQWNLTYEGGSGPATEQGQDT

FTKVKGGSGSANKPMQPITSTANKIVWSDPTRLSTTFSASLLRQRVKVGIAELNNVSG

QYVSVYKRPAPKPEGCADACVIMPNENQSIRTVISGSAENLATLKAEWETHKRNVDT

LFASGNAGLGFLDPTAAIVSSDTTA

>SEQ ID NO: 95: mSA-AP205 DNA
ATGGCAGAAGCAGGTATTACCGGCACCTGGTATAATCAGCATGGTAGCACCTTTA

CCGTTACCGCAGGCGCAGATGGTAATCTGACAGGTCAGTATGAAAATCGTGCACA

GGGCACCGGTTGTCAGAATAGCCCGTATACCCTGACCGGTCGTTATAATGGCACC

AAACTGGAATGGCGTGTTGAATGGAATAATAGCACCGAAAATTGTCATAGCCGT

ACCGAATGGCGTGGTCAGTATCAGGGTGGTGCAGAAGCCCGTATTAATACCCAGT

GGAATCTGACCTATGAAGGTGGTAGCGGTCCGGCAACCGAACAGGGTCAGGATA

CCTTTACCAAAGTTAAAGGTGGCAGCGGTAGCGCAAATAAACCGATGCAGCCGA

TTACCAGCACCGCAAACAAAATTGTTTGGAGCGATCCGACCCGTCTGAGCACCAC

CTTTAGCGCAAGCCTGCTGCGTCAGCGTGTTAAAGTTGGTATTGCAGAACTGAAT

AATGTGAGCGGTCAGTATGTTAGCGTGTATAAACGTCCGGCACCGAAACCGGAA

GGTTGTGCAGATGCATGTGTTATTATGCCGAATGAAAATCAGAGCATTCGTACCG

TTATTAGCGGTAGCGCAGAAAATCTGGCAACCCTGAAAGCAGAATGGGAAACCC

ATAAACGTAATGTGGATACCCTGTTTGCAAGCGGTAATGCAGGTCTGGGTTTTCT

GGACCCGACCGCAGCAATTGTTAGCAGCGATACCACCGCATAA

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Claims

1. A vaccine for use in the prophylaxis and/or treatment of a disease wherein the vaccine comprises:

i. a particle-forming polypeptide comprising a first peptide tag, and

ii. an antigen fused to a second peptide tag,

wherein the antigen and particle-forming polypeptide are linked via an isopeptide bond between the first and second peptide tag, and wherein i-ii form a particle displaying said antigen.