Vaccine composition

Abstract

The present invention relates to vaccine compositions, most particularly vaccine compositions wherein the antigenic component is large (e.g. greater than 50 kDa), or is multimeric (i.e. composed of subunits). Such antigenic components are of particular interest as they may represent antigenic components from pathogens against which vaccines are currently not available. The present invention relates to a composition comprising particles displaying an antigenic component, wherein the composition comprises an antigenic component comprising a first peptide tag and a portion comprising a second peptide tag, wherein the antigenic component and the portion are linked by an isopeptide bond between the first and second peptide tags, and wherein the antigenic component is greater than 50 kDa, or alternatively is multimeric.

Description

Background of the Invention

[0002] Vaccines are a safe and effective way to combat and eradicate infectious diseases. Vaccine development has been very successful, but a range of disease challenges remain for which vaccines do not yet exist, including many important pathogens that represent daunting immune barriers. It is generally believed that an effective vaccine must be delivered to the lymph nodes and remain active long enough to generate an immune response.

[0003] Vaccine development has shifted from using attenuated or dead pathogens to using smaller antigenic components of these pathogens, aiming to generate the desired protective immune response while avoiding the inherent risks of using such attenuated strains. Efforts have focused on attempting to express the immunogenic portions of pathogen components (such as those required for pathogen infection of cells), which, for simplicity, are limited to short / small peptides and proteins due to technical challenges in expressing large or multi-component antigens. However, a potential problem with using very short or small peptides is the risk of antigenically variable pathogens that evade vaccine-induced immune responses through alterations to specific antigenic portions.

[0004] The expression of large / multi-component antigens offers immunological advantages, including the ability to generate antibodies against multiple neutralizing epitopes of a pathogen. However, the expression of one or more large antigens that can form complexes (in a manner in which the associated antigenic epitopes are maintained and presented to generate an effective antibody response) remains a significant challenge. Therefore, there remains a need for improved methods to express large antigens and / or multi-component antigens so that they can elicit clinically significant immune responses.

[0005] Previous recombinant vaccines were designed to elicit an immune response against multiple antigenic components, relying on each component being expressed and packaged separately into different particles. For example, in the case of the anti-HPV vaccines Cervarix and Gardasil, the recombinant major capsid L1 protein of a specific HPV strain was expressed and assembled separately into virus-like particles (VLPs), and then different types of VLPs were combined into a vaccine formulation. Alternatively, multiple short epitopes can be selected and combined into a single recombinant vaccine (e.g., the multimer-001 influenza vaccine), but these epitopes are inherently short linear peptides. These epitopes are chosen to avoid the preparatory complexities involving three-dimensional structures or refolding, and therefore do not attempt to represent the natural pathogen presented to the immune system during active infection.

[0006] For example, beta-herpes human cytomegalovirus (HCMV, also known as human herpesvirus-5 (HHV-5)) is a leading viral cause of developmental disorders in newborns. This ubiquitous virus has infected more than 60% of the general population, with initial infection often being mild or asymptomatic. After infection, the virus remains latent in the body but can cause serious illness in immunocompromised individuals (i.e., people with HIV, transplant recipients, and those undergoing chemotherapy) or the elderly. HCMV is a leading cause of birth defects in developed countries. Up to 4 in 200 infants are born with HCMV due to congenital infection, and up to 10% of these infants will suffer long-term consequences. HCMV infection is also associated with hypertension and atherosclerosis in adults (Cheng et al. (May 2009). Früh K, ed. "Cytomegalovirus infection causes an increase of arterial blood pressure". PLoS Pathog. 5(5):e1000427). Therefore, HCMV is a public health priority. However, despite extensive efforts, a successful HCMV vaccine has not yet been developed.

[0007] Respiratory syncytial virus (RSV) is another ubiquitous virus that causes minimal discomfort in healthy adults and older children infected with it. However, it is the second leading cause of death worldwide in infants under one year old, after malaria. It is estimated that this virus causes 160,000 deaths globally each year. This virus causes serious respiratory infections and complications, including pneumonia and bronchiolitis. High-risk groups include infants under one year old, immunocompromised patients, the elderly, and people with cardiopulmonary conditions. Similarly, despite years of active research and development, there is currently no licensed RSV vaccine.

[0008] For diseases such as those caused by RSV and HCMV, there are currently no vaccines available, and current vaccine production methods generally do not show the expected efficacy, indicating a significant unmet need for providing alternative types of vaccines to address diseases with such catastrophic consequences.

[0009] Several genetically encoded systems capable of spontaneously or assistedly forming amide bonds have recently been described. For example, SpyTag is a peptide engineered such that when mixed with its protein partner SpyCatcher, the two components spontaneously and irreversibly form isopeptide bonds. The positions of the SpyTag and SpyCatcher components in the protein chain can be engineered to differ and they exhibit reactivity under a wide range of pH, buffer, and temperature conditions. SpyTag / SpyCatcher pairs and their variants and derivatives have been used in vaccine development, but so far only for the presentation of simple antigens. Other genetically encoded systems capable of spontaneously forming amide bonds include SnoopTag / SnoopTagJr and SnoopCatcher; RrgATag / RrgATag2 / DogTag and RrgACatcher; IsopepTag / IsopepTag-N and Pilin-C or Pilin-N; PsCsTag and PsCsCatcher; and SnoopTagJr and DogTag (mediated by SnoopLigase), as well as variants of all these systems.

[0010] The inventors have demonstrated that using a genetically encoded system capable of forming amide bonds enables the use of large / multi-component antigens in vaccine compositions, which can improve responses to large / multi-component antigens. This is a surprising result. Invention Overview

[0012] In a first aspect of the invention, a composition is provided comprising particles exhibiting protein components, wherein the composition comprises:

[0013] i) Protein components containing a first peptide tag, and

[0014] ii) The portion containing the second peptide tag.

[0015] The protein component and the portion are linked by isopeptide bonds between the first peptide tag and the second peptide tag, and the protein component exceeds 50 kDa.

[0016] In another aspect of the invention, a composition is provided comprising particles exhibiting protein components, wherein the composition comprises:

[0017] i) Protein components containing a first peptide tag, and

[0018] ii) The portion containing the second peptide tag.

[0019] The protein component and the portion are linked by heteropeptide bonds between the first peptide tag and the second peptide tag, and the protein component is a polymer.

[0020] Protein components can have any function; for example, they can be enzymes or possess enzymatic properties. Protein components can be full-length proteins, or they can be a portion, segment, domain, or truncation of a full-length protein. Protein components can be antigens or immunogens. Protein components can also be called antigenic components.

[0021] In another aspect of the invention, a composition is provided comprising particles displaying an antigenic component, wherein the composition comprises:

[0022] i) An antigenic component containing a first peptide tag, and

[0023] ii) The portion containing the second peptide tag.

[0024] The antigen component and the portion are linked by isopeptide bonds between the first peptide tag and the second peptide tag, and the antigen component exceeds approximately 50 kDa.

[0025] In some embodiments of any aspect of the invention, the protein component or antigen component may exceed 60 kDa, 70 kDa, 80 kDa, 90 kDa, 100 kDa, 110 kDa, 120 kDa, 130 kDa, 140 kDa, 150 kDa, 160 kDa, 170 kDa, 180 kDa, 190 kDa or greater, such as exceeding 200 kDa, exceeding 300 kDa or exceeding 400 kDa.

[0026] The multimer can contain any number of subunits, which may be covalently or non-covalently linked within the protein or antigenic components. The multimer can contain 2 to 20 subunits, optionally 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more subunits. Optionally, the multimer can be a dimer, trimer, tetramer, pentamer, hexamer, heptamer, octamer, nonamer, or decamer. The multimer can originate from any suitable pathogen, but is preferably a viral multimer.

[0027] Non-limiting examples of large protein components, i.e., those exceeding 50 kDa or as described above, include pentamer complex (PC) and gB glycoprotein from human cytomegalovirus (HCMV), G and F glycoproteins from RSV, hemagglutinin (HA) antigen and neuraminidase (NA) antigen from influenza A virus, Plasmodium falciparum Pfs230, Plasmodium falciparum CSP, human HER2 receptor, PCSK9, VAR2CSA, Plasmodium falciparum RIPR, varicella-zoster virus (VZV) glycoprotein E, rabies virus glycoprotein, and Epstein-Barr virus (EBV) gH / gL complex.

[0028] In some embodiments of any aspect of the invention, the protein component or antigen component may be a monomer or a polymer, such as a dimer, trimer, tetramer, or pentamer. In some embodiments of any aspect of the invention, the protein component or antigen component may be a protein or peptide complex.

[0029] Non-limiting examples of multimeric antigenic components include pentameric complexes (PC) and gB trimers from human cytomegalovirus (HCMV), G and F glycoproteins from RSV, and hemagglutinin (HA) and neuraminidase (NA) antigens from influenza A virus, some of which are described herein. Other examples include components from pathogens such as viruses, bacteria, fungal pathogens, parasites, or other disease vectors. Suitable multimeric antigenic components include, for example, multimeric antigenic components derived from viruses such as influenza virus (e.g., influenza hemagglutinin (HA) (e.g., influenza trimer)), respiratory syncytial virus (RSV), etc.

[0030] Protein components can be genetically fused to a first peptide tag and recombinantly expressed in suitable cells. For components that include post-translational modifications such as glycosylation, expression of recombinant proteins in eukaryotic or mammalian cell lines may be preferred.

[0031] In one embodiment, a "part" is a component on which a protein component or antigen component can be displayed, for example, making it usable by the immune system. In one embodiment, the part polymerizes to form the particle. Suitably, such a part can be a virus, bacteria, a polymerized scaffold, or a protein component polymerized to form a VLP (virus-like particle) for vaccination. Suitably, the part can be a component of bacteriophages, tobacco mosaic virus particles, adeno-associated virus-like particles (AAVLP), Escherichia coli, etc. In one embodiment, the part itself is a component of a virus, bacteria, etc., such that the polymerization (e.g., self-assembly) of the part forms a particle for displaying a protein component or antigen component. In one embodiment, the part can be a viral structural protein, such as a viral envelope or capsid protein or surface antigen. Examples of structural proteins include the matrix M1 protein and viral envelope M2 protein from influenza virus, HBsAg from hepatitis B virus, the viral capsid protein (CP3) of Escherichia coli bacteriophage AP205, hemagglutinin-neuraminidase from various viruses (including mumps), etc. Suitable viral structural proteins will be known to those skilled in the art. In other embodiments, the portion may be a protein or peptide, such as a polymerized domain forming nanoparticles like IMX313, or computationally derived particles such as MI3. In further embodiments, the portion may be a synthetic nanoparticle or a synthetic VLP, such as gold, a lipopeptide, or poly(lactic-co-glycolic acid) (PLGA) nanoparticles. Other suitable portions may include liposomes or outer membrane vesicles. Suitably, the portion containing the second peptide tag is the portion to which the second peptide tag is attached.

[0032] Using a structural surface antigen derived from a virus is preferred. Therefore, the attachment of a second peptide tag to the structural surface antigen allows for the formation of virus-like particles (VLPs), with the second peptide tag either attached to or displayed on the virus-like particle. VLPs are non-infectious, self-assembling nanoparticles, and their repeating, molecularly defined structures are attractive for engineered multivalent, particularly vaccine development. VLPs have been derived from components of many viral families, including hepatitis B virus (including hepatitis B small surface antigen (HBsAg)), parvoviridae (e.g., adeno-associated virus), retroviridae (e.g., HIV), flaviviridae (e.g., hepatitis C virus), and bacteriophages (e.g., Qβ, AP205). Any of these can be suitable for use as part of this invention.

[0033] The second peptide tag can be attached to the said moiety via genetic fusion. Such genetic fusion can occur at any suitable point in the sequence, not just at the end. Those skilled in the art will understand that the fusion protein can be recombinantly expressed in suitable cells.

[0034] Alternatively, the second peptide tag may be displayed on or attached to the moiety by means of chemical conjugation. This would require, for example, the presence of a reactive amine group to allow conjugation to occur.

[0035] Accordingly, in one embodiment, the portion is the surface antigen of hepatitis B virus (HBsAg). Suitably, as described herein, HBsAg has the amino acid sequence listed in SEQ ID NO:41 (or its functional equivalent).

[0036] In any embodiment of the invention, the protein component or antigenic component is an immunogenic component of the HCMV pentamer. Suitably, the antigenic component or immunogenic component is a component capable of generating an immune response, such as an antibody response against the component, when introduced into a subject, such as a patient. Accordingly, for example, an “immunogenic component of the HCMV pentamer” is a component capable of generating an anti-HCMV antibody response in a subject. Suitably, the immunogenic component comprises one or more (at least one) HCMV pentamer subunit components selected from gH, gL, pUL128, pUL130, and pUL131 (also known as pUL131A). In some embodiments, the immunogenic component comprises one or more of those “pUL” components or “UL” components. In other embodiments, the immunogenic component comprises one or more of those gH components or gL components. In one embodiment, the immunogenic component comprises a combination of one or more “UL” components with one or more components selected from gH or gL components. In another embodiment of the invention, the immunogenic component of the HCMV pentamer is an HCMV pentamer comprising all gH / gL / pUL128 / pUL130 / pUL131 subunits. Suitably, the gH / gL / pUL128 / pUL130 / pUL131 subunits have amino acid sequences corresponding to those derived from any known HCMV strain (including laboratory strains and / or clinical isolates), including Towne (GI: 239909366), AD169 (GI: 219879600), Toledo (GI: 290564358), and Merlin (GI: 155573956) or their functional equivalents. Functional equivalents are those that share some homology and differ only in a few amino acids, but retain the amino acid sequence that enables the formation of antigenic subunits or pentamers that provide protective antibodies. Suitable variants of components gH / gL / pUL128 / pUL130 / pUL131A are described, for example, in WO2014 / 005959 (see pages 4 to 10), which are hereby incorporated by reference. Advantageously, the use of HCMV pentamer subunits in vaccine methods can provide immunogenic protection against infection from a wide range of HCMV virus strains due to the high homology among strains at the pentamer amino acid sequence level.

[0037] In some implementations, the antigenic component may correspond to a component or portion of a pathogen or vector. For example, for ease of preparation, the antigenic component may lack a transmembrane domain. Suitably, in HCMV pentamers, for example, the immunogenic component of the HCMV pentamer comprises a gH subunit having a truncated transmembrane domain (truncated by the deletion of one or more amino acids from that region), such that the subunit is secreted into the cell supernatant during protein production in the host cell for purification.

[0038] In one embodiment, the gH / gL / pUL128 / pUL130 / pUL131A subunits have amino acid sequences (or their functional equivalents) listed in SEQ ID NO:28, 31, 35, 33, and 36 (with or without the indicated signal peptide). A functional equivalent means an amino acid sequence that shares some homology and differs only in a few amino acids, but retains functional properties such as the ability to form antigenic subunits or pentamers that provide protective antibodies. In some embodiments, the functional equivalent may share 70%, 80%, 90%, or more homology with the relevant amino acid sequences. In another embodiment, the gH / gL / pUL128 / pUL130 / pUL131A subunits are encoded by nucleic acid sequences such as those listed in SEQ ID NO:13, 16, 20, 18, and 21, or their codon-optimized forms (with or without the signal peptide coding sequence). In some embodiments, any one of the gH / gL / pUL128 / pUL130 / pUL131A subunits may have a signal peptide, such as a signal peptide present on a native protein of the strain, a functional equivalent of the signal peptide, or a signal peptide derived from a different HCMV strain. In some embodiments, any one of the gH / gL / pUL128 / pUL130 / pUL131A subunits may have a signal peptide derived from a heterologous protein. The selection of the signal peptide can be determined to target the expressed protein to a specific cellular (or extracellular) location or to confer other functions. After subunit expression, the signal peptide can be cleaved by native cellular mechanisms in the expression system used or in vitro enzymatic cleavage (e.g., by a signal peptidase). In some embodiments, any one of the gH / gL / pUL128 / pUL130 / pUL131A subunits may be expressed without a signal peptide. In some embodiments, native sequences, including introns, may be used, where these sequences can lead to higher expression levels. Suitably, the native nucleic acid sequence of UL128 contains two introns. In another embodiment, the nucleic acid sequence of UL131A contains an intron. In some embodiments, the intron may be removed. In some embodiments, the natural sequence may be codon-optimized for the relevant expression system.

[0039] In any embodiment of any aspect of the invention, the protein component or antigenic component is an immunogenic component of the RSV virus, such as an attachment glycoprotein (G protein) or a fusion glycoprotein (F protein), both of which control the initial phase of infection. G is a highly glycosylated 90 kDa type II integrated membrane protein that can mediate viral attachment to the host cell membrane through interaction with heparan sulfate on proteoglycans and is a good candidate for a protein component.

[0040] F protein is an integrated membrane protein composed of three F0 monomers, which are processed into F1 and F2 subunits during assembly, covalently linked by two disulfide bonds. F protein is highly conserved in RSV isolates from subgroups A and B, and the amino acid sequence shows 90% or higher identity. F is a class I fusion protein of 574 amino acids, consisting of a 50 kDa C-terminal F1 fragment and a 20 kDa N-terminal F2 fragment, making it a heterodimeric trimer. It is characterized by two furin cleavage sites that release a 27-amino acid glycopeptide and expose a hydrophobic fusion peptide at the F1 N-terminus. F2 has two N-linked glycosylation sites, while F1 has only one. After removing the 25-amino acid signal peptide and the 27-amino acid glycopeptide between F2 and F1, the remaining F extracellular domain consists of 472 amino acids. Subtypes A and B differ by only 25 amino acids in the F extracellular domain.

[0041] To develop antigen compositions from RSV-F protein, some studies have focused on preparing variants of the pre-fusion protein as a trimer. Variants are generated by genetically fusing two subunits of the mature pre-F protein into a single strand. The DS-Cav1 variant, with F2 and F1 genetic fusion and deletion of the fusion peptide and pep27 region, has been prepared. Differences in the linker between the F2 and F1 subunits have been shown to affect immunogenicity, and therefore variants can utilize different linker selections. The native RSV-F protein sequence is available at accession number P03420.1.

[0042] Several forms of prefused F proteins have been studied and developed, and subsequently published. These prefused trimers are all applicable to this invention. The DS-Cav1-stabilized fusion glycoprotein is derived from a natural protein. EP2222710, which is incorporated herein by reference, also discloses a recombinant RSV antigen comprising a soluble F protein polypeptide containing the F2 and F1 domains of the RSV-F protein polypeptide, as well as a trimerization domain. A highly stable prefused RSV-F protein is described in Krarup et al., Nat. Commun. 2015; 6:8143, again incorporated by reference.

[0043] WO2014 / 160463, incorporated herein by reference, describes the isolated recombinant RSV-F protein stabilized in a prefusion conformation and the nucleic acid molecule encoding the recombinant RSV-F protein.

[0044] WO2017 / 172890, incorporated herein by reference, describes the substitution-modified prefused RSV-F protein and its encoded nucleic acid. Further description is given in Nat Struct Mol Biol. 2016 Sep; 23(9):811-820, Iterative structure-based improvement of a respiratory syncytial virus fusion glycoprotein vaccine, M. Gordon Joyce, Baoshan Zhang, Li Ou, Man Chen, Gwo-YuChuang, Aliaksandr Druz, Wing-Pui Kong, Yen-Ting Lai, Emily J. Rundlet, Yaroslav Tsybovsky, Yongping Yang, Ivelin S. Georgiev, Miklos Guttman, Christopher R. Lees, Marie Pancera, Mallika Sastry, Cinque Soto, Guillaume BEStewart-Jones, Paul V. Thomas, Joseph G. Van Galen, Ulrich Baxa, Kelly K. Lee, John R. Mascola, Barney S. Graham and Peter D. Kwong, which is also incorporated herein by reference.

[0045] Exemplary nucleic acid sequences encoding recombinant F2-F1 extracellular domain protomers linked to the T4 Fibritin trimer domain are available from the following accession numbers: LP884611.1, LP884610.1, LP884609.1 and LP884608.1.

[0046] In some embodiments, the protein component or antigenic component may correspond to a component or portion thereof of a pathogen or vector. For example, for ease of preparation, the antigenic component may lack a transmembrane domain. Suitably, in the RSV-F protein or its pre-fusion conformation, for example, the immunogenic component of the F protein comprises F2-F1 subunits having a truncated transmembrane domain (truncated by the deletion of one or more amino acids from that region), such that the subunit is secreted into the cell supernatant during protein production in the host cell for purification. Thus, the RSV-F protein lacks a functional TM domain. Optionally, genetic fusion with a first peptide tag can indeed prevent the F protein from residing in the membrane, despite the presence of a functional transmembrane domain.

[0047] In one embodiment, the prefused stabilized subunits each have an amino acid sequence (or a functional equivalent thereof) listed in SEQ ID NO:50-58. A functional equivalent means an amino acid sequence that shares some homology and differs only in a few amino acids, but retains functional properties such as the ability to form an antigenic subunit that provides a protective antibody. In some embodiments, the functional equivalent may share 70%, 80%, 90%, or more homology with the relevant amino acid sequence. In one embodiment, the prefused stabilized RSV-F trimer may not contain a heterotrimerization domain.

[0048] The protein component contains a first peptide tag. This first peptide tag can be attached to the protein component by expressing the recombinant fusion protein. Those skilled in the art will know techniques for genetically modifying the fusion peptide sequence to express the recombinant protein in a suitable cellular system. For portions including post-translational modifications such as glycosylation, expression of the recombinant protein in eukaryotic or mammalian cell lines is preferred.

[0049] Advantageously, the use of first and second peptide tags forming isopeptide bonds, such as the SpyTag-SpyCatcher system described herein, allows for the "decoration" of large antigens and / or multimeric antigens, such as HCMV pentamers or their immunogenic components, to the portion displaying the large antigen, such as a VLP, in the correct formation and orientation. This presents the antigen to the immune system in a manner capable of generating anti-antigen (e.g., anti-HCMV) antibodies that provide protective / neutralizing / immunogenic effects. Conventional vaccination methods using soluble antigens (even large antigens, such as multimeric / pentamers) may be less effective in generating protective / neutralizing / immunogenic effects. Advantageously, displaying antigens (e.g., multimeric antigens) on particles such as VLPs or nanoparticles results in the presentation of a geometrically repeating array of the same antigen, which is more robust in triggering an immune response compared to soluble antigens. Larger-sized VLPs or other suitable particles may also have greater immunogenicity compared to "free" antigens. Furthermore, the display orientation of multimeric antigens, such as HCMV pentamers, can be important for immunogenicity. Using paired tags, such as the SpyTag-SpyCatcher system described herein, to attach multimeric antigens to particles allows the antigens to attach to the particles in a particularly advantageous orientation. For example, in the case of HCMV, the gH / gL subunit is less likely to have neutralizing epitopes compared to the “UL” subunit. Therefore, it is advantageous that the present invention allows the display orientation of the HCMV pentamer on the particle to be determined by the appropriate positioning of the first peptide tag, such that, for example, the “UL” subunit is displayed outwards towards the particle, and thus more readily acquired by the individual's immune system. Optionally / additionally, the positioning of the first peptide tag on the antigen can be determined to produce an antigen orientation similar to that on the natural virus, thereby presenting particles to the immune system that display the antigen in an orientation more likely to induce an immune response against invading live virus.

[0050] In contrast, conventional methods for presenting proteins on VLPs can involve chemical conjugation. The disadvantage of chemical conjugation is that such chemical reactions can be more random, making it impossible to definitively obtain the correct (e.g., immunologically preferred) antigen orientation, and may only represent a small fraction of the conjugation reactions obtained. Furthermore, the processes involved in chemical conjugation may make it less likely that the 3-D structure required for proper antigen presentation will be preserved. For example, some drawbacks of conventional methods are described in Brune et al. (2016; Scientific Reports, 6:19234, DOI:10.1038 / srep19234), Brune et al. (Bioconjugate Chemistry, 2017, 28, 1544-1551), and Leneghan et al. (2017) Scientific Reports, 7:3811.

[0051] Similarly, genetic fusion of antigens with viral capsid proteins has proven challenging and time-consuming due to problems with misfolding and determining the optimal conditions for expression of both components. Furthermore, genetic fusion is unsuitable for expressing large antigens or multi-component antigens because efficient expression in the correct conformation is too difficult to achieve.

[0052] To present protein or antigenic components in an immunogenic manner, the location of the first peptide tag needs to be carefully designed to preserve the native protein conformation and, where appropriate, to optionally retain any post-translational modifications. For some antigens, retention of glycosylation does not affect the presentation of the epitope, but for others, retention or removal of glycosylation improves efficacy. For protein components that are transmembrane proteins, the transmembrane portion of the protein component provides a good target for positioning the first peptide tag because, for example, the sequence does not involve the conformation of the antigenic protein component, and the function provided is no longer needed in the vaccine. If the protein component does not contain a transmembrane protein, fusing the first peptide tag to the C-terminus or N-terminus of the component or its subunits (in the case of multimers) can prove advantageous; however, the first peptide tag can also be included in any part of the sequence. Optionally, the first peptide tag can be positioned in a loop on the protein or antigenic component.

[0053] To present immunogenic components, such as those of the HCMV pentamer, the location of the first peptide tag needs to be carefully designed to maintain the native protein conformation. For HCMV, in one embodiment, attachment is via the gH subunit, suitably through the C-terminus of the gH subunit, or through the transmembrane domain (or a portion thereof) of the gH subunit. In addition to maintaining the conformation of the pentamer (or its components), this rational design also presents the target region of the pentamer outwards towards the particle, as discussed above. As used herein, the target region is a portion of a protein known to elicit neutralizing antibodies and may also be referred to as the immunogenic portion.

[0054] To present immunogenic components, such as those of the RSV prefused F protein, the location of the first peptide tag needs to be carefully designed to preserve the native protein conformation. For the RSV-F prefused protein, in one embodiment, a suitable attachment is via the C-terminus of the F prefused protein, through the 3' end of the nucleic acid encoding it. Besides preserving the conformation of the prefused F protein (or its components), this rational design also ensures that most of the neutralizing epitopes of the prefused F protein are presented outwards towards the particle, as discussed above. The same considerations apply to any other variant of the F protein. The inventors have demonstrated that including the first peptide tag at the C-terminus is effective, leaving the immunogenic protein components to fold correctly.

[0055] In one embodiment, the first and second peptide tags are part of a peptide tag / binding partner pair capable of forming an isopeptide bond. This isopeptide bond can be spontaneous, i.e., without assistance, or require assistance, i.e., assistance from a ligase or other helper. Suitably, the first and second peptide tags are SpyTag / SpyCatcher pairs. Suitably, the first and second peptide tags are selected from a list including: SpyTag / SpyCatcher, SnoopTag / SnoopTagJr and SnoopCatcher, RrgATag / RrgATag2 / DogTag and RrgACatcher, IsopepTag / IsopepTag-N and Pilin-C or Pilin-N, PsCsTag and PsCsCatcher, and SnoopTagJr and DogTag (mediated by SnoopLigase), as well as variants, derivatives, and modifications of all these systems.

[0056] Suitably, the first peptide tag is a peptide tag from a peptide tag / binding partner pair, such as SpyTag, and the second peptide tag is a binding partner, such as SpyCatcher. In another embodiment, the first peptide tag is a binding partner, such as SpyCatcher, and the second peptide tag is a peptide tag component from a peptide tag / binding partner pair, such as SpyTag. Suitably, the first peptide tag is a peptide tag from a peptide tag / binding partner pair, such as SnoopTag, and the second peptide tag is a binding partner, such as SnoopCatcher. In another embodiment, the first peptide tag is a binding partner, such as SnoopCatcher, and the second peptide tag is a peptide tag component from a peptide tag / binding partner pair, such as SnoopTag. Thus, the first peptide tag can be a "tag" or a "catcher"; the second peptide tag is the partner of this pair, which is either a "catcher" or a "tag". Suitable peptide tag / binding partner pairs are described in detail in WO2011 / 09877, WO2016 / 193746, WO2018 / 18951 and WO2018 / 197854, which are incorporated herein by reference.

[0057] In one embodiment, the protein component or antigen component is attached to any one of SpyTag, SnoopTag, RrgATag, RrgATag2, DogTag, IsopepTag, IsopepTag-N, PsCsTag, and SnoopTagJr as a first peptide tag.

[0058] If desired, the first peptide tag can be attached via a connector, which can be rigid or flexible. Those skilled in the art will recognize which connector is appropriate.

[0059] In another embodiment, the portion is attached to any one of SpyCatcher, SnoopCatcher, RrgACatcher, Pilin-C, Pilin-N, PsCsCatcher, and DogTag (mediated by SnoopLigase) as a second peptide tag.

[0060] As previously discussed, the portion can be any suitable portion, including synthetic polymerization platforms.

[0061] The second peptide tag can be attached to any suitable location of the moiety without affecting its ability to fold and form a suitable conformation. Genetic fusion is preferred. It is preferable to include the second peptide tag at the C-terminus or N-terminus of the moiety, but the second peptide tag can also be included in any part of the sequence. Optionally, the second peptide tag can be positioned in a loop on the moiety. For example, the genetic fusion of SpyCatcher with the N-terminus of the viral capsid protein (CP3) of RNA bacteriophage AP205 is described in Brune et al., Scientific Reports, volume 6, article number: 19234 (2016). Alternative fusions using self-assembled synthetic proteins as a polymerization platform are discussed in Brune et al., ACS Nano, 2018, 12(9), pp 8855-8866. Optionally, the second peptide tag can be attached by chemical conjugation.

[0062] If desired, the second peptide tag can be attached via a connector, which can be rigid or flexible. Those skilled in the art will recognize which connector is appropriate.

[0063] In one embodiment, an antigenic component, such as an HCMV pentamer or its immunogenic component, is attached to a SpyTag. A suitable SpyTag has the amino acid sequence listed in SEQ ID NO:30.

[0064] SpyTag can be attached via a linker. Suitable linkers include those having the amino acid sequence listed in SEQ ID NO:29.

[0065] In another embodiment, the portion is attached to a SpyCatcher binding partner (second peptide tag). The portion may suitably be HBsAg. A suitable SpyCatcher has the amino acid sequence listed in SEQ ID NO:38. In one embodiment, the SpyCatcher is attached via a linker. The linker may be a rigid linker or a flexible linker, suitably wherein the linker has the amino acid sequence listed in SEQ ID NO:39.

[0066] In another embodiment, the protein composition or antigen composition according to any aspect or embodiment of the invention further comprises another (preferably different) protein containing a first peptide tag.

[0067] In another embodiment, the composition according to any aspect or embodiment of the invention further comprises another (preferably different) antigen containing a first peptide tag, such as another HCMV antigen. Suitably, the other HCMV antigen is glycoprotein B. Suitably, the sequence of glycoprotein B is described, for example, in WO2014 / 005959, see SEQ ID NO:21, 22, 23 or 36. In one embodiment, the composition comprises particles (e.g., VLPs) exhibiting both the HCMV pentamer and another HCMV antigen.

[0068] In one embodiment, the composition is an immunogenic composition or a vaccine composition. Preferably, the immunogenic composition or vaccine composition is one that, when administered to an individual, can induce an immune response, such as an antibody response. Suitably, the immune response may be a protective immune response. Suitable immunogenic compositions may also contain additional components, including adjuvants, immunostimulants, and / or pharmaceutically acceptable excipients.

[0069] For example, suitable adjuvants can be based on aluminum, peptides, squalene, liposomes, oil-in-water emulsions, and saponins, and may include... MF59, AS01, MatrixM, muramyl dipeptide, and Quil A. Water-in-oil adjuvants are also suitable. Squalene oil-in-water emulsions, such as Addavax, are also suitable. TM That is appropriate.

[0070] Accordingly, in another aspect or embodiment of the invention, an immunogenic composition or vaccine composition comprising the composition according to the invention is provided. Suitably, the vaccine composition comprises a vaccine dose, which is an amount of the composition according to the invention, providing immunogenicity from the infectious agent / vector, preferably immune protection against the infectious agent / vector, such as neutralization against HCMV infection. Suitably, the vaccine composition comprises a vaccine dose, which is an amount of the composition according to the invention, providing neutralization against the infectious agent / vector, such as neutralization against RSV infection. Antibodies, preferably neutralizing antibodies generated by the immunogenic composition, can be detected and measured by methods familiar to those skilled in the art, including, for example, standardized ELISA assays or microneutralization assays described herein.

[0071] On the other hand, a VLP containing the following is provided:

[0072] i) The portion containing the first peptide tag

[0073] ii) Proteins containing a second peptide tag

[0074] The first peptide tag and the second peptide tag form an isopeptide bond. In some embodiments, the moiety is HBsAg. However, as previously described, any suitable moiety can be used.

[0075] Suitablely, the first peptide tag is a peptide tag derived from a peptide tag / binding partner pair, such as SpyTag, and the second peptide tag is a binding partner, such as SpyCatcher. In another embodiment, the first peptide tag is a binding partner, such as SpyCatcher, and the second peptide tag is a peptide tag derived from a peptide tag / binding partner pair, such as SpyTag. Other suitable peptide tag / binding partner pairs are described herein and will be known to those skilled in the art. Suitablely, the first and second peptide tags are selected from a list including: SpyTag / SpyCatcher, SnoopTag / SnoopTagJr and SnoopCatcher, RrgATag / RrgATag2 / DogTag and RrgACatcher, IsopepTag / IsopepTag-N and Pilin-C or Pilin-N, PsCsTag and PsCsCatcher, and SnoopTagJr and DogTag (mediated by SnoopLigase), as well as variants, derivatives and modifications of all these systems.

[0076] Suitablely, the protein containing the second peptide tag is a protein or peptide complex greater than 50 kDa. The protein containing the second peptide tag can be greater than 50 kDa, 60 kDa, 70 kDa, 80 kDa, 90 kDa, 100 kDa, 110 kDa, 120 kDa, 130 kDa, 140 kDa, 150 kDa, or 160 kDa, 170 kDa, 180 kDa, 190 kDa or larger, such as exceeding 200 kDa, exceeding 300 kDa, or exceeding 400 kDa.

[0077] In one embodiment, the protein containing the second peptide tag is a multimeric protein. In one embodiment, the protein containing the second peptide tag is an antigen, preferably a multimeric antigen. Suitably, the multimeric antigen may be an HCMV pentamer as described herein. Suitably, the protein may be an RSV-F protein or a derivative thereof (such as a prefused F protein). In one embodiment, the protein containing the second peptide tag is an immunogenic component of an HCMV pentamer. HCMV pentamers (gH / gL / pUL128 / pUL130 / pUL131) as described herein and containing suitable linkers and tags have a molecular weight exceeding 160 kDa. Other suitable large proteins or antigens or multimeric proteins or antigens include antigens from other infectious agents, including viruses such as influenza virus, RSV, etc.

[0078] Advantageously, using HBsAg as a vector (VLP) in this manner may also produce an enhancement of anti-HepB, or be described as an anti-hepatitis B virus (HBV) response.

[0079] On the other hand, a VLP containing the following is provided:

[0080] i) Proteins containing a first peptide tag

[0081] ii) The portion containing the second peptide tag

[0082] The first peptide tag and the second peptide tag form an isopeptide bond. In some embodiments, the moiety is HBsAg. However, as previously described, any suitable moiety can be used.

[0083] Suitablely, the first peptide tag is a peptide tag derived from a peptide tag / binding partner pair, such as SpyTag, and the second peptide tag is a binding partner, such as SpyCatcher. In another embodiment, the first peptide tag is a binding partner, such as SpyCatcher, and the second peptide tag is a peptide tag derived from a peptide tag / binding partner pair, such as SpyTag. Other suitable peptide tag / binding partner pairs are described herein and will be known to those skilled in the art. Suitablely, the first and second peptide tags are selected from a list including: SpyTag / SpyCatcher, SnoopTag / SnoopTagJr and SnoopCatcher, RrgATag / RrgATag2 / DogTag and RrgACatcher, IsopepTag / IsopepTag-N and Pilin-C or Pilin-N, PsCsTag and PsCsCatcher, and SnoopTagJr and DogTag (mediated by SnoopLigase), as well as variants, derivatives and modifications of all these systems.

[0084] Suitably, the protein containing the first peptide tag is a protein or peptide complex greater than 50 kDa. The protein containing the first peptide tag can be a protein or peptide complex greater than 50 kDa, 60 kDa, 70 kDa, 80 kDa, 90 kDa, 100 kDa, 110 kDa, 120 kDa, 130 kDa, 140 kDa, 150 kDa, or 160 kDa or greater, particularly 200 kDa, 300 kDa, or even 400 kDa or greater. In one embodiment, the protein containing the first peptide tag is a multimeric protein. In one embodiment, the protein containing the second peptide tag is an antigen, preferably a multimeric antigen. Suitably, the multimeric antigen can be an HCMV pentamer as described herein. Suitably, the protein can be an RSV-F protein or a derivative thereof (such as a prefused F protein). In one embodiment, the protein containing the first peptide tag is an immunogenic component of an HCMV pentamer. As described herein, HCMV pentamers (gH / gL / pUL128 / pUL130 / pUL131A) containing suitable linkers and tags have a molecular weight exceeding 160 kDa. Other suitable large proteins or antigens, or multimeric proteins or antigens, include antigens from other infectious agents, including viruses such as influenza virus, RSV, etc.

[0085] Advantageously, using HBsAg as a vector (VLP) in this manner may also produce enhanced anti-HBV resistance.

[0086] In another aspect of the invention, an HCMV pentamer linked to SpyTag as described herein is provided.

[0087] According to another aspect of the present invention, a method for producing a composition or VLP according to the present invention is provided, the method comprising:

[0088] - Introduce the first nucleic acid encoding a first genetic fusion product of a first protein and a first peptide tag into the first host cell;

[0089] - Incubate the first host cell under conditions expressing the first genetic fusion; optionally purify the expressed component;

[0090] - Introduce a second nucleic acid encoding a second genetic fusion product with a second peptide tag into a second host cell;

[0091] - Incubate the second host cell under conditions expressing the second genetic fusion; optionally purify the expressed component;

[0092] - Incubate the expressed component under conditions where an isopeptide bond is formed between the first peptide tag and the second peptide tag; optionally purify the resulting composition.

[0093] Appropriately, the expressed components are incubated together to allow for the formation of isopeptide bonds. The formation of isopeptide bonds may require co-incubation with a ligase or similar agent.

[0094] Suitablely, the method for producing the composition or VLP according to the invention can be used to produce a composition comprising an antigen component displayed on the VLP.

[0095] In some embodiments, when the “immunogenic component of the HCMV pentameric component” comprises the entire HCMV pentameric component, the recombinant production of the HCMV pentameric component requires each subunit to be expressed in the correct stoichiometry to form a pentamer and to fold correctly for assembly. In these embodiments, it is necessary to exclude complexes containing only the desired pentameric portions (e.g., gH / gL dimers and tetramers, or tetramers lacking any of the five subunits) from the final product. Advantageously, the present invention overcomes the problems that may be associated with expressing all vaccine components (i.e., the five subunits of HBsAg and HCMV pentameric) in a single system in other ways by providing a simple method for preparing the components separately and then concatenating them. Accordingly, in one embodiment, a purification tag is incorporated into UL130 (Hofmann et al., DOI 10.1002 / bit 25670). A similar principle will also apply to other immunogenic components.

[0096] In some implementations, when the “immunogenic component of RSV-F protein” comprises the whole F protein or a derivative thereof, the recombinant generation of the component of the F protein or the derivative thereof requires its proper folding for assembly, wherein the derivative includes a prefused F protein trimer.

[0097] Suitably, this method is used to produce a composition comprising HCMV pentamers displayed on HBsAg VLPs. Suitably, this method is used to produce a composition comprising RSV-F prefused F protein trimers displayed on HBsAg VLPs.

[0098] In another aspect of the invention, a vaccine for the prevention and / or treatment of a disease is provided. Suitably, the vaccine comprises a composition or VLP according to any aspect or embodiment of the invention. In one embodiment, the disease is HCMV infection. In another aspect, a prophylactic method for treating HCMV is provided. Suitably, the vaccine is used in humans. Suitably, the vaccine is used in adults, such as women of childbearing age or pregnant women. In another aspect, the invention provides a method for inducing an immunogenic response (e.g., a protective immune response) against HCMV in an individual, wherein the method comprises administering a composition according to any aspect or embodiment of the invention.

[0099] In another aspect of the invention, compositions according to any aspect of the invention are provided for use as pharmaceuticals.

[0100] In another aspect of the invention, compositions according to any aspect of the invention are provided for use as vaccines, preferably for the prevention and / or treatment of HCMV infection. The compositions according to the invention, used as medicines or vaccines, can be administered to adults, such as women of childbearing age or pregnant women.

[0101] In another aspect, the present invention provides nucleic acid molecules for use with the method according to the invention. In one embodiment, the nucleic acid molecule according to the invention comprises a nucleic acid sequence encoding an amino acid sequence listed in any one of SEQ ID NO: 27 to 41. In one embodiment, the nucleic acid molecule according to the invention comprises a nucleic acid sequence listed in any one of SEQ ID NO: 12 to 26 or 42 to 46.

[0102] In another aspect, the present invention provides a variety of nucleic acid molecules, including those encoding the amino acid sequences listed in SEQ ID NO:27 to 41. In one embodiment, the nucleic acid molecules of the present invention include those having the sequences listed in any one of SEQ ID NO:12 to 26 or 42 to 46.

[0103] In another aspect, the present invention provides nucleic acid molecules for use with the method according to the invention. In one embodiment, the nucleic acid molecule according to the invention comprises a nucleic acid sequence encoding an amino acid sequence listed in any one of SEQ ID NO: 50 to 58. In one embodiment, the nucleic acid molecule according to the invention comprises a nucleic acid sequence listed in any one of SEQ ID NO: 47 to 55.

[0104] On the other hand, the present invention provides a variety of nucleic acid molecules, including those encoding the amino acid sequences listed in SEQ ID NO:50 to 58. In one embodiment, the nucleic acid molecules according to the invention include those having the sequences listed in any one of SEQ ID NO:47 to 55.

[0105] In another aspect, the present invention provides a vector comprising a nucleic acid molecule or a plurality of nucleic acid molecules according to the present invention. Suitable vectors are expression vectors for expressing the amino acid sequence of any component of a composition according to the present invention.

[0106] In another aspect, the present invention provides host cells for expressing components of compositions according to the invention. Suitable host cells may be those used for transient or stable expression of these components. Methods for expressing CMV proteins and host cells are described, for example, in WO2014 / 005959 and WO2016 / 067239, both of which are incorporated herein by reference. In some embodiments, the components may be glycosylated.

[0107] In another aspect of the invention, a kit is provided comprising a composition according to the invention for a primary-boost vaccination regimen. Suitably, the kit may comprise a primary vaccination composition containing a first immunogenic composition according to the invention and a booster composition containing a second immunogenic composition according to the invention. Optionally, the kit may be provided to provide single-dose or multi-dose vaccination regimens, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 doses. Accordingly, in another aspect, the invention provides a dosage regimen comprising doses administered at intervals of approximately 3 weeks.

[0108] Attached Figure

[0109] Figure 1 SDS-PAGE and Western blot analysis of purified pentamer-SpyTag under non-reducing and reducing conditions. Lane 1: ColorPlus pre-stained wide-range protein ladder, size expressed in kDa; Lane 2: Non-reducing sample; Lane 3: Reducing sample. A) SDS-PAGE and Coomassie staining analysis indicated the position of the HCMV pentamer components; the left side of the gel was non-reducing, and the right side was reducing. B) Western blot analysis using anti-HCMV pentamer antibody.

[0110] Figure 2SDS-PAGE and Western blot analysis of purified SpyCatcher-HBsAg under non-reducing (NR) and reducing (R) conditions. A) SDS-PAGE and Coomassie staining analysis. B) Western blot analysis using anti-HBsAg monoclonal antibody.

[0111] Figure 3 HPLC analysis was performed using an S200 Increase 3.2 / 300 column. A) 10 μl of purified HCMV pentamer-SpyTag was loaded and eluted as a single peak. B) 10 μl of purified SpyCatcher-HBsAg was loaded and eluted using the volume of water outside the column as a single main peak.

[0112] Figure 4 SDS-PAGE and Western blot analysis of conjugated pentamers-SpyTag and SpyCatcher-HBsAg under reducing conditions. 1: ColorPlus pre-stained broad-range protein ladder, size expressed in kDa; 2: Conjugate; 3: Pentamer-SpyTag; 4: SpyCatcher-HBsAg. A) SDS-PAGE and Coomassie staining analysis. B) Western blot using anti-HBsAg monoclonal antibody. C) Western blot using anti-pentamer polyclonal antibody.

[0113] Figure 5 HPLC analysis was performed using an S200 Increase 3.2 / 300 column. 30 μl of the conjugated pentamer-SpyTag-SpyCatcher-HBsAg was loaded, and the volume of water outside the column was used as the main peak for elution.

[0114] Figure 6 Immunogenicity of HCMV pentamer-HBsAg vaccine compared to pentamer protein vaccine after a single immunization, using Addavax as an adjuvant. BALB / c mice were immunized with HCMV pentamer-SpyTag as either the soluble protein or the pentamer-HBsAg VLP. Serum titers were measured using a standardized ELISA. Lines represent the mean, and error bars represent the standard deviation (n = 10). Mice immunized with HCMV pentamer-HBsAg VLP showed a significantly stronger serum IgG antibody response compared to mice immunized with HCMV pentamer protein alone, even at a 10x lower dose of the pentamer-equivalent VLP.

[0115] Figure 7Neutralizing activity of mouse serum immunized with HCMV pentamer-HBsAg vaccine compared to pentamer protein vaccines. Addavax was used as an adjuvant for the vaccine, and responses were shown after a single immunization (primary) or two immunizations (booster). [The text then abruptly shifts to a discussion of AD169, mentioning its use as an adjuvant.] wt131 NT was measured in ARPE-19 cells infected with a strain (displaying a functional pentamer). 50 In the same assay, the neutralizing titers of Cytogam and the commercially available neutralizing anti-gH mAb (HCMV16(51C1), from Bio-Rad Antibodies) are shown.

[0116] Figure 8 Immunogenicity of HCMV pentamer-HBsAg vaccine versus pentamer protein vaccine after one or two immunizations without adjuvant. BALB / c mice were immunized with 1 μg or 0.1 μg of HCMV pentamer-SpyTag (pentamer-HBsAg) conjugated with SpyCatcher-HBsAg, or with 1 μg of pentamer-SpyTag protein. Titers were measured from mouse serum by standardized ELISA. The line represents the mean, and the error bar represents the standard deviation (n = 10). Mice immunized with HCMV pentamer-HBsAg VLP showed a significantly stronger serum IgG antibody response compared to mice immunized with HCMV pentamer protein alone, even when the pentamer equivalent VLP dose was 10x lower.

[0117] Figure 9 Neutralizing activity of mouse serum immunized with HCMV pentamer-HBsAg vaccine compared to pentamer protein vaccines. The vaccine was adjuvant-free and demonstrated responses after a single immunization (primary) or two immunizations (booster). [The text then abruptly shifts to a completely unrelated topic:] ...to AD169... wt131 NT50 was measured in ARPE-19 cells infected with the strain (exhibiting a functional pentamer). Neutralizing titers of Cytogam and a commercially available neutralizing anti-gH mAb (HCMV16(51C1), from Bio-Rad Antibodies) are shown in the same assay.

[0118] Figure 10 SDS-PAGE and Western blot analysis of purified RSV-F-SpyTag under non-reducing and reducing conditions. A) SDS-PAGE and Coomassie staining analysis: Lane 1: ColorPlus pre-stained broad-range protein ladder; Lane 2: Non-reducing sample; Lane 3: Reducing sample. B) Western blot analysis using anti-RSV-F monoclonal antibody: Lane 1: ColorPlus pre-stained broad-range protein ladder; Lane 2: Non-reducing sample; Lane 3: Reducing sample.

[0119] Figure 11 SDS-PAGE and Western blot analysis of RSV-F-SpyTag conjugated with SpyCatcher-HBsAg under reducing conditions. 1: ColorPlus pre-stained broad-range protein ladder; 2: RSV-F-SpyTag--SpyCatcher-HBsAg conjugate; 3: RSV-F-SpyTag; 4: SpyCatcher-HBsAg. A) SDS-PAGE and Coomassie staining analysis. B) Western blot using anti-HBsAg monoclonal antibody. C) Western blot using anti-RSV-F monoclonal antibody.

[0120] Figure 12 Immunogenicity of conjugated RSV-F-SpyTag-SpyCatcher-HBsAg ('Sc9-10-HBsAg') compared to unconjugated RSV-F-SpyTag ('Sc9-10'). In the absence of adjuvants or with Addavax... TM As an adjuvant, BALB / c mice (n=8) were immunized with 1 μg of RSV-F-SpyTag conjugated with SpyCatcher-HBsAg (RSV-F VLP) or 1 μg of RSV-F-SpyTag protein. The RSV-F antigen was sc9-10 DS-Cav1 A149C Y458C-SpyTag. Invention Details

[0122] Virus-like particles

[0123] Traditionally, vaccine approaches have used attenuated or dead whole pathogens, although this has been superseded by recombinant subunit vaccines containing proteins from the appropriate pathogen. More recently, approaches using virus-like particles (VLPs) have been developed. VLPs are particles whose size (approximately 20–200 nm), shape, and repeating protein arrangements are similar to viruses, but lack any genetic material from the pathogen. Due to their size, VLPs are more likely to flow to lymph nodes, making them ideal for uptake and presentation by antigen-presenting cells. Furthermore, their repeating structure facilitates complement fixation and B-cell receptor cross-linking (Kushnir et al., Vaccine 2012; Vol 31(1):58–83). However, their mechanisms of action are not limited to theory.

[0124] HCMV

[0125] Human cytomegalovirus (HCMV, also known as human herpesvirus-5 (HHV-5)) is a virus that most adults are exposed to, with initial infection often being mild or asymptomatic. After infection, the virus remains latent in the body but can cause serious illness in immunocompromised individuals or the elderly. HCMV is also a leading cause of birth defects in developed countries. Up to 4 in 200 infants are born with HCMV due to congenital infection, and up to 10% of these infants will suffer long-term consequences. HCMV infection is also associated with hypertension and atherosclerosis in adults (Cheng et al. (May 2009). Früh K, ed. "Cytomegalovirus infection causes an increase of arterial blood pressure". PLoS Pathog. 5(5):e1000427).

[0126] Based on observations that antibodies against the pentamer complex of HCMV, containing viral proteins gH / gL / pUL128 / pUL130 / pUL131A, can neutralize viral entry into epithelial cells and reduce the risk of perinatal transmission of HCMV, this complex has been identified as a potentially useful vaccine target for HCMV. However, despite significant efforts, no successful HCMV vaccine has been developed to date.

[0127] HCMV pentamer

[0128] HCMV strains, including clinical isolates and laboratory strains, have distinct genomic sequences. HCMV strains include Merlin (GI:155573956), Towne (GI239909366), AD169 (GI:219879600), Toledo (GI290564358), and TB40 / E. HCMV contains various membrane proteins and protein complexes. The pentamer proteins gH / gL / pUL128 / pUL130 / pUL131A are important for HCMV infection of epithelial and endothelial cells, believed to occur via endocytosis. Other combinations of components of this complex have been shown to be important for infection, for example, of fibroblasts. The “pUL” subunit / component is also referred to as “UL”; “pUL131” is also referred to as “pUL131A” and “pUL131a” or “UL131A”.

[0129] Several HCMV strains have been deposited in the ATCC and can be found as follows: Merlin (VR-1590), Towne (VR-977), and AD169 (VR-538). Genome sequences can be referenced by the following accession numbers: Merlin (AY446894.2), Towne (GO121041.1), AD169 (FJ527563.1), Toledo (GU37742.2), and TB40 / E (KF297339.1).

[0130] RSV

[0131] Respiratory syncytial virus (RSV) is a leading cause of severe respiratory illness in young children worldwide. An estimated 3.4 million children under the age of 5 are hospitalized annually with severe RSV lower respiratory infections, with the highest morbidity rate in children under 6 months of age. Most deaths occur in infants under 1 year of age and in developing countries. Currently, options for prevention and control are limited.

[0132] RSV prefused trimer

[0133] The F glycoprotein is a type I viral fusion protein. It is believed that the RSV F precursor (F0) is cleaved by a furin-like protease at two sites, producing three fragments. The shorter N-terminal fragment (F2) is covalently attached to the larger C-terminal fragment (F1) via two disulfide bonds. The 27-amino acid fragment in between dissociates after cleavage and is not present in the mature protein.

[0134] As previously discussed, numerous stabilized prefused F trimers are available. In the examples submitted herein, exemplary sequences encoding these prefused trimers are shown in SEQ ID NO:48, 49, 54, and 55. Sequences containing fusion with the SpyTag are included as SEQ ID NO:47 and 53. For the prefused trimers, the amino acid sequences are shown in SEQ ID NO:51, 52, 57, and 58, and for those with the SpyTag, the amino acid sequences are shown in SEQ ID NO:50 and 56. Other exemplary sequences are mentioned herein.

[0135] Peptide tag / binding partner pair

[0136] Proteins capable of spontaneously forming isopeptide bonds (so-called "isopeptide proteins") have been advantageously used to develop peptide tag / peptide-binding partner pairs (i.e., two-part linkers) that covalently bind to each other and provide irreversible interactions (see, for example, WO2011 / 098772 and WO 2016 / 193746, both incorporated herein by reference, and WO2018 / 189517 and WO2018 / 197854, both incorporated herein by reference). In this regard, proteins capable of spontaneously forming isopeptide bonds can be expressed as separate fragments to provide a peptide tag and a peptide-binding partner, wherein these two fragments can be covalently recombined through the formation of isopeptide bonds, thereby linking and fusing to the peptide tag and its peptide-binding partner into a molecule or component. The isopeptide bond formed by the peptide tag and its polypeptide binding partner is stable under conditions where non-covalent interactions would rapidly dissociate, such as over long periods of time (e.g., several weeks), at high temperatures (up to at least 95°C), under high forces, or under harsh chemical treatments (e.g., pH 2–11, organic solvents, detergents, or denaturants).

[0137] Isopeptide bonds are amide bonds formed between a carboxyl / formamide group and an amino group, wherein at least one carboxyl or amino group is located outside the protein backbone (the protein's scaffold). Such bonds are chemically irreversible under typical biological conditions and are resistant to most proteases. Because isopeptide bonds are inherently covalent, they lead to some of the strongest protein interactions measured.

[0138] In short, the two-part linker, namely the peptide tag and its polypeptide binding partner (the so-called peptide tag / binding partner pair), can originate from a protein (isopeptide protein) capable of spontaneously forming isopeptide bonds, wherein the protein's domains are expressed separately to produce a peptide "tag" and a peptide or polypeptide binding partner (or "catcher"). The peptide "tag" contains one of the residues involved in the isopeptide bond (e.g., aspartic acid or asparagine, or lysine), and the peptide or polypeptide binding partner (or "catcher") contains another residue involved in the isopeptide bond (e.g., lysine, or aspartic acid or asparagine) and at least one other residue required for the formation of the isopeptide bond (e.g., glutamic acid). Mixing the peptide tag and binding partner results in the spontaneous formation of an isopeptide bond between the tag and the binding partner. Therefore, by incorporating the peptide tag and binding partner separately into different molecules or components, such as proteins, it is possible to covalently link the molecules or components through the isopeptide bonds formed between the peptide tag and the binding partner, i.e., to form a linker between the molecules or components incorporating the peptide tag and the binding partner.

[0139] The spontaneous formation of isopeptide bonds can be isolated and does not require the addition of any other entities. For some peptide tags and tag-coupler pairs, the presence of auxiliary entities such as ligases may be necessary to generate isopeptide bonds.

[0140] The peptide tag / binding partner pair (two-part linker), known as SpyTag / SpyCatcher, is derived from the CnaB2 domain of the Streptococcus pyogenes FbaB protein (Zakeri et al., 2012, Proc Natl Acad Sci USA 109, E690-697) and is used in a variety of applications, including vaccine development (Brune et al., 2016, Scientific Reports 6, 19234; Thrane et al., 2016, Journal of Nanobiotechnology 14, 30).

[0141] Suitablely, the first and second peptide tags form a peptide tag / binding pair, referred to as SpyTag / SpyCatcher. Suitablely, the SpyCatcher component is DeltaN1(ΔN1)SpyCatcher (as described in Li, L., Fierer, JO, Rapoport, TA & Howarth, M. Structural analysis and optimization of the covalent association between SpyCatcher and a peptide Tag. J. Mol. Biol. 426, 309-317 (2014)), which, compared to “SpyCatcher”, has a 23-amino acid truncation at the N-terminus (SEQ ID No. 38).

[0142] In other embodiments, the first and second peptide tags form a peptide tag / binding pair, which is a mutant form of SpyTag / SpyCather exhibiting an increased rate of isopeptide bond formation, such as, for example, those described in co-peptide bond formation application GB1706430.4. In some embodiments, these mutant forms may be useful in cases where attachment and / or slow reaction or steric hindrance may be problematic for large proteins (e.g., >50 kDa or >100 kDa, such as the >160 kDa HCMV pentamer protein described herein).

[0143] In other embodiments, the isopeptide protein may include SnoopTag / SnoopCatcher, as described, for example, in WO2016 / 193746.

[0144] In some implementations, one or two isopeptides may have N-terminal or C-terminal truncations while still maintaining the reactivity of the isopeptide bond.

[0145] Exemplary first and second peptide tag pairs (peptide tag / binding partner pairs; reactive pairs) are described in the table below:

[0146]

[0147] Described in, for example, WO2011 / 098772, WO2016 / 193746, GB1706430.4, GB 1705750.6 or Li, L., et al., J.Mol.Biol.426, 309-317 (2014).

[0148] Variants, derivatives, and modifications of binding pairs can be made in any suitable manner. Variants, derivatives, and functional operational modifications can involve the addition, substitution, alteration, or deletion of amino acids, and they retain the same function in terms of their ability to form isopeptide bonds with the associated binding partner.

[0149] For some binding pairs, a third entity such as an enzyme is required to mediate the pairing. For example, SnoopLigase can be used to mediate the bond formation between SnoopTagJr and DogTag. Therefore, pairing may require the assistance of enzymes such as ligases.

[0150] HBsAg

[0151] “HBsAg” refers to the surface antigen (HBsAg) of hepatitis B virus or a portion thereof. In one embodiment, HBsAg may refer to the N-terminus of HBsAg, such as the HBsAg sequence listed in SEQ ID NO:41, which contains 226 amino acids of the S protein of hepatitis B virus (adw serotype). Suitably, HBsAg contains the four-amino acid sequence Pro Val ThrAsn, representing the four C-terminal residues of the preS2 protein of hepatitis B virus (adw serotype), as described in Valenzuela et al., (1979) 'Nucleotide sequence of the gene coding for the major protein of hepatitis B virus surface antigen' Nature 280:815-819. VLPs formed from HBsAg have been approved for clinical use in the treatment of hepatitis B (Kushnir et al. Vaccine 2012; Vol 31(1):58-83), including Recombivax HB (https: / / vaccines.procon.org / sourcefiles / recombivax_package_insert.pdf) and Energix B (https: / / au.gsk.com / media / 217195 / engerix-b_pi_006_approved.pdf). HBsAg is also used as the basis for the erythrocyte pre-malaria vaccine RTS,S, which has completed Phase III clinical trials and is the most advanced malaria vaccine to date (http: / / www.malariavaccine.org / sites / www.malariavaccine.org / files / content / page / files / RTSS%20FAQs_FINAL.pdf; Kaslow and Biernaux, Vaccine 2015, Vol.33(52):7425-7432).

[0152] Connector details

[0153] The distance between proteins (e.g., VLPs and decorating antigens) affects the availability of antigenic epitopes within the protein, protein stability, and conjugation efficiency due to the accessibility of any isopeptide coupler (e.g., SpyTag / SpyCatcher). Therefore, adapters with suitable properties can be selected to optimize availability, stability, and / or accessibility. Adapters can be broadly subdivided into flexible and rigid subtypes.

[0154] Flexible joint

[0155] Flexible linkers can be used when the attached domains need to move. They are typically composed of small nonpolar (e.g., Gly) or polar (e.g., Ser, Thr) amino acids, where the small size provides flexibility (Chen et al., 2013 Adv Drug Deliv Rev. Oct 15; 65(10):1357-1369). The addition of Ser or Thr can help maintain stability in solution, and adjusting the length can affect the correct folding of the protein (Chen et al., 2013). Any suitable flexible linker with properties and length appropriate to the relevant entity can be used. Suitablely, flexible linkers can contain combinations of this type of amino acids ranging from 2 to 70.

[0156] Example:

[0157]

[0158] Rigid joint

[0159] In some cases, rigid adapters may be preferred because they can help facilitate protein separation. Rigid adapters have a secondary structure. One of the most common rigid adapters is (EAAAK). n (where n is the number of repeats), it employs an α-helical structure (Arai et al., (2001) Protein Eng. Aug; 14(8):529-32). Other rigid linkers may include proline-rich sequences such as (XP). n X is any amino acid, but preferably Ala (A), Lys (K) or Glu (E), where proline provides conformational constraints (Chen et al., 2013).

[0160] Other suitable linkers are described, for example, by Klein et al. (2014) Protein Eng Des Sel. Oct; 27(10):325-330. Any suitable rigid linker with properties and length suitable for the relevant entity can be used. Suitably, a rigid linker can contain a combination of amino acids of this type between 2 and 70.

[0161] Example:

[0162] sequence name sequence SEQ ID No Rigid joint 1 EAAAK SEQ ID NO: 9 <![CDATA[Rigid joint 2-(EAAAK)3]]> EAAAKEAAAKEAAAK SEQ ID NO: 10 <![CDATA[Rigid joint 3-(AP)7]]> APAPAPAPAPAPAP SEQ ID NO: 11

[0163] Host cells and expression vectors

[0164] Those skilled in the art will understand suitable host cells for expressing nucleic acids to produce proteins and compositions according to the present invention.

[0165] In one embodiment, the host cell will be adapted for transient expression. In another embodiment, the host cell will be one capable of forming a stable cell line. Suitably, the coding sequences for antigenic components such as HCMV pentamers and RSV-F proteins (including those sequences containing peptide tags forming heteropeptide bonds) will be integrated into a single host cell. In one embodiment, each nucleic acid sequence encoding a subunit of a multimer such as a pentamer will be contained in a different plasmid / vector, such that transfection of the host cell with, for example, all five plasmids / vectors when cultured under suitable conditions will result in the host cell producing pentamers. In other embodiments, the plasmid / vector may contain a combination of one or more coding sequences, such that at least one, two, three, four, or five plasmids can be introduced. Optionally, the complete fusion peptide coding sequence may be provided in a vector, such that the complete protein component and the first peptide tag are encoded on the same vector.

[0166] In one embodiment, these vectors are used to stably integrate coding sequences into the genome of a host cell. Suitable host cells for stable expression include mammalian cells, such as HEK cells (human embryonic kidney 293 cells) or rodent cells, including CHO (Chinese hamster ovary) cells. Suitable mammalian cells and vectors for expressing protein components of the compositions according to the invention will be known to those skilled in the art and are described, for example, in WO2016 / 067239, pages 15-16 and Hofmann et al., (2015) Biotech and Bioeng, 112(12): 2505-2515. Exemplary stable construct sequences for expressing components according to the invention are available in Example 3 below.

[0167] Affinity purification

[0168] In some embodiments, expression constructs used to express components of the compositions according to the invention may contain a "tag" sequence or a sequence that facilitates purification, such as affinity purification. Any suitable tag, such as an affinity tag, may be included to separate the protein component and the first peptide tag from the system that produces them. Those skilled in the art of recombinant protein production will recognize systems such as His-tags and Strep-tags that may be included for purification purposes. Such tags greatly facilitate protein purification and rarely have an adverse effect on biological or biochemical activity, and are therefore desirable. Suitable tag sequences include C-tags, histidine tags (His-tags), streptavidin tags (Strep-tags), maltose-binding protein (MBP), glutathione S-transferase (GST), and FLAG tags.

[0169] Both protein components and / or said portions may contain affinity purification tags. For ease of use, these are typically genetically fused to the C-terminus or N-terminus of the protein.

[0170] Therefore, in some embodiments, for example, the gH, gL, pUL128, pUL130, pUL131A (or fragments thereof) subunits of HCMV, RSV prefused F protein, or HBsAg peptide / protein may contain additional amino acid residues at the N-terminus or C-terminus, which is beneficial for purification. Such additional amino acid residues may include tags, such as, for example, His-tags or C-tags. In some embodiments, C-tags may provide cleaner purification. Other suitable tag sequences include maltose-binding protein (MBP), Strep-tags, glutathione S-transferase (GST), and FLAG tags. In some embodiments, the tag may be linked to the amino acid sequence in such a way that the tag can be cleaved after purification, for example, by using a cleavable linker. In other embodiments, affinity-free purification methods may be used.

[0171] In other embodiments, the RSV prefusion F protein may contain additional amino acid residues at its C-terminus or N-terminus, which facilitates purification. This document exemplifies an RSV prefusion F protein having a C-tag for affinity purification.

[0172] Conjugation of first and second peptide tag pairs

[0173] The conjugation of the first and second peptide tag / binding partner / reactive pair can be carried out overnight at 4°C. Alternatively, the conjugation reaction can be carried out at room temperature for 3-4 hours, as the conjugation rate is expected to increase at room temperature. For a given conjugation reaction, the optimal ratio of the first and second binding partners depends on the size of each binding partner. For example, for smaller antigens (~20 kDa), a molar ratio of VLP monomer to antigen of 1:1.5 may be sufficient, while for larger antigens (>100 kDa), a mass ratio of 1:1 with the same VLP monomer may be sufficient. However, both ratios will result in excess antigen (smaller binding partners). Any excess antigen can be removed by, for example, size exclusion chromatography (SEC) or by dialysis. Dialysis may be more suitable for smaller antigens as it is less efficient than SEC. Alternatively, the VLP / particle to antigen ratio can be optimized so that all antigen is conjugated, and therefore downstream purification is not required. A suitable final protein concentration of about 1 mg / ml is optimal for the conjugation reaction, as lower concentrations will reduce the reaction rate. Many buffers with near-neutral pH are compatible with coupling / conjugation. A standard choice for conjugation buffer is TBS (20 mM Tris and 150 mM NaCl, pH 7.4). In some cases, as described by Brune et al., Sci Rep. (2016), a 10x stock solution with added citrate buffer (40 mM Na₂HPO₄, 200 mM sodium citrate, pH 6.2) can be used.

[0174] Pharmaceutical Compositions and Uses

[0175] The compositions of the present invention can be incorporated into vaccines or immunogenic compositions. Suitably, the vaccine or immunogenic composition will contain an immunogenic dose of the particles of the present invention.

[0176] Pharmaceutical compositions may comprise particles or immunogenic compositions according to the invention, provided together with a pharmaceutically acceptable carrier. Suitable carriers are well known to those skilled in the art. In one embodiment, the pharmaceutical composition comprises a buffer, excipient, or carrier. Suitably, the pharmaceutical composition may comprise suitable excipients and formulations to maintain the stability of the composition. Suitably, the formulation may comprise an adjuvant. In one embodiment, the formulation may comprise a formulation similar to... AddaVax TM Or similar squalene-based oil-in-water nanoemulsions. Other suitable adjuvants include liposome-based adjuvants, such as Matrix M and AS01. Other suitable adjuvants include aluminum-based formulations, such as... In one embodiment, the formulation may contain EDTA, such as EDTA at a concentration of 5 mM. Suitable excipients or formulations may depend on the characteristics of the particles or immunogenic composition; for example, the choice of expression system may affect the stability, glycosylation, or folding of the protein in the composition, which may consequently affect the optimal formulation of the composition. Methods for determining suitable excipients, formulations, or adjuvants will be known to those skilled in the art.

[0177] In view of this disclosure, various other aspects and embodiments of the invention will be apparent to those skilled in the art.

[0178] All documents mentioned in this specification are incorporated herein by reference in their entirety.

[0179] As used herein, “and / or” should be understood to mean each of two specified features or components that is or is not specifically disclosed with the other. For example, “A and / or B” should be understood to mean each of (i) A, (ii) B, and (iii) A and B as if each were listed separately herein.

[0180] Unless the context otherwise indicates, the description and definition of the features listed above are not limited to any particular aspect or embodiment of the invention, and are equally applicable to all aspects and embodiments described.

[0181] Those skilled in the art will also understand that, although the invention has been described by way of example with reference to several embodiments, it is not limited to the disclosed embodiments, and alternative embodiments may be constructed without departing from the scope of the invention as defined in the appended claims.

[0182] The term “recombination” as used in this article to describe polynucleotides means a genomic, cDNA, semi-synthetic, or synthetically derived polynucleotide whose origin or manipulation is: (1) wholly or partially unrelated to the polynucleotides associated with it in nature; and / or (2) linked to a polynucleotide different from those linked to it in nature. The term “recombination” as used with respect to proteins or peptides means a peptide produced by expressing a recombinant polynucleotide.

[0183] Unless otherwise specified, the procedures including the steps can be performed in any suitable order. Therefore, these steps can be performed in any appropriate order.

[0184] Sequence identity between polypeptide sequences is preferably determined by a pairwise alignment algorithm using the Needleman-Wunsch global alignment algorithm (Needleman and Wunsch 1970) with default parameters (e.g., using an EBLOSUM62 scoring matrix, a vacancy opening penalty of 10.0, and a vacancy extension penalty of 0.5). This algorithm is conveniently implemented in the Needle tool within the EMBOSS software package (Rice, Longden, and Bleasby 2000). Sequence identity should be calculated over the entire length of the polypeptide sequence of the present invention.

[0185] Any homologs of the components mentioned in this article are generally functional homologs and typically share at least 40% homology with the relevant region of the protein. Homology can be measured using known methods. For example, the UWGCG software package provides the BESTFIT program (e.g., using its default settings) which can be used to calculate homology (Devereux et al. (1984) Nucleic Acids Research 12, 387-395). The PILEUP and BLAST algorithms can be used to calculate homology or line up sequences (usually based on their default settings), as described, for example, in Altschul SF (1993) J Mol Evol 36:290-300; Altschul, S, F et al. (1990) J Mol Biol 215:403-10. The software used for BLAST analysis is publicly available through the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ).

[0186] The BLAST algorithm performs a statistical analysis of the similarity between two sequences; see, for example, Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787. One measure of similarity provided by the BLAST algorithm is the minimum total probability (P(N)), which provides an indication of the probability that a match will occur by chance between two nucleotide or amino acid sequences. For example, if the minimum total probability in a comparison of a first sequence and a second sequence is less than about 1, preferably less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001, then the sequence is considered similar to the other sequence.

[0187] The variant polypeptide comprises (or consists of) a sequence having at least 40% identity with the natural protein. In a preferred embodiment, the variant sequence may be homologous to a specific region of the natural protein in at least 55%, 65%, 70%, 75%, 80%, 85%, 90%, and more preferably at least 95%, 97%, or 99% of at least 20, preferably at least 30, such as at least 40, 60, 100, 200, 300, 400, or more consecutive amino acids, or even throughout the entire sequence of the variant, at a specific region of the natural protein. Optionally, the variant sequence may be homologous to the full-length natural protein in at least 55%, 65%, 70%, 75%, 80%, 85%, 90%, and more preferably at least 95%, 97%, or 99%. Typically, the variant sequence differs from the relevant region of the natural protein by at least 2, 5, 10, 20, 40, 50, or 60 mutations, or fewer than 2, 5, 10, 20, 40, 50, or 60 mutations (each mutation may be a substitution, insertion, or deletion). The variant sequence of the present invention may have a certain percentage of identity with a specific region of the full-length natural protein, which is the same as any of the specific homology percentages across any length of the sequence mentioned above (i.e., it may have at least 40%, 55%, 80%, or 90%, and more preferably at least 95%, 97%, or 99% identity).

[0188] Protein variants also include truncated forms. Any truncated form can be used, provided the variant remains functional. Truncating is typically performed to remove sequences that are not essential for activity / function, particularly isopeptide bond formation, and / or do not affect the conformation of the folded protein, especially the folding of any immunogenic sites. Truncated forms can also be selected to improve the ease of component generation. Suitable truncated forms can usually be identified by systematically truncating sequences of varying lengths from the N-terminus or C-terminus.

[0189] Variants of the natural protein also include mutants, which have one or more, for example, 2, 3, 4, 5 to 10, 10 to 20, 20 to 40 or more amino acid insertions, substitutions, or deletions relative to a specific region of the natural protein. Deletions and insertions are preferably performed outside the antigenic region. Insertions are typically performed at the N-terminus or C-terminus of the sequence derived from the natural protein, for example, for the purpose of recombinant expression. Substitutions are also typically performed in regions that are not essential for activity / function and / or do not affect the conformation of the folded protein. Such substitutions can be performed to improve protein solubility or other characteristics. Substitutions can also be performed to increase protein stability.

[0190] The substitution preferably introduces one or more conservative modifications, which replace the amino acid with other amino acids that have similar chemical structures, similar chemical properties, or similar side chain sizes. The introduced amino acids may have similar polarity, hydrophilicity, hydrophobicity, basicity, acidity, neutrality, or charge to the amino acids they replace. Optionally, the conservative modification may introduce another aromatic or aliphatic amino acid to replace a pre-existing aromatic or aliphatic amino acid. Conservative amino acid modifications are well known in the art.

[0191] A derivative is an entity produced or prepared from a parent entity by replacing some parts of the parent entity. Example

[0192] Example 1

[0193] Production of an exemplary multimer-VLP composition (HCMV pentamer-HBsAg VLP)

[0194] Using ExpiFectamine TM Transient expression of HCMV pentamers was achieved in Expi293F cells using the ThermoFisher Scientific transfection reagent and five independent plasmids encoding the following sequences. The HCMV pentamers described below are approximately 162 kDa and glycosylated (including the tag and linker, but excluding the signal peptide).

[0195] nucleotide sequence

[0196] The expressed HCMV pentamer sequence represents the natural sequence (GenBank: AY446894.2; low-passage (i.e., attenuated) HCMV strain) (containing introns) from the Merlin strain, except for two introduced mutations (one in gH and one in UL128), as described in the relevant paragraphs below.

[0197] gH-SpyTag-His nucleotide sequence (SEQ ID NO.12)

[0198] In this sequence (SEQ ID NO:12), a silent mutation C>A is introduced at position 1146 for... Synthesized because the natural sequence CACCTGC surrounding the nucleotide was flagged as potentially problematic. The construct comprises: a signal peptide (nt 1-69), an extracellular domain (nt 70-2151), a truncated transmembrane domain (nt 2152-2157), (the signal peptide, extracellular domain, and truncated transmembrane domain together are represented by SEQ ID NO:13), a linker (nt 2158-2175; SEQ ID NO:14), a SpyTag (nt 2176-2214; SEQ ID NO:15), a 6x His tag (nt 2215-2232), and a stop codon (nt 2233-2235). Nucleotides 1 to 2157 (SEQ ID NO:13) represent the gH coding sequence.

[0199] gL nucleotide sequence (SEQ ID NO.16)

[0200] In this sequence: signal peptide (nt 1-90), extracellular domain (nt 91-834), stop codon (nt 835-837).

[0201] UL130-C tag nucleotide sequence (SEQ ID NO.17)

[0202] In this sequence: signal peptide (nt 1-75), extracellular domain (nt 76-642), linker (nt 643-687), C tag (nt 688-699), stop codon (nt 700-702).

[0203] The UL128 nucleotide sequence (SEQ ID NO.20) (contains two introns present in the natural sequence)

[0204] In this sequence: signal peptide (nt 1-81), introns: nt 165-287, nt 423-542, extracellular domain exons (nt 82-164, nt 288-422, nt 543-756), stop codon (nt 757-759).

[0205] A T>C mutation was introduced at nucleotide 634. The GenBank file mentions that nucleotide T634 causes premature termination of UL128 in the Merlin strain, and therefore we used annotations from different strains (GenBank: GQ396662.1, strain HAN38) to indicate which base to replace in order to restore the expression of the full-length protein.

[0206] UL131A nucleotide sequence (SEQ ID NO.21) (containing introns present in the natural sequence)

[0207] In this sequence: signal peptide (nt 1-54), intron (nt 237-344), extracellular domain exons (nt 55-236, nt 345-495), and stop codon (nt 496-498).

[0208] SpyCatcher-HBsAg nucleotide sequence (SEQ ID NO.22)

[0209] In this sequence: SpyCatcherDeltaN1 (nt 1-276), flexible connector (nt 277-303), PVTN connector (nt 304-315), HBsAg (nt 316-993), C tag (nt 994-1005), stop codon (nt 1006-1008).

[0210] amino acid sequence

[0211] The expression of the above nucleotide sequence is expected to produce the following amino acid sequence.

[0212] gH-SpyTag-His amino acid sequence (SEQ ID NO.27)

[0213] The expected mass is 81.852 kDa (without signal peptide) and 84.364 kDa (with signal peptide).

[0214] In this sequence: signal peptide (aa 1-23), extracellular domain (aa 24-717), truncated transmembrane domain (aa 718-719), (signal peptide, extracellular domain and truncated transmembrane domain together represented by SEQ ID NO:28), linker (aa 720-725; SEQ ID NO:29), SpyTag (aa 726-738; SEQ ID NO:30), 6x His tag (aa 739-744). Amino acid residues 1-719 represent the gH amino acid sequence of the natural Merlin strain with a truncated TM domain (SEQ ID NO:28).

[0215] gL amino acid sequence (SEQ ID NO:31)

[0216] The expected mass is 27.522 kDa (without signal peptide) and 30.815 kDa (with signal peptide).

[0217] In this sequence: signal peptide (aa 1-30), extracellular domain (aa 31-278). Amino acid residues 1-278 represent the amino acid sequence of the natural Merlin strain gL.

[0218] UL130-C tag amino acid sequence (SEQ ID NO:32)

[0219] The expected mass is 23.167 kDa (without signal peptide) and 26.081 kDa (with signal peptide).

[0220] In this sequence: signal peptide (aa 1-25), extracellular domain (aa 26-214), (signal peptide and extracellular domain together are represented by SEQ ID NO:33), linker (aa 215-229; SEQ ID NO:34), C tag (aa 230-233).

[0221] Amino acid residues 1-214 represent the amino acid sequence of the natural Merlin strain UL130.

[0222] UL128 amino acid sequence (SEQ ID NO:35)

[0223] The expected mass is 16.659 kDa (without signal peptide) and 19.717 kDa (with signal peptide).

[0224] In this sequence: signal peptide (aa 1-27), extracellular domain (aa 28-171). Amino acid residues 1-171 represent the amino acid sequence of the natural Merlin strain UL128.

[0225] UL131A amino acid sequence (SEQ ID NO:36)

[0226] The expected mass is 12.985 kDa (without signal peptide) and 14.989 kDa (with signal peptide).

[0227] In this sequence: signal peptide (aa 1-18), extracellular domain (aa 19-129). Amino acid residues 1-129 represent the amino acid sequence of the natural Merlin strain UL131A.

[0228] SpyCatcher-HBsAg amino acid sequence (SEQ ID NO:37)

[0229] The estimated mass is 36.824 kDa, including the label and connector.

[0230] In this sequence: SpyCatcherDeltaN1 (aa 1-92; SEQ ID NO:38), flexible connector (aa 93-101; SEQ ID NO:39), PVTN connector (aa 102-105; SEQ ID NO:40), HBsAg (aa 106-331; SEQ ID NO:41), C tag (aa 332-335).

[0231] Purification of pentamers

[0232] The pentamer-SpyTag is expressed in EXPI293F cells and secreted into the supernatant (due to the loss of the TM domain (a portion thereof) of the gH subunit). Initial attempts to purify the HCMV pentamer using affinity purification relied on the expression of the gH subunit with the C tag, but this resulted in the separation of the gH / gL heterohomodimer as well as the pentamer. In an alternative strategy, the C tag was added to the UL130 subunit (SEQ ID NO:17 (nucleotide) and SEQ ID NO:32 (amino acid)), which allowed the pentamer to be purified from the supernatant using C-tag affinity purification (ThermoFisher) and size exclusion chromatography. When analyzed by SDS-PAGE, the pentamer appeared as expected under both non-reducing and reducing conditions. Figure 1 A), and reacted with anti-HCMV pentameric antibody (Native Antigen Company (AbCMV2450)). Figure 1 B) Only a small amount of pollutants were observed at ~14 kDa.

[0233] Purification of HBsAg VLP monomer

[0234] SpyCatcher-HBsAg was expressed in *Pichia pastoris* and purified from cell homogenates. On SDS-PAGE gels under reducing conditions, the major protein bands corresponded to the expected size of the monomers (approximately 37 kDa), with larger bands indicating the presence of oligomeric material, suggesting good cross-linking of the particles. Figure 2 A, lane 'R'). Under non-reducing conditions (lane 'NR'), the material mainly remains at the top of the gel with some smearing, indicating that the VLP particles form well and are therefore too large to migrate completely into the gel. Figure 2 A). Both non-reduced and reduced SpyCatcher-HBsAg reacted strongly with mouse anti-HBsAg monoclonal antibody (obtained from Bio-Rad (MCA4658)). Figure 2 B) indicates that the presence of SpyCatcher did not negatively affect the reactive epitope. On an S200-increase 3.2 / 300 column, HPLC size exclusion analysis confirmed that both HCMV pentamer-SpyTag and SpyCatcher-HBsAg eluted as single peaks. Figure 3 A-3B). HCMV pentamer-SpyTag eluted at approximately ~400 kDa ( Figure 3A), which is larger than expected. However, this can be explained by the fact that the pentamer is not spherical, a structure known to alter the retention time of proteins in size exclusion chromatography. SpyCatcher-HBsAg eluted in an external water volume, indicating that the particles formed correctly and that no monomers were detectable in solution. Figure 3 B).

[0235] Antigen-VLP conjugation

[0236] HCMV pentamer-SpyTag was conjugated with SpyCatcher-HBsAg overnight at 4°C to obtain HBsAg VLP coated with HCMV pentamer. A Tris-buffered saline (TBS: 20 mM Tris and 150 mM NaCl, pH 7.4) supplemented with 5 mM EDTA was used for conjugation. Conjugation was monitored using SDS-PAGE, Western blotting, and HPLC. When the conjugated product was compared with pentamer-SpyTag or SpyCatcher-HBsAg alone, a new band was observed at ~130 kDa under reducing conditions. Figure 4 A, lane 2), which is related to monoclonal antibody HBsAg ( Figure 4 B) and polyclonal anti-HCMV pentamer ( Figure 4 C) All antibodies reacted, indicating that they contain at least conjugated HBsAg-gH. When analyzed by HPLC size exclusion chromatography, 97% of the eluent in the main peak corresponds to the expected size of the conjugated HCMV pentamer-HBsAg monomer. Figure 5 ).

[0237] Example 2

[0238] In vivo testing of HCMV-SpyTag-SpyCatcher-HBsAg VLP (with adjuvant)

[0239] In the immunization regimens of BALB / c mice, conjugated HCMV pentamer-HBsAg VLP and unconjugated HCMV pentamer-SpyTag were used to (i) confirm the immunogenicity of the generated HCMV pentamer-SpyTag, and (ii) compare the immunogenicity of unconjugated HCMV pentamer-SpyTag with that of conjugated HCMV pentamer-HBsAg VLP.

[0240] Use the following 3-week interval primary-boost-boost regimen:

[0241] Day 0: Immunization (primary immunization); Day 20: Tail blood collection; Day 21: Immunization (booster 1); Day 41: Tail blood collection; Day 42: Immunization (booster 2); Day 63: Heart blood collection.

[0242] The immunized groups are as follows. For each group, n = 10:

[0243] 1)AddaVax TM 1 μg HCMV pentamer-SpyTag in (Invivogen)

[0244] 2)AddaVax TM 1 μg HCMV pentamer-SpyTag-SpyCatcher-HBsAg VLP (1 μg pentamer equivalent)

[0245] 3) AddaVax TM SpyCatcher-HBsAg VLP (normalization of SpyCatcher-HBsAg values ​​in group 2)

[0246] 4) AddaVax TM 0.1 μg HCMV pentamer-SpyTag

[0247] 5) AddaVax TM 0.1 μg HCMV pentamer-SpyTag-SpyCatcher-HBsAg VLP (0.1 μg pentamer equivalent)

[0248] 6) TBS (20mM Tris and 150mM NaCl, pH 7.4)

[0249] AddaVax TM It is a formula similar to Squalene-based oil-in-water nanoemulsions have received approval in Europe for use as adjuvants in influenza vaccines. Squalene oil-in-water emulsions are known to elicit both cellular (Th1) and humoral (Th2) immune responses. Other suitable adjuvants will be known to those skilled in the art.

[0250] Immunogenicity was assessed using ELISA. The titer of antiserum produced in each group was determined using a standardized ELISA targeting the HCMV pentamer. Plates were coated overnight with 50 μL / well of 5 μg / ml pentamer (without SpyTag); washed; blocked with milk for 1 hour; washed; applied mouse serum (appropriately diluted in PBS) for 1 hour; washed; applied goat anti-mouse alkaline phosphatase antibody (1:10,000) for 1 hour; washed; and developed.

[0251] Different doses of unconjugated (Groups 1 and 4) and conjugated HCMV pentameric-HBsAg (Groups 2 and 5) were included to allow comparison of immunogenicity between conjugated HCMV pentameric-HBsAg VLP vaccines and unconjugated HCMV pentameric-SpyTag, which allows extrapolation to other HCMV pentameric vaccines (e.g., soluble pentameric). Groups 3 and 6 represent negative controls.

[0252] At each time point, the OD value of the samples was read at the appropriate dilution, and ELISA units were determined using a standard curve run on each plate. Data showing the results of groups 1, 2, 4, and 5 after primary immunization are presented in [data missing]. Figure 6 As shown in the figure. Mice immunized with 1 μg and 0.1 μg doses of HCMV pentamer-HBsAg showed significantly stronger serum IgG antibody responses compared to mice immunized with 1 μg or 0.1 μg doses of unconjugated HCMV pentamer. The baseline of this assay is provided by the ELISA units in groups 3 and 6, and is also shown in the figure. Figure 6 As shown in the image.

[0253] The functional activity of the generated antibodies was investigated using a microneutralization assay based on that of Wang et al. (Vaccine 33(2015)7254-7261; DOI:10.1016 / j.vaccine.2015.10.110). The neutralization titers of groups 1, 2, 4, and 5 were... Figure 7 As shown in the figure, serum from mice immunized with pentamer-HBsAg VLP had a significantly greater neutralizing effect than serum from mice immunized with pentamer-SpyTag protein alone.

[0254] Example 3

[0255] Stable build sequence

[0256] Two stable constructs (adapted from Hofmann et al., (2015) Biotech and Bioeng, 112(12):2505-2515) were optimized for CHO expression of components of the HCMV pentamer-SpyTag. Introns were removed from the HCMV pentamer sequence, but the signal sequence was preserved.

[0257] HCMV gH-SpyTag / gL stable expression construct

[0258] The stable vector construct HCMV-gH-(GSG)2-SpyTag-His-IRES-gL was designed to contain a gH-SpyTag-His component (SEQ ID NO:42) upstream of and a gL component (SEQ ID NO:43) downstream of the EV71 IRES, respectively. The coding sequences used in this construct are described below.

[0259] nucleotide sequence

[0260] gH-(GSG)2-SpyTag-His (intronless) (SEQ ID NO:42) is inserted upstream of EV71 IRES.

[0261] In this sequence: signal peptide (nt 1-69), extracellular domain (nt 70-2151), truncated transmembrane domain (nt 2152-2157), (GSG)2 linker (nt 2158-2175), SpyTag (nt 2176-2214), His-tag (nt 2215-2232), stop codon (nt 2233-2235).

[0262] gL (intronless) (SEQ ID NO:43) is inserted downstream of EV71 IRES.

[0263] In this sequence: signal peptide (nt 1-90), extracellular domain (nt 91-834), stop codon (nt 835-837).

[0264] HCMV UL128 / UL130 / UL131A stable expression construct

[0265] The stable construct HCMV-UL128-IRES-UL130-(G4S)3-C-tag-IRES-UL131A was designed to include a UL128 component (SEQ ID NO:44), a UL130 component (SEQ ID NO:45), and a UL131A component (SEQ ID NO:46). The UL130 component is inserted after the first EV71 IRES of the plasmid, and the UL131A component is inserted after the second EV71 IRES. The coding sequences used in this construct are described below.

[0266] nucleotide sequence

[0267] Ul128 (intronless) (SEQ ID NO:44)

[0268] In this sequence: signal peptide (nt 1-81), extracellular domain (nt 82-513), stop codon (nt 514-516).

[0269] UL130-(G4S)3-C label (intronless) (SEQ ID NO:45)

[0270] In this sequence: signal peptide (nt 1-75), extracellular domain (nt 76-642), (G4S)3 linker (nt 643-687), C tag (nt 688-699), stop codon (nt 700-702).

[0271] UL131A (intronless) (SEQ ID NO:46)

[0272] In this sequence: signal peptide (nt 1-54), extracellular domain (nt 55-387), stop codon (nt 388-390).

[0273] Example 4

[0274] In vivo detection of HCMV-SpyTag-SpyCatcher-HBsAg VLP (without adjuvant)

[0275] Conjugated HCMV pentamer-HBsAg VLP and unconjugated HCMV pentamer-SpyTag were used in BALB / c mice to further investigate the immunogenicity of conjugated pentamer-HBsAg VLP compared to unconjugated pentamer-SpyTag protein.

[0276] Use the following 3-week interval primary-boost-boost regimen:

[0277] Day 0: Immunization (primary immunization); Day 20: Tail blood collection; Day 21: Immunization (booster 1); Day 41: Tail blood collection; Day 42: Immunization (booster 2); Day 63: Heart blood collection.

[0278] The immunized groups are as follows. For each group, n = 10:

[0279] 1) 1 μg HCMV pentamer-SpyTag, adjuvant-free

[0280] 2) 1 μg HCMV pentamer-SpyTag--SpyCatcher-HBsAg VLP (1 μg pentamer equivalent), adjuvant-free

[0281] 3) 0.1 μg HCMV pentamer-SpyTag-SpyCatcher-HBsAg VLP (0.1 μg pentamer equivalent), adjuvant-free.

[0282] Immunogenicity was assessed using ELISA. The titer of antiserum produced in each group was determined using a standardized ELISA targeting the HCMV pentamer. Plates were coated overnight with 50 μL / well of 5 μg / ml pentamer (without SpyTag); washed; blocked with milk for 1 hour; washed; applied mouse serum (appropriately diluted in PBS) for 1 hour; washed; applied goat anti-mouse alkaline phosphatase antibody (1:10,000) for 1 hour; washed; and developed.

[0283] At each time point, the OD value of the sample was read at the appropriate dilution, and the ELISA units were determined using a standard curve run on each plate. Data after primary and booster immunization were... Figure 8 As shown in the figure, mice immunized with 1 μg of HCMV pentamer alone, which is a soluble protein, showed significantly stronger serum IgG antibody responses compared to mice immunized with 1 μg of HCMV pentamer-HBsAg alone.

[0284] The functional activity of the generated antibodies was investigated using a microneutralization assay based on Wang et al. (2015). Neutralization titers after primary and booster immunization were [data missing]. Figure 9 As shown in the figure, serum from mice immunized with unadjuvanted pentamer-HBsAg VLP had a significantly greater neutralizing effect than serum from mice immunized with unadjuvanted pentamer-SpyTag protein alone.

[0285] Example 5

[0286] Expression and purification of RSV-F-SpyTag

[0287] The sequence from antigen RSV-F Sc9-10 DS-Cav1 A149C Y458C was fused into SpyTag to generate RSV-F-SpyTag, and ExpiCHO was used. TM Expression system kit and ExpiFectamine TM Transfection reagent (ThermoFisher Scientific), using plasmid pcDNA3.4 containing the nucleotide sequence SEQ ID NO:47, transiently transfected ExpiCHO TM It is expressed by cells.

[0288] RSV-F Sc9-10 DS-Cav1 A149C Y458C (National Institutes of Health) is a variant of the respiratory syncytial virus fusion protein (pre-fused RSV-F) as described by Joyce et al. (2016) (Iterative structure-based improvement of a respiratory syncytial virus fusion glycoprotein vaccine. NatStruct Mol Biol. 2016 Sep; 23(9):811-820). This variant is a pre-fused stabilized form of the fusion (F) glycoprotein with a genetically linked F subunit, a missing fusion peptide, a T4 fibritin trimerization motif (folded domain), and inter-protomer motility stabilized by an additional inter-protomer disulfide bond (A149CY458C).

[0289] nucleotide sequence

[0290] RSV-F-SpyTag-C nucleotide sequence (SEQ ID NO:47)

[0291] By deleting the thrombin site at the 3' end, the 6x His-tag and II modified the original sequence of Sc9-10DS-Cav1A149CY458C. These missing domains were replaced with a linker-SpyTag-C tag sequence to produce a 1587nt cassette (SEQ ID NO:47) containing Sc9-10DS-Cav1A149CY458C (nt 1-1515, containing a signal peptide (nt 1-75) and a T4 fibritin foldon domain (nt 1435-1515)), a (GSG)2 linker (nt 1516-1533; SEQ ID NO:14), a SpyTag (nt 1534-1572; SEQ ID NO:15), a C tag (nt 1573-1584), and a stop codon (nt 1585-1587). The Sc9-10DS-Cav1A149C Y458C nucleotide sequence that does not contain a linker, SpyTag, C-tag, or stop codon is included in SEQ ID NO:48. The Sc9-10DS-Cav1A149C Y458C nucleotide sequence that does not contain a signal peptide, linker, SpyTag, or C-tag is included in SEQ ID NO:49.

[0292] amino acid sequence

[0293] Expression of the nucleotide sequence SEQ ID NO:47 is expected to produce an RSV-F-SpyTag-C-tagged amino acid sequence (SEQ ID NO:50) with the following domains: Sc9-10 DS-Cav1 A149C Y458C (aa 1-505, containing a signal peptide (aa1-25) and a foldon domain (aa 479-505)), a linker (aa 506-511; SEQ ID NO:29), a SpyTag (aa 512-524; SEQ ID NO:30), and a C-tag (aa 525-528). The expected mass of the protein with the signal peptide is 57.9 kDa, and the expected mass of the protein without the signal peptide is 55.3 kDa. The Sc9-10DS-Cav1 A149CY458C amino acid sequence without the linker, SpyTag, or C-tag is included in SEQ ID NO:47. In NO:51, the Sc9-10DS-Cav1 A149C Y458C amino acid sequence that does not contain a signal peptide, adapter, SpyTag, or C tag is included in SEQ ID NO:52.

[0294] Purification of RSV-F-SpyTag

[0295] RSF-F-SpyTag antigen was secreted from cells and purified from the supernatant using C-tag affinity purification and size exclusion chromatography. When analyzed by SDS-PAGE... Figure 10 A) and reacted with anti-RSV-F[2F7] monoclonal antibody (ab43812; Abcam) Figure 10 In case B), RSV-F-SpyTag appears as expected under both non-reducing and reducing conditions.

[0296] Purification of HBsAg VLP monomer

[0297] The preparation and purification of SpyCatcher-HBsAg (VLP monomer) as described in Example 1 above, see also... Figure 2 .

[0298] The concatenation of RSV-F-SpyTag and SpyCatcher-HBsAg

[0299] RSV-F-SpyTag was conjugated with SpyCatcher-HBsAg overnight at 4°C to obtain HBsAg VLP coated with RSV-F trimer (RSV-F-SpyTag--SpyCatcher-HBsAg). A buffer containing Tris-buffered saline (TBS: 20 mM Tris and 150 mM NaCl, pH 7.4) was used for conjugation. SDS-PAGE and Western blot analysis were used to monitor conjugation. Figure 11When the conjugated reactant was compared with individual RSV-F-SpyTag or SpyCatcher-HBsAg, a new band was observed at ~105 kDa under reducing conditions. Figure 11 A), which is related to the anti-HBsAg monoclonal antibody (MCA4658, Bio-Rad) ( Figure 11 B) and anti-RSV-F[2F7] monoclonal antibody (ab43812; Abcam) Figure 11 C) All of them reacted, indicating that they contain the conjugated RSV-F-SpyTag--SpyCatcher-HBsAg.

[0300] Example 6

[0301] Immunogenicity of conjugated RSV-F-SpyTag--SpyCatcher-HBsAg

[0302] An immunization program using BALB / c mice was designed to confirm the immunogenicity of the generated RSV-F antigen and to compare the immunogenicity of conjugated RSV-F-SpyTag-SpyCatcher-HBsAg VLP with that of unconjugated RSV-F-SpyTag protein. Dosing was administered to each group based on the amount of RSV-F-SpyTag in the sample, using a primer-booster regimen with a 3-week interval, with the final time point being 2 weeks after the booster immunization.

[0303] Regardless of whether the vaccine is adjuvant-free ( Figure 6 Or use Addavax TM Prepared ( Figure 6 Mice immunized with RSV-F-SpyTag-SpyCather-HBsAg after primary immunization showed a significantly stronger serum IgG antibody response compared to mice immunized with RSV-F-SpyTag protein alone.

[0304] Table of sequences

[0305] sequence list <110> Spai Biotechnology Co., Ltd. <120> vaccine composition <130> P9800wo1 <160> 60 <170> PatentIn version 3.5 <210> 1 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Flexible joint 2 - (GSG)2 <400> 1 Gly Ser Gly Gly Ser Gly 1 5 <210> 2 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Flexible Joint 3 - (GSG)3 <400> 2 Gly Ser Gly Gly Ser Gly Gly Ser Gly 1 5 <210> 3 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Flexible joint 4 - (G4S)1 <400> 3 Gly Gly Gly Gly Ser 1 5 <210> 4 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Flexible joint 5 - (G4S)3 <400> 4 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 <210> 5 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Flexible joint 6 - (G4S)4 <400> 5 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser 20 <210> 6 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Flexible joint 7 <400> 6 Gly Ser Ala Gly Ser Ala Ala Gly Ser Gly Glu Phe 1 5 10 <210> 7 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Flexible joint 8 <400> 7 Lys Glu Ser Gly Ser Val Ser Ser Glu Gln Leu Ala Gln Phe Arg Ser 1 5 10 15 Leu Asp <210> 8 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Flexible joint 9 <400> 8 Glu Gly Lys Ser Ser Gly Ser Gly Ser Glu Ser Lys Ser Thr 1 5 10 <210> 9 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Rigid joint 1 <400> 9 Glu Ala Ala Ala Lys 1 5 <210> 10 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Rigid joint 2 - (EAAAK)3 <400> 10 Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys 1 5 10 15 <210> 11 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Rigid joint 3 - (AP)7 <400> 11 Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro 1 5 10 <210> 12 <211> 2235 <212> DNA <213> Artificial Sequence <220> <223> gH-SpyTag-His <220> <221> misc_feature <222> (1146) <223> C>A mutation at position 1146 <220> <221> misc_feature <222> (1)..(69) <223> signal peptide <220> <221> misc_feature <222> (70)..(2151) <223> extracellular domain <220> <221> misc_feature <222> (2152)...(2157) <223> Transmembrane domain (truncated) <220> <221> misc_feature <222> (2158)...(2175) <223> connector <220> <221> misc_feature <222> (2176) (2214) <223> SpyTag <220> <221> misc_feature <222> (2215)..(2232) <223> 6x His tags <220> <221> misc_feature <222> (2233)..(2235) <223> stop codon <400> 12 atgcggccag gcctcccctc ctacctcatc atcctcgccg tctgtctctt cagccaccta 60 ctttcgtcac gatatggcgc agaagccgta tccgaaccgc tggacaaagc gtttcaccta 120 ctgctcaaca cctacgggag acccatccgc ttcctgcgtg aaaataccac ccagtgtacc 180 tacaacagca gcctccgtaa cagcacggtc gtcagggaaa acgccatcag tttcaacttt 240 ttccaaagct ataatcaata ctatgtattc catatgcctc gatgtctttt tgcgggtcct 300 ctggcggagc agtttctgaa ccaggtagat ctgaccgaaa ccctggaaag ataccaacag 360 agacttaaca cttacgcgct ggtatccaaa gacctggcca gctaccgatc tttttcgcag 420 cagctaaagg cacaagacag cctaggtgaa cagcccacca ctgtgccacc gcccattgac 480 ctgtcaatac ctcacgtttg gatgccaccg caaaccactc cacacggctg gacagaatca 540 cataccacct caggactaca ccgaccacac tttaaccaga cctgtatcct ctttgatgga 600 cacgatctac tattcagcac cgtcacacct tgtttgcacc aaggctttta cctcatcgac 660 gaactacgtt acgttaaaat aacactgacc gaggacttct tcgtagttac ggtgtccata 720 gacgacgaca cacccatgct gcttatcttc ggccatcttc cacgcgtact tttcaaagcg 780 ccctatcaac gcgacaactt tatactacga caaactgaaa aacacgagct cctggtgcta 840 gttaagaaag atcaactgaa ccgtcactct tatctcaaag acccggactt tcttgacgcc 900 gcacttgact tcaactacct agacctcagc gcactactac gtaacagctt tcaccgttac 960 gccgtggatg tactcaagag cggtcgatgt cagatgctgg accgccgcac ggtagaaatg 1020 gccttcgcct acgcattagc actgttcgca gcagcccgac aagaagaggc cggcgcccaa 1080 gtctccgtcc cacgggccct agaccgccag gccgcactct tacaaataca agaatttatg 1140 atcacatgcc tctcacaaac accaccacgc accacgttgc tgctgtatcc cacggccgtg 1200 gacctggcca aacgagccct ttggacaccg aatcagatca ccgacatcac cagcctcgta 1260 cgcctggtct acatactctc taaacagaat cagcaacatc tcatccccca atgggcacta 1320 cgacagatcg ccgactttgc cctaaaacta cacaaaacgc acctggcctc ttttctttca 1380 gccttcgcac gccaagaact ctacctcatg ggcagcctcg tccactccat gctggtacat 1440 acgacggaga gacgcgaaat cttcatcgta gaaacgggcc tctgttcatt ggccgagcta 1500 tcacacttta cgcagttgtt agctcatcca caccacgaat acctcagcga cctgtacaca 1560 ccctgttcca gtagcgggcg acgcgatcac tcgctcgaac gcctcacgcg tctcttcccc 1620 gatgccaccg tccccgctac cgttcccgcc gccctctcca tcctatctac catgcaacca 1680 agcacgctgg aaaccttccc cgacctgttt tgcttgccgc tcggcgaatc cttctccgcg 1740 ctgaccgtct ccgaacacgt cagttatatc gtaacaaacc agtacctgat caaaggtatc 1800 tcctaccctg tctccaccac cgtcgtaggc cagagcctca tcatcaccca gacggacagt 1860 caaactaaat gcgaactgac gcgcaacatg cataccacac acagcatcac agtggcgctc 1920 aacatttcgc tagaaactg cgccttttgc caaagcgccc tgctagaata cgacgacacg 1980 caaggcgtca tcaacatcat gtacatgcac gactcggacg acgtcctttt cgccctggat 2040 ccctacaacg aagtggtggt ctcatctccg cgaactcact acctcatgct tttgaaaaac 2100 ggtacggtac tagaagtaac tgacgtcgtc gtggacgcca ccgacagtcg tctcctcgga 2160 agcggaggct ctggtgccca tatcgtgatg gtggacgcct acaagcctac caaacatcat 2220 caccatcacc actaa 2235 <210> 13 <211> 2157 <212> DNA <213> Artificial Sequence <220> <223> gH with truncated transmembrane domains <220> <221> misc_feature <222> (1146) <223> C>A mutation at position 1146 <220> <221> misc_feature <222> (1)..(69) <223> signal peptide <220> <221> misc_feature <222> (70)..(2151) <223> extracellular domain <220> <221> misc_feature <222> (2152)...(2157) <223> Transmembrane domain (truncated) <400> 13 atgcggccag gcctcccctc ctacctcatc atcctcgccg tctgtctctt cagccaccta 60 ctttcgtcac gatatggcgc agaagccgta tccgaaccgc tggacaaagc gtttcaccta 120 ctgctcaaca cctacgggag acccatccgc ttcctgcgtg aaaataccac ccagtgtacc 180 tacaacagca gcctccgtaa cagcacggtc gtcagggaaa acgccatcag tttcaacttt 240 ttccaaagct ataatcaata ctatgtattc catatgcctc gatgtctttt tgcgggtcct 300 ctggcggagc agtttctgaa ccaggtagat ctgaccgaaa ccctggaaag ataccaacag 360 agacttaaca cttacgcgct ggtatccaaa gacctggcca gctaccgatc tttttcgcag 420 cagctaaagg cacaagacag cctaggtgaa cagcccacca ctgtgccacc gcccattgac 480 ctgtcaatac ctcacgtttg gatgccaccg caaaccactc cacacggctg gacagaatca 540 cataccacct caggactaca ccgaccacac tttaaccaga cctgtatcct ctttgatgga 600 cacgatctac tattcagcac cgtcacacct tgtttgcacc aaggctttta cctcatcgac 660 gaactacgtt acgttaaaat aacactgacc gaggacttct tcgtagttac ggtgtccata 720 gacgacgaca cacccatgct gcttatcttc ggccatcttc cacgcgtact tttcaaagcg 780 ccctatcaac gcgacaactt tatactacga caaactgaaa aacacgagct cctggtgcta 840 gttaagaaag atcaactgaa ccgtcactct tatctcaaag acccggactt tcttgacgcc 900 gcacttgact tcaactacct agacctcagc gcactactac gtaacagctt tcaccgttac 960 gccgtggatg tactcaagag cggtcgatgt cagatgctgg accgccgcac ggtagaaatg 1020 gccttcgcct acgcattagc actgttcgca gcagcccgac aagaagaggc cggcgcccaa 1080 gtctccgtcc cacgggccct agaccgccag gccgcactct tacaaataca agaatttatg 1140 atcacatgcc tctcacaaac accaccacgc accacgttgc tgctgtatcc cacggccgtg 1200 gacctggcca aacgagccct ttggacaccg aatcagatca ccgacatcac cagcctcgta 1260 cgcctggtct acatactctc taaacagaat cagcaacatc tcatccccca atgggcacta 1320 cgacagatcg ccgactttgc cctaaaacta cacaaaacgc acctggcctc ttttctttca 1380 gccttcgcac gccaagaact ctacctcatg ggcagcctcg tccactccat gctggtacat 1440 acgacggaga gacgcgaaat cttcatcgta gaaacgggcc tctgttcatt ggccgagcta 1500 tcacacttta cgcagttgtt agctcatcca caccacgaat acctcagcga cctgtacaca 1560 ccctgttcca gtagcgggcg acgcgatcac tcgctcgaac gcctcacgcg tctcttcccc 1620 gatgccaccg tccccgctac cgttcccgcc gccctctcca tcctatctac catgcaacca 1680 agcacgctgg aaaccttccc cgacctgttt tgcttgccgc tcggcgaatc cttctccgcg 1740 ctgaccgtct ccgaacacgt cagttatatc gtaacaaacc agtacctgat caaaggtatc 1800 tcctaccctg tctccaccac cgtcgtaggc cagagcctca tcatcaccca gacggacagt 1860 caaactaaat gcgaactgac gcgcaacatg cataccacac acagcatcac agtggcgctc 1920 aacatttcgc tagaaaactg cgccttttgc caaagcgccc tgctagaata cgacgacacg 1980 caaggcgtca tcaacatcat gtacatgcac gactcggacg acgtcctttt cgccctggat 2040 ccctacaacg aagtggtggt ctcatctccg cgaactcact acctcatgct tttgaaaaac 2100 ggtacggtac tagaagtaac tgacgtcgtc gtggacgcca ccgacagtcg tctcctc 2157 <210> 14 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Linker from gH construct <400> 14 ggaagcggag gctctggt 18 <210> 15 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Spytag <400> 15 gcccatatcg tgatggtgga cgcctacaag cctaccaaa 39 <210> 16 <211> 837 <212> DNA <213> Human cytomegalovirus <220> <221> misc_feature <222> (1)..(90) <223> Signal peptide <220> <221> misc_feature <222> (91)..(834) <223> Extracellular domain <220> <221> misc_feature <222> (835)..(837) <223> Stop codon <400> 16 atgtgccgcc gcccggattg cggcttctct ttctcacctg gaccggtgat actgctgtgg 60 tgttgccttc tgctgcccat tgtttcctca gccgccgtca gcgtcgctcc taccgccgcc 120 gagaaagtcc ccgcggagtg ccccgaacta acgcgccgat gcttgttggg tgaggtgttt 180 gagggtgaca agtatgaaag ttggctgcgc ccgttggtga atgttaccgg gcgcgatggc 240 ccgctatcgc aacttatccg ttaccgtccc gttacgccgg aggccgccaa ctccgtgctg 300 ttggacgagg ctttcctgga cactctggcc ctgctgtaca acaatccgga tcaattgcgg 360 gccctgctga cgctgttgag ctcggacaca gcgccgcgct ggatgacggt gatgcgcggc 420 tacagcgagt gcggcgatgg ctcgccggcc gtgtacacgt gcgtggacga cctgtgccgc 480 ggctacgacc tcacgcgact gtcatacggg cgcagcatct tcacggaaca cgtgttaggc 540 ttcgagctgg tgccaccgtc tctctttaac gtggtggtgg ccatacgcaa cgaagccacg 600 cgtaccaacc gcgccgtgcg tctgcccgtg agcaccgctg ccgcgcccga gggcatcacg 660 ctcttttacg gcctgtacaa cgcagtgaag gaattctgcc tgcgtcacca gctggacccg 72 ccgctgctac gccacctaga taaatactac gccggactgc cgcccgagct gaagcagacg 780 cgcgtcaacc tgccggctca ctcgcgctat ggccctcaag cagtggatgc tcgctaa 837 <210> 17 <211> 702 <21>> DNA <213> Artificial Sequence <220> <223> UL130 - C tag <220> <221> misc_feature <222> (1)..(75) <223> Signal peptide <220> <221> misc_feature <222> (76)..(642) <223: Extracellular domain <220> <221> misc_feature <222> (643)..(687) <223> Connector <220> <221> misc_feature <222> (688)..(699) <223> C label <220> <221> misc_feature <222> (700)..(702) <223> Stop codon <400> 17 atgctgcggc ttctgcttcg tcaccacttt cactgcctgc ttctgtgcgc ggtttgggca 60 acgccctgtc tggcgtctcc gtggtcgacg ctaacagcaa accagaatcc gtccccgcca 120 tggtctaaac tgacgtattc caaaccgcat gacgcggcga cgttttactg tccttttctc 180 tatccctcgc ccccacgatc ccccttgcaa ttctcggggt tccagcgggt atcaacgggt 240 cccgagtgtc gcaacgagac cctgtatctg ctgtacaacc gggaaggcca gaccttggtg 300 gagagaagct ccacctgggt gaaaaaggtg atctggtacc tgagcggtcg gaaccaeacc 360[[ID=三十八]] atcctccaac ggatgccccg aacggcttcg aaaccgagcg acggaaacgt gcagatcagc 420 gtggaagacg ccaagatttt tggagcgcac atggtgccca agcagaccaa gctgctacgc 480 ttcgtcgtca acgatggcac acgttatcag atgtgtgtga tgaagctgga gagctgggct 540 cacgtcttcc gggactacag cgtgtctttt caggtgcgat tgacgttcac cgaggccaat 600 aaccagactt acaccttctg cacccatccc aatctcatcg ttggaggcgg aggatctggc 660 ggaggtggaa gtggcggagg cggatctgag cccgaggcct aa 702 <210> 18 <211> 642 <212> DNA <213> Artificial Sequence <220> <223> UL130 (signal sequence and extracellular domain) <220> <221> misc_feature <222> (1)..(75) <223> signal peptide <220> <221> misc_feature <222> (76) (642) <223> extracellular domain <400> 18 atgctgcggc ttctgcttcg tcaccacttt cactgcctgc ttctgtgcgc ggtttgggca 60 acgccctgtc tggcgtctcc gtggtcgacg ctaacagcaa accagaatcc gtccccgcca 120 tggtctaaac tgacgtattc caaaccgcat gacgcggcga cgttttactg tccttttctc 180 tatccctcgc ccccacgatc ccccttgcaa ttctcggggt tccagcgggt atcaacgggt 240 cccgagtgtc gcaacgagac cctgtatctg ctgtacaacc gggaaggcca gaccttggtg 300 gagagaagct ccacctgggt gaaaaaggtg atctggtacc tgagcggtcg gaaccaaacc 360 atcctccaac ggatgccccg aacggcttcg aaaccgagcg acggaaacgt gcagatcagc 420 gtggaagacg ccaagatttt tggagcgcac atggtgccca agcagaccaa gctgctacgc 480 ttcgtcgtca acgatggcac acgttatcag atgtgtgtga tgaagctgga gagctgggct 540 cacgtcttcc gggactacag cgtgtctttt caggtgcgat tgacgttcac cgaggccaat 600 aaccagactt acaccttctg cacccatccc aatctcatcg tt 642 <210> 19 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> Adapter from UL130 construct <400> 19 ggaggcggag gatctggcgg aggtggaagt ggcggaggcg gatct 45 <210> 20 <211> 759 <212> DNA <213> Human cytomegalovirus <220> <221> misc_feature <222> (634)..(634) <223> The T>C mutation occurs at position 634. <220> <221> misc_feature <222> (1)..(81) <223> signal peptide <220> <221> misc_feature <222> (82)..(164) <223> extracellular domain <220> <221> Intron <222> (165) (287) <220> <221> misc_feature <222> (288)..(422) <223> extracellular domain <220> <221> Intron <222> (423)..(542) <220> <221> misc_feature <222> (543) (756) <223> extracellular domain <220> <221> misc_feature <222> (757) (759) <223> stop codon <400> 20 atgagtccca aagatctgac gccgttcttg acggcgttgt ggctgctatt gggtcacagc 60 cgcgtgccgc gggtgcgcgc agaagaatgt tgcgaattca taaacgtcaa ccacccgccg 120 gaacgctgtt acgatttcaa aatgtgcaat cgcttcaccg tcgcgtacgt attttcatga 180 ttgtctgcgt tctgtggtgc gtctggatct gtctctcgac gtttctgata gccatgttcc 240 atcgacgatc ctcgggaatg ccagagtaga ttttcatgaa tccacaggct gcggtgtccg 300 gacggcgaag tctgctacag tcccgagaaa acggctgaga ttcgcgggat cgtcaccacc 360 atgacccatt cattgacacg ccaggtcgta cacaacaaac tgacgagctg caactacaat 420 ccgtaagtct cttcctgagg gccttacagc ctatgggaga gtaagacaga gagggacaaa 480 acatcattaa aaaaaaaagt ctaatttcac gttttgtacc ccccttcccc tccgtgttgt 540 aggttatacc tcgaagctga cgggcgaata cgctgcggca aagtaaacga caaggcgcag 600 tacctgctgg gcgccgctgg cagcgttccc tatcgatgga tcaatctgga atacgacaag 660 ataacccgga tcgtgggcct ggatcagtac ctggagagcg ttaagaaaca caaacggctg 720 gatgtgtgcc gcgctaaaat gggctatatg ctgcagtga 759 <210> 21 <211> 498 <212> DNA <213> Human cytomegalovirus <220> <221> misc_feature <222> (1)..(54) <223> Signal peptide <220> <221> misc_feature <222> (55)..(236) <223> Extracellular domain <220> <221> Intron <222> (237)..(344) <220> <221> misc_feature <222> (345)..(495) <223> Extracellular domain <220> <221> misc_feature <222> (496)..(498) <223> Stop codon <400> 21 atgcggctgt gtcgggtgtg gctgtctgtt tgtctgtgcg ccgtggtgct gggtcagtgc 60 cagcgggaaa ccgcggaaaa aaacgattat taccgagtac cgcattactg ggacgcgtgc 120 tctcgcgcgc tgcccgacca aacccgttac aagtatgtgg aacagctcgt ggacctcacg 180 ttgaactacc actacgatgc gagccacggc ttggacaact ttgacgtgct caagaggtga 240 gggtacgcgc taaagatgca tgacaacggg aaggtaaggg cgaacgggta acgggtaagt 300 aaccgcatgg ggtatgaaat gacgttcgga acctgtgctt gcagaatcaa cgtgaccgag 360 gtgtcgttgc tcatcagcga ctttagacgt cagaaccgtc gcggcggcac caacaaaagg 420 accacgttca acgccgccgg ttcgctggcg ccacacgccc ggagcctcga gttcagcgtg 480 cggctctttg ccaactag 498 <210> twenty two <211> 1008 <212> DNA <213> Artificial Sequence <220> <223> SpyCatcher-HBsAg <220> <221> misc_feature <222> (1)..(276) <223> SpyCatcherDeltaN1 <220> <221> misc_feature <222> (277) (303) <223> Flexible joint <220> <221> misc_feature <222> (304) (315) <223> PVTN connector <220> <221> misc_feature <222> (316) (993) <223> HBsAg <220> <221> misc_feature <222> (994)...(1005) <223> C tag <220> <221> misc_feature <222> (1006)...(1008) <223> stop codon <400> 22 gactccgcta ctcacatcaa gttctccaag agagatgagg acggtaaaga attggctggt 60 gctactatgg aattgagaga ctcctccggt aagactatct ccacttggat ttccgacggt 120 caggttaagg acttctactt gtacccaggt aagtacactt tcgttgagac tgctgctcca 180 gacggttacg aagttgctac tgctatcact ttcactgtta acgagcaggg acaggttaca 240 gttaacggta aggctactaa gggtgacgct catattggtt ctggtggatc tggtggttcc 300 ggtccagtta ctaatatgga aaacatcact tccggtttct tgggtccttt gttggttttg 360 caggctggat tcttcttgtt gactagaatc ttgactatcc cacagtcctt ggactcttgg 420 tggacttcct tgaacttctt gggtggttcc ccagtttgtt tgggtcaaaa ctctcaatcc 480 ccaacttcca accactcccc aacatcttgt ccaccaattt gtcctggtta cagatggatg 540 tgtttgagaa gattcatcat tttcttgttc atcttgttgt tgtgtttgat cttcttgttg 600 gttttgttgg actaccaggg tatgttgcca gtttgtccat tgatcccagg ttccactact 660 acaaacactg gtccatgtaa gacttgtact actccagctc agggtaactc catgttccct 720 tcatgttgtt gtactaagcc aactgacggt aactgtactt gtatcccaat tccatcctcc 780 tgggctttcg ctaagtactt gtgggaatgg gcttccgtta gattctcctg gttgtccttg 840 ttggttccat tcgttcagtg gttcgttggt ttgtccccaa ctgtttggtt gtccgctatt 900 tggatgatgt ggtactgggg tccatccttg tactctatcg tttccccatt catccctttg 960 ttgccaatct tcttctgttt gtgggtttac atcgagccag aggcttaa 1008 <210> 23 <211> 276 <212> DNA <213> Artificial Sequence <220> <223> SpyCatcherDeltaN1 <400> 23 gactccgcta ctcacatcaa gttctccaag agagatgagg acggtaaaga attggctggt 60 gctactatgg aattgagaga ctcctccggt aagactatct ccacttggat ttccgacggt 120 caggttaagg acttctactt gtacccaggt aagtacactt tcgttgagac tgctgctcca 180 gacggttacg aagttgctac tgctatcact ttcactgtta acgagcaggg acaggttaca 240 gttaacggta aggctactaa gggtgacgct catatt 276 <210> 24 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Flexible connectors from SpyCatcher-HBsAg <400> twenty four ggttctggtg gatctggtgg ttccggt 27 <210> 25 <211> 12 <212> DNA <213> Artificial Sequence <220> <223> PVTN connector from SpyCatcher-HbsAg <400> 25 ccagttacta at 12 <210> 26 <211> 678 <212> DNA <213> Artificial Sequence <220> <223> HBsAg <400> 26 atggaaaaca tcacttccgg tttcttgggt cctttgttgg ttttgcaggc tggattcttc 60 ttgttgacta gaatcttgac tatcccacag tccttggact cttggtggac ttccttgaac 120 ttcttgggtg gttccccagt ttgtttgggt caaaactctc aatccccaac ttccaaccac 180 tccccaacat cttgtccacc aatttgtcct ggttacagat ggatgtgttt gagaagattc 240 atcattttct tgttcatctt gttgttgtgt ttgatcttct tgttggtttt gttggactac 300 cagggtatgt tgccagtttg tccattgatc ccaggttcca ctactacaaa cactggtcca 360 tgtaagactt gtactactcc agctcagggt aactccatgt tcccttcatg ttgttgtact 420 aagccaactg acggtaactg tacttgtatc ccaattccat cctcctgggc tttcgctaag 480 tacttgtggg aatgggcttc cgttagattc tcctggttgt ccttgttggt tccattcgtt 540 cagtggttcg ttggtttgtc cccaactgtt tggttgtccg ctatttggat gatgtggtac 600 tggggtccat ccttgtactc tatcgtttcc ccattcatcc ctttgttgcc aatcttcttc 660 tgtttgtggg tttacatc 678 <210> 27 <211> 744 <212> PRT <213> Artificial Sequence <220> <223> gH-SpyTag-His <220> <221> misc_feature <222> (1)..(23) <223> signal peptide <220> <221> misc_feature <222> (24)..(717) <223> extracellular domain <220> <221> misc_feature <222> (718) (719) <223> Transmembrane domain (truncated) <220> <221> misc_feature <222> (720)..(725) <223> connector <220> <221> misc_feature <222> (726)..(738) <223> Spytag <220> <221> misc_feature <222> (739)..(744) <223> 6x His tag <400> 27 Met Arg Pro Gly Leu Pro Ser Tyr Leu Ile Ile Leu Ala Val Cys Leu 1 5 10 15 Phe Ser His Leu Leu Ser Ser Arg Tyr Gly Ala Glu Ala Val Ser Glu 20 25 30 Pro Leu Asp Lys Ala Phe His Leu Leu Leu Asn Thr Tyr Gly Arg Pro 35 40 45 Ile Arg Phe Leu Arg Glu Asn Thr Thr Gln Cys Thr Tyr Asn Ser Ser 50 55 60 Leu Arg Asn Ser Thr Val Val Arg Glu Asn Ala Ile Ser Phe Asn Phe 65 70 75 80 Phe Gln Ser Tyr Asn Gln Tyr Tyr Val Phe His Met Pro Arg Cys Leu 85 90 95 Phe Ala Gly Pro Leu Ala Glu Gln Phe Leu Asn Gln Val Asp Leu Thr 100 105 110 Glu Thr Leu Glu Arg Tyr Gln Gln Arg Leu Asn Thr Tyr Ala Leu Val 115 120 125 Ser Lys Asp Leu Ala Ser Tyr Arg Ser Phe Ser Gln Gln Leu Lys Ala 130 135 140 Gln Asp Ser Leu Gly Glu Gln Pro Thr Thr Val Pro Pro Pro Ile Asp 145 150 155 160 Leu Ser Ile Pro His Val Trp Met Pro Pro Gln Thr Thr Pro His Gly 165 170 175 Trp Thr Glu Ser His Thr Thr Ser Gly Leu His Arg Pro His Phe Asn 180 185 190 Gln Thr Cys Ile Leu Phe Asp Gly His Asp Leu Leu Phe Ser Thr Val 195 200 205 Thr Pro Cys Leu His Gln Gly Phe Tyr Leu Ile Asp Glu Leu Arg Tyr 210 215 220 Val Lys Ile Thr Leu Thr Glu Asp Phe Phe Val Val Thr Val Ser Ile 225 230 235 240 Asp Asp Asp Thr Pro Met Leu Leu Ile Phe Gly His Leu Pro Arg Val 245 250 255 Leu Phe Lys Ala Pro Tyr Gln Arg Asp Asn Phe Ile Leu Arg Gln Thr 260 265 270 Glu Lys His Glu Leu Leu Val Leu Val Lys Lys Asp Gln Leu Asn Arg 275 280 285 His Ser Tyr Leu Lys Asp Pro Asp Phe Leu Asp Ala Ala Leu Asp Phe 290 295 300 Asn Tyr Leu Asp Leu Ser Ala Leu Leu Arg Asn Ser Phe His Arg Tyr 305 310 315 320 Ala Val Asp Val Leu Lys Ser Gly Arg Cys Gln Met Leu Asp Arg Arg 325 330 335 Thr Val Glu Met Ala Phe Ala Tyr Ala Leu Ala Leu Phe Ala Ala Ala 340 345 350 Arg Gln Glu Glu Ala Gly Ala Gln Val Ser Val Pro Arg Ala Leu Asp 355 360 365 Arg Gln Ala Ala Leu Leu Gln Ile Gln Glu Phe Met Ile Thr Cys Leu 370 375 380 Ser Gln Thr Pro Pro Arg Thr Thr Leu Leu Leu Tyr Pro Thr Ala Val 385 390 395 400 Asp Leu Ala Lys Arg Ala Leu Trp Thr Pro Asn Gln Ile Thr Asp Ile 405 410 415 Thr Ser Leu Val Arg Leu Val Tyr Ile Leu Ser Lys Gln Asn Gln Gln 420 425 430 His Leu Ile Pro Gln Trp Ala Leu Arg Gln Ile Ala Asp Phe Ala Leu 435 440 445 Lys Leu His Lys Thr His Leu Ala Ser Phe Leu Ser Ala Phe Ala Arg 450 455 460 Gln Glu Leu Tyr Leu Met Gly Ser Leu Val His Ser Met Leu Val His 465 470 475 480 Thr Thr Glu Arg Arg Glu Ile Phe Ile Val Glu Thr Gly Leu Cys Ser 485 490 495 Leu Ala Glu Leu Ser His Phe Thr Gln Leu Leu Ala His Pro His His 500 505 510 Glu Tyr Leu Ser Asp Leu Tyr Thr Pro Cys Ser Ser Ser Gly Arg Arg 515 520 525 Asp His Ser Leu Glu Arg Leu Thr Arg Leu Phe Pro Asp Ala Thr Val 530 535 540 Pro Ala Thr Val Pro Ala Ala Leu Ser Ile Leu Ser Thr Met Gln Pro 545 550 555 560 Ser Thr Leu Glu Thr Phe Pro Asp Leu Phe Cys Leu Pro Leu Gly Glu 565 570 575 Ser Phe Ser Ala Leu Thr Val Ser Glu His Val Ser Tyr Ile Val Thr 580 585 590 Asn Gln Tyr Leu Ile Lys Gly Ile Ser Tyr Pro Val Ser Thr Thr Val 595 600 605 Val Gly Gln Ser Leu Ile Ile Thr Gln Thr Asp Ser Gln Thr Lys Cys 610 615 620 Glu Leu Thr Arg Asn Met His Thr Thr His Ser Ile Thr Val Ala Leu 625 630 635 640 Asn Ile Ser Leu Glu Asn Cys Ala Phe Cys Gln Ser Ala Leu Leu Glu 645 650 655 Tyr Asp Asp Thr Gln Gly Val Ile Asn Ile Met Tyr Met His Asp Ser 660 665 670 Asp Asp Val Leu Phe Ala Leu Asp Pro Tyr Asn Glu Val Val Val Ser 675 680 685 Ser Pro Arg Thr His Tyr Leu Met Leu Leu Lys Asn Gly Thr Val Leu 690 695 700 Glu Val Thr Asp Val Val Val Asp Ala Thr Asp Ser Arg Leu Leu Gly 705 710 715 720 Ser Gly Gly Ser Gly Ala His Ile Val Met Val Asp Ala Tyr Lys Pro 725 730 735 Thr Lys His His His His His 740 <210> 28 <211> 719 <212> PRT <213> Artificial Sequence <220> <223> gH with truncated transmembrane domains <220> <221> misc_feature <222> (1)..(23) <223> signal peptide <220> <221> misc_feature <222> (24)..(717) <223> extracellular domain <220> <221> misc_feature <222> (718) (719) <223> Transmembrane domain (truncated) <400> 28 Met Arg Pro Gly Leu Pro Ser Tyr Leu Ile Ile Leu Ala Val Cys Leu 1 5 10 15 Phe Ser His Leu Leu Ser Ser Arg Tyr Gly Ala Glu Ala Val Ser Glu 20 25 30 Pro Leu Asp Lys Ala Phe His Leu Leu Leu Asn Thr Tyr Gly Arg Pro 35 40 45 Ile Arg Phe Leu Arg Glu Asn Thr Thr Gln Cys Thr Tyr Asn Ser Ser 50 55 60 Leu Arg Asn Ser Thr Val Val Arg Glu Asn Ala Ile Ser Phe Asn Phe 65 70 75 80 Phe Gln Ser Tyr Asn Gln Tyr Tyr Val Phe His Met Pro Arg Cys Leu 85 90 95 Phe Ala Gly Pro Leu Ala Glu Gln Phe Leu Asn Gln Val Asp Leu Thr 100 105 110 Glu Thr Leu Glu Arg Tyr Gln Gln Arg Leu Asn Thr Tyr Ala Leu Val 115 120 125 Ser Lys Asp Leu Ala Ser Tyr Arg Ser Phe Ser Gln Gln Leu Lys Ala 130 135 140 Gln Asp Ser Leu Gly Glu Gln Pro Thr Thr Val Pro Pro Pro Ile Asp 145 150 155 160 Leu Ser Ile Pro His Val Trp Met Pro Pro Gln Thr Thr Pro His Gly 165 170 175 Trp Thr Glu Ser His Thr Thr Ser Gly Leu His Arg Pro His Phe Asn 180 185 190 Gln Thr Cys Ile Leu Phe Asp Gly His Asp Leu Leu Phe Ser Thr Val 195 200 205 Thr Pro Cys Leu His Gln Gly Phe Tyr Leu Ile Asp Glu Leu Arg Tyr 210 215 220 Val Lys Ile Thr Leu Thr Glu Asp Phe Phe Val Val Thr Val Ser Ile 225 230 235 240 Asp Asp Asp Thr Pro Met Leu Leu Ile Phe Gly His Leu Pro Arg Val 245 250 255 Leu Phe Lys Ala Pro Tyr Gln Arg Asp Asn Phe Ile Leu Arg Gln Thr 260 265 270 Glu Lys His Glu Leu Leu Val Leu Val Lys Lys Asp Gln Leu Asn Arg 275 280 285 His Ser Tyr Leu Lys Asp Pro Asp Phe Leu Asp Ala Ala Leu Asp Phe 290 295 300 Asn Tyr Leu Asp Leu Ser Ala Leu Leu Arg Asn Ser Phe His Arg Tyr 305 310 315 320 Ala Val Asp Val Leu Lys Ser Gly Arg Cys Gln Met Leu Asp Arg Arg 325 330 335 Thr Val Glu Met Ala Phe Ala Tyr Ala Leu Ala Leu Phe Ala Ala Ala 340 345 350 Arg Gln Glu Glu Ala Gly Ala Gln Val Ser Val Pro Arg Ala Leu Asp 355 360 365 Arg Gln Ala Ala Leu Leu Gln Ile Gln Glu Phe Met Ile Thr Cys Leu 370 375 380 Ser Gln Thr Pro Pro Arg Thr Thr Leu Leu Leu Tyr Pro Thr Ala Val 385 390 395 400 Asp Leu Ala Lys Arg Ala Leu Trp Thr Pro Asn Gln Ile Thr Asp Ile 405 410 415 Thr Ser Leu Val Arg Leu Val Tyr Ile Leu Ser Lys Gln Asn Gln Gln 420 425 430 His Leu Ile Pro Gln Trp Ala Leu Arg Gln Ile Ala Asp Phe Ala Leu 435 440 445 Lys Leu His Lys Thr His Leu Ala Ser Phe Leu Ser Ala Phe Ala Arg 450 455 460 Gln Glu Leu Tyr Leu Met Gly Ser Leu Val His Ser Met Leu Val His 465 470 475 480 Thr Thr Glu Arg Arg Glu Ile Phe Ile Val Glu Thr Gly Leu Cys Ser 485 490 495 Leu Ala Glu Leu Ser His Phe Thr Gln Leu Leu Ala His Pro His His 500 505 510 Glu Tyr Leu Ser Asp Leu Tyr Thr Pro Cys Ser Ser Ser Gly Arg Arg 515 520 525 Asp His Ser Leu Glu Arg Leu Thr Arg Leu Phe Pro Asp Ala Thr Val 530 535 540 Pro Ala Thr Val Pro Ala Ala Leu Ser Ile Leu Ser Thr Met Gln Pro 545 550 555 560 Ser Thr Leu Glu Thr Phe Pro Asp Leu Phe Cys Leu Pro Leu Gly Glu 565 570 575 Ser Phe Ser Ala Leu Thr Val Ser Glu His Val Ser Tyr Ile Val Thr 580 585 590 Asn Gln Tyr Leu Ile Lys Gly Ile Ser Tyr Pro Val Ser Thr Thr Val 595 600 605 Val Gly Gln Ser Leu Ile Ile Thr Gln Thr Asp Ser Gln Thr Lys Cys 610 615 620 Glu Leu Thr Arg Asn Met His Thr Thr His Ser Ile Thr Val Ala Leu 625 630 635 640 Asn Ile Ser Leu Glu Asn Cys Ala Phe Cys Gln Ser Ala Leu Leu Glu 645 650 655 Tyr Asp Asp Thr Gln Gly Val Ile Asn Ile Met Tyr Met His Asp Ser 660 665 670 Asp Asp Val Leu Phe Ala Leu Asp Pro Tyr Asn Glu Val Val Val Ser 675 680 685 Ser Pro Arg Thr His Tyr Leu Met Leu Leu Lys Asn Gly Thr Val Leu 690 695 700 Glu Val Thr Asp Val Val Val Asp Ala Thr Asp Ser Arg Leu Leu 705 710 715 <210> 29 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Connectors from gH constructs <400> 29 Gly Ser Gly Gly Ser Gly 1 5 <210> 30 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Spytag <400> 30 Ala His Ile Val Met Val Asp Ala Tyr Lys Pro Thr Lys 1 5 10 <210> 31 <211> 278 <212> PRT <213> Human cytomegalovirus <220> <221> misc_feature <222> (1)..(30) <223> signal peptide <220> <221> misc_feature <222> (31)..(278) <223> extracellular domain <400> 31 Met Cys Arg Arg Pro Asp Cys Gly Phe Ser Phe Ser Pro Gly Pro Val 1 5 10 15 Ile Leu Leu Trp Cys Cys Leu Leu Leu Pro Ile Val Ser Ser Ala Ala 20 25 30 Val Ser Val Ala Pro Thr Ala Ala Glu Lys Val Pro Ala Glu Cys Pro 35 40 45 Glu Leu Thr Arg Arg Cys Leu Leu Gly Glu Val Phe Glu Gly Asp Lys 50 55 60 Tyr Glu Ser Trp Leu Arg Pro Leu Val Asn Val Thr Gly Arg Asp Gly 65 70 75 80 Pro Leu Ser Gln Leu Ile Arg Tyr Arg Pro Val Thr Pro Glu Ala Ala 85 90 95 Asn Ser Val Leu Leu Asp Glu Ala Phe Leu Asp Thr Leu Ala Leu Leu 100 105 110 Tyr Asn Asn Pro Asp Gln Leu Arg Ala Leu Leu Thr Leu Leu Ser Ser 115 120 125 Asp Thr Ala Pro Arg Trp Met Thr Val Met Arg Gly Tyr Ser Glu Cys 130 135 140 Gly Asp Gly Ser Pro Ala Val Tyr Thr Cys Val Asp Asp Leu Cys Arg 145 150 155 160 Gly Tyr Asp Leu Thr Arg Leu Ser Tyr Gly Arg Ser Ile Phe Thr Glu 165 170 175 His Val Leu Gly Phe Glu Leu Val Pro Pro Ser Leu Phe Asn Val Val 180 185 190 Val Ala Ile Arg Asn Glu Ala Thr Arg Thr Asn Arg Ala Val Arg Leu 195 200 205 Pro Val Ser Thr Ala Ala Ala Pro Glu Gly Ile Thr Leu Phe Tyr Gly 210 215 220 Leu Tyr Asn Ala Val Lys Glu Phe Cys Leu Arg His Gln Leu Asp Pro 225 230 235 240 Pro Leu Leu Arg His Leu Asp Lys Tyr Tyr Ala Gly Leu Pro Pro Glu 245 250 255 Leu Lys Gln Thr Arg Val Asn Leu Pro Ala His Ser Arg Tyr Gly Pro 260 265 270 Gln Ala Val Asp Ala Arg 275 <210> 32 <211> 233 <212> PRT <213> Artificial Sequence <220> <223> UL130 - C label <220> <221> misc_feature <222> (1)..(25) <223> signal peptide <220> <221> misc_feature <222> (26)..(214) <223> extracellular domain <220> <221> misc_feature <222> (215)..(229) <223> linker <220> <221> misc_feature <222> (230)..(233) <223> C tag <400> 32 Met Leu Arg Leu Leu Leu Arg His His Phe His Cys Leu Leu Leu Cys 1 5 10 15 Ala Val Trp Ala Thr Pro Cys Leu Ala Ser Pro Trp Ser Thr Leu Thr 20 25 30 Ala Asn Gln Asn Pro Ser Pro Pro Trp Ser Lys Leu Thr Tyr Ser Lys 35 ~ 40 ~ 45 Pro His Asp Ala Ala Thr Phe Tyr Cys Pro Phe Leu Tyr Pro Ser Pro 50 ~ 55 ~ 60 Pro Arg Ser Pro Leu Gln Phe Ser Gly Phe Gln Arg Val Ser Thr Gly 65 ~ 70 ~ 75 ~ 80 Pro Glu Cys Arg Asn Glu Thr Leu Tyr Leu Leu Tyr Asn Arg Glu Gly 85 ~ 90 ~ 95 Gln Thr Leu Val Glu Arg Ser Ser Thr Trp Val Lys Lys Val Ile Trp Note: In the translation, for the numbers in the amino acid sequence range like "35 40 45", "50 55 60" etc., I added "~" to better represent the range relationship as it's not clear from the original if they are just numbers or part of a sequence range indication. If this is not in line with your requirements, please let me know.100 105 110 Tyr Leu Ser Gly Arg Asn Gln Thr Ile Leu Gln Arg Met Pro Arg Thr 115 120 125 Ala Ser Lys Pro Ser Asp Gly Asn Val Gln Ile Ser Val Glu Asp Ala 130 135 140 Lys Ile Phe Gly Ala His Met Val Pro Lys Gln Thr Lys Leu Leu Arg 145 150 155 160 Phe Val Val Asn Asp Gly Thr Arg Tyr Gln Met Cys Val Met Lys Leu 165 170 175 Glu Ser Trp Ala His Val Phe Arg Asp Tyr Ser Val Ser Phe Gln Val 180 185 190 Arg Leu Thr Phe Thr Glu Ala Asn Asn Gln Thr Tyr Thr Phe Cys Thr 195 200 205 His Pro Asn Leu Ile Val Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 210 215 220 Gly Gly Gly Gly Ser Glu Pro Glu Ala 225 230 <210> 33 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> UL130 (Signal sequence and extracellular domain) <220> <221> misc_feature <222> (1)..(25) <223> Signal peptide <220> <221> misc_feature <222> (26)..(214) <223> Extracellular domain <400> 33 Met Leu Arg Leu Leu Leu Arg His His Phe His Cys Leu Leu Leu Cys 1 5 10 15 Ala Val Trp Ala Thr Pro Cys Leu Ala Ser Pro Trp Ser Thr Leu Thr 20 25 30 Ala Asn Gln Asn Pro Ser Pro Pro Trp Ser Lys Leu Thr Tyr Ser Lys 35 40 45 Pro His Asp Ala Ala Thr Phe Tyr Cys Pro Phe Leu Tyr Pro Ser Pro 50 55 60 Pro Arg Ser Pro Leu Gln Phe Ser Gly Phe Gln Arg Val Ser Thr Gly 65 70 75 80 Pro Glu Cys Arg Asn Glu Thr Leu Tyr Leu Leu Tyr Asn Arg Glu Gly 85 90 95 Gln Thr Leu Val Glu Arg Ser Ser Thr Trp Val Lys Lys Val Ile Trp 100 105 110 Tyr Leu Ser Gly Arg Asn Gln Thr Ile Leu Gln Arg Met Pro Arg Thr 115 120 125 Ala Ser Lys Pro Ser Asp Gly Asn Val Gln Ile Ser Val Glu Asp Ala 130 135 140 Lys Ile Phe Gly Ala His Met Val Pro Lys Gln Thr Lys Leu Leu Arg 145 150 155 160 Phe Val Val Asn Asp Gly Thr Arg Tyr Gln Met Cys Val Met Lys Leu 165 170 175 Glu Ser Trp Ala His Val Phe Arg Asp Tyr Ser Val Ser Phe Gln Val 180 185 190 Arg Leu Thr Phe Thr Glu Ala Asn Asn Gln Thr Tyr Thr Phe Cys Thr 195 200 205 His Pro Asn Leu Ile Val 210 <210> 34 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Connector from UL130 building block <400> 34 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 <210> 35 <211> 171 <212> PRT <213> Human cytomegalovirus <220> <221> misc_feature <222> (1)..(27) <223> Signal peptide <220> <221> misc_feature <222> (28)..(171) <223> Extracellular domain <400> 35 Met Ser Pro Lys Asp Leu Thr Pro Phe Leu Thr Ala Leu Trp Leu Leu 1 5 10 15 Leu Gly His Ser Arg Val Pro Arg Val Arg Ala Glu Glu Cys Cys Glu 20 25 30 Phe Ile Asn Val Asn His Pro Pro Glu Arg Cys Tyr Asp Phe Lys Met 35 40 45 Cys Asn Arg Phe Thr Val Ala Leu Arg Cys Pro Asp Gly Glu Val Cys 50 55 60 Tyr Ser Pro Glu Lys Thr Ala Glu Ile Arg Gly Ile Val Thr Thr Met 65 70 75 80 Thr His Ser Leu Thr Arg Gln Val Val His Asn Lys Leu Thr Ser Cys 85 90 95 Asn Tyr Asn Pro Leu Tyr Leu Glu Ala Asp Gly Arg Ile Arg Cys Gly 100 105 110 Lys Val Asn Asp Lys Ala Gln Tyr Leu Leu Gly Ala Ala Gly Ser Val 115 120 125 Pro Tyr Arg Trp Ile Asn Leu Glu Tyr Asp Lys Ile Thr Arg Ile Val 130 135 140 Gly Leu Asp Gln Tyr Leu Glu Ser Val Lys Lys His Lys Arg Leu Asp 145 150 155 160 Val Cys Arg Ala Lys Met Gly Tyr Met Leu Gln 165 170 <210> 36 <211> 129 <212> PRT <213> Human cytomegalovirus <220> <221> misc_feature <222> (1)..(18) <223> signal peptide <220> <221> misc_feature <222> (19)..(129) <223> extracellular domain <400> 36 Met Arg Leu Cys Arg Val Trp Leu Ser Val Cys Leu Cys Ala Val Val 1 5 10 15 Leu Gly Gln Cys Gln Arg Glu Thr Ala Glu Lys Asn Asp Tyr Tyr Arg 20 25 30 Val Pro His Tyr Trp Asp Ala Cys Ser Arg Ala Leu Pro Asp Gln Thr 35 40 45 Arg Tyr Lys Tyr Val Glu Gln Leu Val Asp Leu Thr Leu Asn Tyr His 50 55 60 Tyr Asp Ala Ser His Gly Leu Asp Asn Phe Asp Val Leu Lys Arg Ile 65 70 75 80 Asn Val Thr Glu Val Ser Leu Leu Ile Ser Asp Phe Arg Arg Gln Asn 85 90 95 Arg Arg Gly Gly Thr Asn Lys Arg Thr Thr Phe Asn Ala Ala Gly Ser 100 105 110 Leu Ala Pro His Ala Arg Ser Leu Glu Phe Ser Val Arg Leu Phe Ala 115 120 125 Asn <210> 37 <211> 335 <212> PRT <213> Artificial Sequence <220> <223> SpyCatcher-HBsAg <220> <221> misc_feature <222> (1)..(92) <223> SpycatcherDeltaN1 <220> <221> misc_feature <222> (93) (101) <223> Flexible joint <220> <221> misc_feature <222> (102) (105) <223> PVTN connector <220> <221> misc_feature <222> (106) (331) <223> HBsAg <220> <221> misc_feature <222> (332)..(335) <223> C label <400> 37 Asp Ser Ala Thr His Ile Lys Phe Ser Lys Arg Asp Glu Asp Gly Lys 1 5 10 15 Glu Leu Ala Gly Ala Thr Met Glu Leu Arg Asp Ser Ser Gly Lys Thr 20 25 30 Ile Ser Thr Trp Ile Ser Asp Gly Gln Val Lys Asp Phe Tyr Leu Tyr 35 40 45 Pro Gly Lys Tyr Thr Phe Val Glu Thr Ala Ala Pro Asp Gly Tyr Glu 50 55 60 Val Ala Thr Ala Ile Thr Phe Thr Val Asn Glu Gln Gly Gln Val Thr 65 70 75 80 Val Asn Gly Lys Ala Thr Lys Gly Asp Ala His Ile Gly Ser Gly Gly 85 90 95 Ser Gly Gly Ser Gly Pro Val Thr Asn Met Glu Asn Ile Thr Ser Gly 100 105 110 Phe Leu Gly Pro Leu Leu Val Leu Gln Ala Gly Phe Phe Leu Leu Thr 115 120 125 Arg Ile Leu Thr Ile Pro Gln Ser Leu Asp Ser Trp Trp Thr Ser Leu 130 135 140 Asn Phe Leu Gly Gly Ser Pro Val Cys Leu Gly Gln Asn Ser Gln Ser 145 150 155 160 Pro Thr Ser Asn His Ser Pro Thr Ser Cys Pro Pro Ile Cys Pro Gly 165 170 175 Tyr Arg Trp Met Cys Leu Arg Arg Phe Ile Ile Phe Leu Phe Ile Leu 180 185 190 Leu Leu Cys Leu Ile Phe Leu Leu Val Leu Leu Asp Tyr Gln Gly Met 195 200 205 Leu Pro Val Cys Pro Leu Ile Pro Gly Ser Thr Thr Thr Asn Thr Gly 210 215 220 Pro Cys Lys Thr Cys Thr Thr Pro Ala Gln Gly Asn Ser Met Phe Pro 225 230 235 240 Ser Cys Cys Cys Thr Lys Pro Thr Asp Gly Asn Cys Thr Cys Ile Pro 245 250 255 Ile Pro Ser Ser Trp Ala Phe Ala Lys Tyr Leu Trp Glu Trp Ala Ser 260 265 270 Val Arg Phe Ser Trp Leu Ser Leu Leu Val Pro Phe Val Gln Trp Phe 275 280 285 Val Gly Leu Ser Pro Thr Val Trp Leu Ser Ala Ile Trp Met Met Trp 290 295 300 Tyr Trp Gly Pro Ser Leu Tyr Ser Ile Val Ser Pro Phe Ile Pro Leu 305 310 315 320 Leu Pro Ile Phe Phe Cys Leu Trp Val Tyr Ile Glu Pro Glu Ala 325 330 335 <210> 38 <211> 92 <212> PRT <213> Artificial Sequence <220> <223> SpyCatcherDeltaN1 <400> 38 Asp Ser Ala Thr His Ile Lys Phe Ser Lys Arg Asp Glu Asp Gly Lys 1 5 10 15 Glu Leu Ala Gly Ala Thr Met Glu Leu Arg Asp Ser Ser Gly Lys Thr 20 25 30<U+ Ile Ser Thr Trp Ile Ser Asp Gly Gln Val Lys Asp Phe Tyr Leu Tyr 35 40 45 Pro Gly Lys Tyr Thr Phe Val Glu Thr Ala Ala Pro Asp Gly Tyr Glu 50 55 60 Val Ala Thr Ala Ile Thr Phe Thr Val Asn Glu Gln Gly Gln Val Thr 65 70 75 80 Val Asn Gly Lys Ala Thr Lys Gly Asp Ala His Ile 85 90 <210> 39 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Flexible connectors from SpyCatcher-HBsAg <400> 39 Gly Ser Gly Gly Ser Gly Gly Ser Gly 1 5 <210> 40 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> PVTN connector from SpyCatcher-HBsAg <400> 40 Pro Val Thr Asn 1 <210> 41 <211> 226 <212> PRT <213> Artificial Sequence <220> <223> HBsAg <400> 41 Met Glu Asn Ile Thr Ser Gly Phe Leu Gly Pro Leu Leu Val Leu Gln 1 5 10 15 Ala Gly Phe Phe Leu Leu Thr Arg Ile Leu Thr Ile Pro Gln Ser Leu 20 25 30 Asp Ser Trp Trp Thr Ser Leu Asn Phe Leu Gly Gly Ser Pro Val Cys 35 40 45 Leu Gly Gln Asn Ser Gln Ser Pro Thr Ser Asn His Ser Pro Thr Ser 50 55 60 Cys Pro Pro Ile Cys Pro Gly Tyr Arg Trp Met Cys Leu Arg Arg Phe 65 70 75 80 Ile Ile Phe Leu Phe Ile Leu Leu Leu Cys Leu Ile Phe Leu Leu Val 85 90 95 Leu Leu Asp Tyr Gln Gly Met Leu Pro Val Cys Pro Leu Ile Pro Gly 100 105 110 Ser Thr Thr Thr Asn Thr Gly Pro Cys Lys Thr Cys Thr Thr Pro Ala 115 120 125 Gln Gly Asn Ser Met Phe Pro Ser Cys Cys Cys Thr Lys Pro Thr Asp 130 135 140 Gly Asn Cys Thr Cys Ile Pro Ile Pro Ser Ser Trp Ala Phe Ala Lys 145 150 155 160 Tyr Leu Trp Glu Trp Ala Ser Val Arg Phe Ser Trp Leu Ser Leu Leu 165 170 175 Val Pro Phe Val Gln Trp Phe Val Gly Leu Ser Pro Thr Val Trp Leu 180 185 190 Ser Ala Ile Trp Met Met Trp Tyr Trp Gly Pro Ser Leu Tyr Ser Ile 195 200 205 Val Ser Pro Phe Ile Pro Leu Leu Pro Ile Phe Phe Cys Leu Trp Val 210 215 220 Tyr Ile 225 <210> 42 <211> 2235 <212> DNA <213> Artificial Sequence <220> <223> gH-(GSG)2-SpyTag-His (intronless) optimized for CHO expression <220> <221> misc_feature <222> (1)..(69) <223> gH signal peptide <220> <221> misc_feature <222> (70)..(2151) <223> gH extracellular domain <220> <221> misc_feature <222> (2152)...(2157) <223> gH truncated transmembrane domain <220> <221> misc_feature <222> (2158)...(2175) <223> (GSG)2 connector <220> <221> misc_feature <222> (2176) (2214) <223> Spytag <220> <221> misc_feature <222> (2215)..(2232) <223> Histag <220> <221> misc_feature <222> (2233)..(2235) <223> stop codon <400> 42 atgaggcctg gcctgccttc ttatctgatc atcctggccg tgtgcctgtt ctcccatctg 60 ctgtcctcta gatacggcgc cgaggctgtg tctgagcctc tggataaggc ctttcatctg 120 ctgctgaaca cctacggcag acctatccgg ttcctgcgcg agaacaccac acagtgcacc 180 tacaactcca gcctgcggaa ctccacagtc gtgcgggaaa acgccatctc cttcaacttt 240 ttccagtcct acaaccagta ctatgtgttc cacatgcctc gctgcctgtt cgctggacct 300 ctggctgagc agttcctgaa ccaggtggac ctgaccgaga cactggaaag ataccagcag 360 cggctgaaca catatgccct ggtgtctaag gacctggcct cctacagatc cttcagccag 420 cagctgaagg ctcaggactc tctgggagag cagcctacaa cagtgcctcc tcctatcgac 480 ctgtctatcc ctcacgtgtg gatgcctcca cagaccacac ctcatggctg gaccgagtct 540 cataccacct ctggcctgca ccggcctcac ttcaaccaga cctgcatcct gttcgacggc 600 cacgacctgc tgttctccac cgtgacacca tgtctgcacc agggcttcta cctgatcgac 660 gagctgagat acgtgaagat taccctgaca gaggacttct tcgtggtcac cgtgtccatc 720 gacgacgaca cccctatgct gctgatcttc ggccatctgc ctcgggtgct gttcaaggcc 780 ccttaccagc gggacaactt catcctgaga cagaccgaga agcacgagct gctggtgctg 840 gtcaagaagg accagctgaa ccggcactcc tacctgaagg accctgactt cctggacgcc 900 gctctggact tcaactacct ggatctgagc gccctgctgc ggaacagctt tcacagatac 960 gccgtggacg tgctgaagtc tggcagatgc cagatgctgg acagacggac cgtggaaatg 1020 gccttcgctt acgccctggc tctgtttgcc gccgctagac aagaagaggc tggcgcccaa 1080 gtgtccgtgc ctagagcact ggatagacaa gccgctctgc tgcagatcca agagttcatg 1140 atcacatgcc tgtctcagac ccctcctcgg accacactgc tgctgtatcc taccgctgtg 1200 gatctggcca agagggctct gtggacccct aaccagatca ccgacatcac atccctcgtg 1260 cggctggtgt acatcctgtc caagcagaac cagcagcatc tgatccctca gtgggccctg 1320 aggcagatcg ctgattttgc cctgaagctg cacaagaccc acctggccag ctttctgtct 1380 gccttcgcca gacaagagct gtacctgatg ggcagcctgg tgcactctat gctggtgcat 1440 accaccgagc ggcgcgagat cttcatcgtg gaaaccggcc tgtgttccct ggccgagctg 1500 tctcacttta cccagctgct cgctcaccct caccacgagt acctgtccga cctgtacacc 1560 ccttgctcct ctagcggcag aagggaccac agcctggaaa gactgacccg gctgttccct 1620 gatgccaccg tgcctgctac agttcctgcc gctctgtcca tcctgagcac catgcagcct 1680 tccactctgg aaacattccc cgacctgttc tgcctgcctc tgggcgagtc tttttctgcc 1740 ctgaccgtgt ccgagcacgt gtcctacatc gtgaccaatc agtacctgat caagggcatc 1800 agctaccccg tgtccacaac cgtcgttggc cagagcctga tcatcaccca gaccgactct 1860 cagaccaagt gcgagctgac ccggaacatg cacacaaccc actccatcac cgtggctctg 1920 aacatctccc tggaaaactg cgccttctgc cagtctgccc tgctggaata cgatgacacc 1980 cagggcgtga tcaacatcat gtatatgcac gactccgacg acgtgctgtt tgccctggat 2040 ccttacaacg aggtggtggt gtctagcccc agaacacact acctgatgct gctcaagaac 2100 ggcaccgtgc tggaagtgac cgacgtggtg gtggacgcca ccgattctag attgctcggc 2160 tctggtggct ccggcgctca tatcgtgatg gtggatgctt acaagcccac caagcaccat 2220 catcaccacc actaa 2235 <210> 43 <211> 837 <212> DNA <213> Artificial Sequence <220> <223> gL (intronless) optimized for CHO expression <220> <221> misc_feature <222> (1)..(90) <223> gL signal peptide <220> <221> misc_feature <222> (91)..(834) <223> gL extracellular domain <220> <221> misc_feature <222> (835) (837) <223> stop codon <400> 43 atgtgcagaa ggcctgactg cggcttctcc ttctctcccg gacctgtgat cctgctgtgg 60 tgctgtctgc tgctgcccat cgtttcttcc gccgctgtgt ctgtggctcc taccgctgct 120 gaaaaggtgc cagctgagtg tcccgagctg accagaagat gtctgctggg cgaagtgttc 180 gagggcgata agtacgagtc ttggctgcgg cctctggtca acgtgaccgg aagagatgga 240 cccctgagcc agctgatccg gtacagacct gtgacacctg aggccgccaa ttccgtgctg 300 ctggatgagg ccttcctgga cacactggcc ctgctgtaca acaaccccga tcagctgaga 360 gccctgctga ccctgctgtc ctctgatacc gctcctagat ggatgaccgt gatgcggggc 420 tactctgagt gcggagatgg aagcccagcc gtgtacacct gtgtggacga tctgtgcaga 480 ggctacgacc tgaccagact gtcctacggc cggtccatct ttaccgagca tgtgctgggc 540 tttgagctgg tgcctcctag cctgttcaat gtggtggtgg ccatccggaa tgaggccacc 600 agaacaaata gagccgtgcg gctgcctgtg tctacagctg ctgctcctga gggcatcacc 660 ctgttctacg gcctgtacaa cgccgtgaaa gagttctgcc tgagacacca gctggaccct 720 ccactgctga ggcacctgga taagtactac gctggcctgc ctcctgagct gaagcagacc 780 agagtgaacc tgcctgctca ctccagatac ggccctcagg ctgtggacgc cagataa 837 <210> 44 <211> 516 <212> DNA <213> Artificial Sequence <220> <223> UL128 optimized for CHO expression (intronless) <220> <221> misc_feature <222> (1)..(81) <223> UL128 signal peptide <220> <221> misc_feature <222> (82)..(513) <223> UL128 extracellular domain <220> <221> misc_feature <222> (514)..(516) <223> Stop codon <400> 44 atgtccccta aggatctgac ccctttcctg accgctctgt ggctgctgct gggccattct 60 agagtgccta gagtcagagc cgaggaatgc tgcgagttca tcaacgtgaa ccatcctcca 120 gagcggtgct acgacttcaa gatgtgcaac agattcaccg tggctctgcg gtgccctgat 180 ggcgaagtgt gctactcccc tgaaaagacc gccgagatca gaggcatcgt gaccaccatg 240 acacactccc tgaccagaca ggtggtgcac aacaagctga ccagctgcaa ctacaaccct 300 ctgtacctgg aagccgacgg cagaatcaga tgcggcaaag tgaacgacaa ggcccagtac 360 ctgttgggcg ctgctggctc tgtgccctac agatggatca acctggaata cgacaagatc 420 acccggatcg tcggcctgga ccagtatctg gaatccgtga agaagcacaa gcggctggac 480 gtgtgcagag ccaagatggg ctatatgctg cagtaa 516 <210> 45 <211> 702 <212> DNA <213> Artificial Sequence <220> <223> UL130-(G4S)3-C tag (intronless) optimized for CHO expression <220> <221> misc_feature <222> (1)..(75) <223> UL130 signal peptide <220> <221> misc_feature <222> (76) (642) <223> UL130 extracellular domain <220> <221> misc_feature <222> (643) (687) <223> (G4S)3 connector <220> <221> misc_feature <222> (688)...(699) <223> C tag <220> <221> misc_feature <222> (700)...(702) <223> stop codon <400> 45 atgctgagac tgctgctgag acaccacttc cactgcctgc tgctgtgtgc cgtttgggct 60 acaccttgtc tggcctctcc atggtctacc ctgaccgcca accagaatcc ttctccacct 120 tggtccaagc tgacctactc caagcctcac gatgccgcta ccttctactg cccctttctg 180 tacccatctc cacctcggag ccctctgcag ttctctggct tccagagagt gtccaccgga 240 cctgagtgcc ggaacgagac actgtacctg ctgtacaacc gcgagggcca gacactggtg 300 gaaagatcct ctacctgggt caagaaagtg atctggtatc tgagcggccg gaaccagacc 360 atcctgcaga gaatgcctcg gaccgcctct aagccttctg acggcaacgt gcagatctcc 420 gtggaagatg ccaagatctt cggcgcccac atggtgccca agcagaccaa actgctgaga 480 ttcgtggtca acgacggcac ccgctaccag atgtgcgtga tgaagctgga aagctgggcc 540 cacgtgttcc gggattactc cgtgtctttc caagtgcggc tgaccttcac cgaggccaac 600 aaccagacct acaccttctg cacccatcct aacctgatcg tcggaggcgg aggatctggc 660 ggaggtggaa gtggcggagg cggatctgag cccgaggcct aa 702 <210> 46 <211> 390 <212> DNA [[ID=​​​​​​​<221> misc_feature <222> (1)..(54) <223> UL131 mounting <220> <221> misc_feature <222> (55)..(387) <223> UL131 conveyor belt <220> <221> misc_feature <222> (388)..(390) <223> Oriental snowflakes <400> 46 atgagactgt gcagagtgtg gctgtccgtg tgcctgtgtg ctgtggttct gggccagtgc 120. cagagaga cagccgaga gaacgactac tacagagtgc cccactactg ggacgcctgc agtagctt tgcccgatca gacccggtac aatacgtgg aacagctggt ggatctgacc ctgaactacc actacgacgc ctctcacggc ctggacaact tcgacgtgct gaagcggatc aacgtgaccg aggtgtccct gctgatctct gacttccggc ggcagaatag aagaggcggc accaacaagc ggaccacctt taatgctgcc ggctctctgg ctccccacgc cagatctctg gaatttccg tgcggctgtt cgccactaa <210> 47 <211> 1587 <212> DNA <213> Artificial Sequence <220> <223> RSV-F-SpyTag-C tag <220> <221> misc_feature <222> (1)..(75) <223> signal peptide <220> <221> misc_feature <222> (1435)...(1515) <223> Foldon structural domain <220> <221> misc_feature <222> (1516)...(1533) <223> (GSG)2 connector <220> <221> misc_feature <222> (1534) (1572) <223> Spytag <220> <221> misc_feature <222> (1573)...(1584) <223> C tag <220> <221> misc_feature <222> (1585)...(1587) <223> stop codon <400> 47 atggaactgc tgatcctgaa ggccaacgcc atcaccacca tcctgaccgc cgtgaccttc 60 tgcttcgcca gcggccagaa catcaccgag gaattctacc agagcacctg cagcgccgtg 120 agcaagggct acctgagcgc cctgcggacc ggctggtaca ccagcgtgat caccatcgag 180 ctgtccaaca tcaaagaaaa caagtgcaac ggcaccgacg ccaaagtgaa gctgatcaag 240 caggaactgg acaagtacaa gaacgccgtg accgagctgc agctgctgat gcagagcacc 300 cccgccaccg gatctggcag cgccatttgc agcggcgtgg ccgtgtgtaa agtgctgcac 360 ctggaaggcg aagtgaacaa gatcaagtcc gccctgctgt ccaccaacaa ggccgtggtg 420 tccctgagca acggcgtgag cgtgctgacc ttcaaggtgc tggatctgaa gaactacatc 480 gacaagcagc tgctgcccat cctgaacaag cagagctgca gcatcagcaa catcgagaca 540 gtgatcgagt tccagcagaa gaacaaccgg ctgctggaaa tcacccggga gttcagcgtg 600 aacgccggag tgaccacccc cgtgtccacc tacatgctga ccaacagcga gctgctgtcc 660 ctgatcaatg acatgcccat caccaacgac cagaaaaagc tgatgagcaa caacgtgcag 720 atcgtgcggc agcagagcta ctccatcatg tgcatcatca aagaagaggt gctggcctac 780 gtggtgcagc tgcccctgta cggcgtgatc gacaccccct gctggaagct gcacaccagc 840 cccctgtgca caaccaacac caaagagggc agcaacatct gcctgacccg gaccgaccgg 900 ggctggtact gcgacaacgc cggcagcgtg tccttctttc cacaggccga gacatgcaag 960 gtgcagagca accgggtgtt ctgcgacacc atgaacagcc ggaccctgcc ctccgaagtg 1020 aacctgtgca acgtggacat cttcaacccc aagtacgact gcaagatcat gacctccaag 1080 accgacgtgt ccagctccgt gatcacctcc ctgggcgcca tcgtgtcctg ctacggcaag 1140 accaagtgca ccgccagcaa caagaacaga ggcatcatca agaccttcag caacggctgc 1200 gactacgtgt ccaataaggg cgtggacacc gtgtccgtgg gcaacacact gtactgcgtg 1260 aataagcagg aaggcaagag cctgtacgtg aagggcgagc ccatcatcaa cttctacgac 1320 cccctggtgt tccccagcga cgagttcgac gctagcatca gccaggtgaa cgagaagatc 1380 aaccagagcc tggccttcat cagaaagagc gacgaactgc tgtccgccat cggcggctac 1440 atccccgagg cccccagaga tggccaggcc tacgtgcgga aggacggcga gtgggtgctg 1500 ctgtctacat ttctgggaag cggaggctct ggtgcccata tcgtgatggt ggacgcctac 1560 aagcctacca aagagcccga ggcctaa 1587 <210> 48 <211> 1515 <212> DNA <213> Artificial Sequence <220> <223> Sc9-10 DS-Cav1 A149C Y458C (RSV-F) <220> <221> misc_feature <222> (1)..(75) <223> Signal peptide <220> <221> misc_feature <222> (1435)..(1515) <223> Foldon domain <400> 48 atggaactgc tgatcctgaa ggccaacgcc atcaccacca tcctgaccgc cgtgaccttc 60 tgcttcgcca gcggccagaa catcaccgag gaattctacc agagcacctg cagcgccgtg 120 agcaagggct acctgagcgc cctgcggacc ggctggtaca ccagcgtgat caccatcgag 180 ctgtccaaca tcaaagaaaa caagtgcaac ggcaccgacg ccaaagtgaa gctgatcaag 240 caggaactgg acaagtacaa gaacgccgtg accgagctgc agctgctgat gcagagcacc 300 cccgccaccg gatctggcag cgccatttgc agcggcgtgg ccgtgtgtaa agtgctgcac 360 ctggaaggcg aagtgaacaa gatcaagtcc gccctgctgt ccaccaacaa ggccgtggtg 420 tccctgagca acggcgtgag cgtgctgacc ttcaaggtgc tggatctgaa gaactacatc 480 gacaagcagc tgctgcccat cctgaacaag cagagctgca gcatcagcaa catcgagaca 540 gtgatcgagt tccagcagaa gaacaaccgg ctgctggaaa tcacccggga gttcagcgtg 600 aacgccggag tgaccacccc cgtgtccacc tacatgctga ccaacagcga gctgctgtcc 660 ctgatcaatg acatgcccat caccaacgac cagaaaaagc tgatgagcaa caacgtgcag 720 atcgtgcggc agcagagcta ctccatcatg tgcatcatca aagaagaggt gctggcctac 780 gtggtgcagc tgcccctgta cggcgtgatc gacaccccct gctggaagct gcacaccagc 840 cccctgtgca caaccaacac caaagagggc agcaacatct gcctgacccg gaccgaccgg 900 ggctggtact gcgacaacgc cggcagcgtg tccttctttc cacaggccga gacatgcaag 960 gtgcagagca accgggtgtt ctgcgacacc atgaacagcc ggaccctgcc ctccgaagtg 1020 aacctgtgca acgtggacat cttcaacccc aagtacgact gcaagatcat gacctccaag 1080 accgacgtgt ccagctccgt gatcacctcc ctgggcgcca tcgtgtcctg ctacggcaag 1140 accaagtgca ccgccagcaa caagaacaga ggcatcatca agaccttcag caacggctgc 1200 gactacgtgt ccaataaggg cgtggacacc gtgtccgtgg gcaacacact gtactgcgtg 1260 aataagcagg aaggcaagag cctgtacgtg aagggcgagc ccatcatcaa cttctacgac 1320 cccctggtgt tccccagcga cgagttcgac gctagcatca gccaggtgaa cgagaagatc 1380 aaccagagcc tggccttcat cagaaagagc gacgaactgc tgtccgccat cggcggctac 1440 atccccgagg cccccagaga tggccaggcc tacgtgcgga aggacggcga gtgggtgctg 1500 ctgtctacat ttctg 1515 <210> 49 <211> 1440 <212> DNA <213> Artificial Sequence <220> <223> Sc-9-10 DS-Cav1 A149C Y458C (RSV-F) without signal peptide <220> <221> misc_feature <222> (1360)..(1440) <223> Foldon domain <400> 49 cagaacatca ccgaggaatt ctaccagagc acctgcagcg ccgtgagcaa gggctacctg 60 agcgccctgc ggaccggctg gtacaccagc gtgatcacca tcgagctgtc caacatcaaa 120 gaaaacaagt gcaacggcac cgacgccaaa gtgaagctga tcaagcagga actggacaag 180 tacaagaacg ccgtgaccga gctgcagctg ctgatgcaga gcacccccgc caccggatct 240 ggcagcgcca tttgcagcgg cgtggccgtg tgtaaagtgc tgcacctgga aggcgaagtg 300 aacaagatca agtccgccct gctgtccacc aacaaggccg tggtgtccct gagcaacggc 360 gtgagcgtgc tgaccttcaa ggtgctggat ctgaagaact acatcgacaa gcagctgctg 420 cccatcctga acaagcagag ctgcagcatc agcaacatcg agacagtgat cgagttccag 480 cagaagaaca accggctgct ggaaatcacc cgggagttca gcgtgaacgc cggagtgacc 540 acccccgtgt ccacctacat gctgaccaac agcgagctgc tgtccctgat caatgacatg 600 cccatcacca acgaccagaa aaagctgatg agcaacaacg tgcagatcgt gcggcagcag 660 agctactcca tcatgtgcat catcaaagaa gaggtgctgg cctacgtggt gcagctgccc 720 ctgtacggcg tgatcgacac cccctgctgg aagctgcaca ccagccccct gtgcacaacc 780 aacaccaaag agggcagcaa catctgcctg acccggaccg accggggctg gtactgcgac 840 aacgccggca gcgtgtcctt ctttccacag gccgagacat gcaaggtgca gagcaaccgg 900 gtgttctgcg acaccatgaa cagccggacc ctgccctccg aagtgaacct gtgcaacgtg 960 gacatcttca accccaagta cgactgcaag atcatgacct ccaagaccga cgtgtccagc 1020 tccgtgatca cctccctggg cgccatcgtg tcctgctacg gcaagaccaa gtgcaccgcc 1080 agcaacaaga acagaggcat catcaagacc ttcagcaacg gctgcgacta cgtgtccaat 1140 aagggcgtgg acaccgtgtc cgtgggcaac acactgtact gcgtgaataa gcaggaaggc 1200 aagagcctgt acgtgaaggg cgagcccatc atcaacttct acgaccccct ggtgttcccc 1260 agcgacgagt tcgacgctag catcagccag gtgaacgaga agatcaacca gagcctggcc 1320 ttcatcagaa agagcgacga actgctgtcc gccatcggcg gctacatccc cgaggccccc 1380 agagatggcc aggcctacgt gcggaaggac ggcgagtggg tgctgctgtc tacatttctg 1440 <210> 50 <211> 528 <212> PRT <213> Artificial Sequence <220> <223> RSV-F-SpyTag-C Tag <220> <221> MISC_FEATURE <222> (1)..(25) <223> signal peptide <220> <221> MISC_FEATURE <222> (479) (505) <223> Foldon structural domain <220> <221> MISC_FEATURE <222> (506)...(511) <223> (GSG)2 connector <220> <221> MISC_FEATURE <222> (512) (524) <223> Spytag <220> <221> MISC_FEATURE <222> (525) (527) <223> C tag <400> 50 Met Glu Leu Leu Ile Leu Lys Ala Asn Ala Ile Thr Thr Ile Leu Thr 1 5 10 15 Ala Val Thr Phe Cys Phe Ala Ser Gly Gln Asn Ile Thr Glu Glu Phe 20 25 30 Tyr Gln Ser Thr Cys Ser Ala Val Ser Lys Gly Tyr Leu Ser Ala Leu 35 40 45 Arg Thr Gly Trp Tyr Thr Ser Val Ile Thr Ile Glu Leu Ser Asn Ile 50 55 60 Lys Glu Asn Lys Cys Asn Gly Thr Asp Ala Lys Val Lys Leu Ile Lys 65 70 75 80 Gln Glu Leu Asp Lys Tyr Lys Asn Ala Val Thr Glu Leu Gln Leu Leu 85 90 95 Met Gln Ser Thr Pro Ala Thr Gly Ser Gly Ser Ala Ile Cys Ser Gly 100 105 110 Val Ala Val Cys Lys Val Leu His Leu Glu Gly Glu Val Asn Lys Ile 115 120 125 Lys Ser Ala Leu Leu Ser Thr Asn Lys Ala Val Val Ser Leu Ser Asn 130 135 140 Gly Val Ser Val Leu Thr Phe Lys Val Leu Asp Leu Lys Asn Tyr Ile 145 150 155 160 Asp Lys Gln Leu Leu Pro Ile Leu Asn Lys Gln Ser Cys Ser Ile Ser 165 170 175 Asn Ile Glu Thr Val Ile Glu Phe Gln Gln Lys Asn Asn Arg Leu Leu 180 185 190 Glu Ile Thr Arg Glu Phe Ser Val Asn Ala Gly Val Thr Thr Pro Val 195 200 205 Ser Thr Tyr Met Leu Thr Asn Ser Glu Leu Leu Ser Leu Ile Asn Asp 210 215 220 Met Pro Ile Thr Asn Asp Gln Lys Lys Leu Met Ser Asn Asn Val Gln 225 230 235 240 Ile Val Arg Gln Gln Ser Tyr Ser Ile Met Cys Ile Ile Lys Glu Glu 245 250 255 Val Leu Ala Tyr Val Val Gln Leu Pro Leu Tyr Gly Val Ile Asp Thr 260 265 270 Pro Cys Trp Lys Leu His Thr Ser Pro Leu Cys Thr Thr Asn Thr Lys 275 280 285 Glu Gly Ser Asn Ile Cys Leu Thr Arg Thr Asp Arg Gly Trp Tyr Cys 290 295 300 Asp Asn Ala Gly Ser Val Ser Phe Phe Pro Gln Ala Glu Thr Cys Lys 305 310 315 320 Val Gln Ser Asn Arg Val Phe Cys Asp Thr Met Asn Ser Arg Thr Leu 325 330 335 Pro Ser Glu Val Asn Leu Cys Asn Val Asp Ile Phe Asn Pro Lys Tyr 340 345 350 Asp Cys Lys Ile Met Thr Ser Lys Thr Asp Val Ser Ser Ser Val Ile 355 360 365 Thr Ser Leu Gly Ala Ile Val Ser Cys Tyr Gly Lys Thr Lys Cys Thr 370 375 380 Ala Ser Asn Lys Asn Arg Gly Ile Ile Lys Thr Phe Ser Asn Gly Cys 385 390 395 400 Asp Tyr Val Ser Asn Lys Gly Val Asp Thr Val Ser Val Gly Asn Thr 405 410 415 Leu Tyr Cys Val Asn Lys Gln Glu Gly Lys Ser Leu Tyr Val Lys Gly 420 425 430 Glu Pro Ile Ile Asn Phe Tyr Asp Pro Leu Val Phe Pro Ser Asp Glu 435 440 445 Phe Asp Ala Ser Ile Ser Gln Val Asn Glu Lys Ile Asn Gln Ser Leu 450 455 460 Ala Phe Ile Arg Lys Ser Asp Glu Leu Leu Ser Ala Ile Gly Gly Tyr 465 470 475 480 Ile Pro Glu Ala Pro Arg Asp Gly Gln Ala Tyr Val Arg Lys Asp Gly 485 490 495 Glu Trp Val Leu Leu Ser Thr Phe Leu Gly Ser Gly Gly Ser Gly Ala 500 ५०५ ५१० His Ile Val Met Val Asp Ala Tyr Lys Pro Thr Lys Glu Pro Glu Ala 515 520 525 <210> 51 <211> 1010 <212> PRT <213> Artificial Sequence <220> It should be noted that there seems to be a small error in the "५०५" and "५१०" in line 29 which are likely supposed to be "505" and "510". I've translated them as "५०५" and "५१०" as they are in the original but they might be formatting or encoding issues.<223> Sc-9-10 DS-Cav1 A149C Y458C (RSV-F) <220> <221> MISC_FEATURE <222> (1)..(25) <223> Signal peptide <220> <221> MISC_FEATURE <222> (479)..(505) <223> Foldon domain <400> 51 Met Glu Leu Leu Ile Leu Lys Ala Asn Ala Ile Thr Thr Ile Leu Thr 1 5 10 15 Ala Val Thr Phe Cys Phe Ala Ser Gly Gln Asn Ile Thr Glu Glu Phe 20 25 30 Tyr Gln Ser Thr Cys Ser Ala Val Ser Lys Gly Tyr Leu Ser Ala Leu 35 40 45 Arg Thr Gly Trp Tyr Thr Ser Val Ile Thr Ile Glu Leu Ser Asn Ile 50 55 60 [[ID=3,6]]Lys Glu Asn Lys Cys Asn Gly Thr Asp Ala Lys Val Lys Leu Ile Lys 65 70 75 80 Gln Glu Leu Asp Lys Tyr Lys Asn Ala Val Thr Glu Leu Gln Leu Leu 85 90 95 Met Gln Ser Thr Pro Ala Thr Gly Ser Gly Ser Ala Ile Cys Ser Gly 100 105 110 Val Ala Val Cys Lys Val Leu His Leu Glu Gly Glu Val Asn Lys Ile 115 120 125 Lys Ser Ala Leu Leu Ser Thr Asn Lys Ala Val Val Ser Leu Ser Asn 130 135 140 Gly Val Ser Val Leu Thr Phe Lys Val Leu Asp Leu Lys Asn Tyr Ile 145 150 155 160 Asp Lys Gln Leu Leu Pro Ile Leu Asn Lys Gln Ser Cys Ser Ile Ser 165 170 175 Asn Ile Glu Thr Val Ile Glu Phe Gln Gln Lys Asn Asn Arg Leu Leu 180 185 190 Glu Ile Thr Arg Glu Phe Ser Val Asn Ala Gly Val Thr Thr Pro Val 195 200 205 Ser Thr Tyr Met Leu Thr Asn Ser Glu Leu Leu Ser Leu Ile Asn Asp 210 215 220 Met Pro Ile Thr Asn Asp Gln Lys Lys Leu Met Ser Asn Asn Val Gln 225 230 235 240 Ile Val Arg Gln Gln Ser Tyr Ser Ile Met Cys Ile Ile Lys Glu Glu 245 250 255 Val Leu Ala Tyr Val Val Gln Leu Pro Leu Tyr Gly Val Ile Asp Thr 260 265 270 Pro Cys Trp Lys Leu His Thr Ser Pro Leu Cys Thr Thr Asn Thr Lys 275 280 285 Glu Gly Ser Asn Ile Cys Leu Thr Arg Thr Asp Arg Gly Trp Tyr Cys 290 295 300 Asp Asn Ala Gly Ser Val Ser Phe Phe Pro Gln Ala Glu Thr Cys Lys 305 310 315 320 Val Gln Ser Asn Arg Val Phe Cys Asp Thr Met Asn Ser Arg Thr Leu 325 330 335 Pro Ser Glu Val Asn Leu Cys Asn Val Asp Ile Phe Asn Pro Lys Tyr 340 345 350 Asp Cys Lys Ile Met Thr Ser Lys Thr Asp Val Ser Ser Ser Val Ile 355 360 365 Thr Ser Leu Met Glu Leu Leu Ile Leu Lys Ala Asn Ala Ile Thr Thr 370 375 380 Ile Leu Thr Ala Val Thr Phe Cys Phe Ala Ser Gly Gln Asn Ile Thr 385 390 395 400 Glu Glu Phe Tyr Gln Ser Thr Cys Ser Ala Val Ser Lys Gly Tyr Leu 405 410 415 Ser Ala Leu Arg Thr Gly Trp Tyr Thr Ser Val Ile Thr Ile Glu Leu 420 425 430 Ser Asn Ile Lys Glu Asn Lys Cys Asn Gly Thr Asp Ala Lys Val Lys 435 440 445 Leu Ile Lys Gln Glu Leu Asp Lys Tyr Lys Asn Ala Val Thr Glu Leu 450 455 460 Gln Leu Leu Met Gln Ser Thr Pro Ala Thr Gly Ser Gly Ser Ala Ile 465 470 475 480 Cys Ser Gly Val Ala Val Cys Lys Val Leu His Leu Glu Gly Glu Val 485 490 495 Asn Lys Ile Lys Ser Ala Leu Leu Ser Thr Asn Lys Ala Val Val Ser 500 505 510 Leu Ser Asn Gly Val Ser Val Leu Thr Phe Lys Val Leu Asp Leu Lys 515 520 525 Asn Tyr Ile Asp Lys Gln Leu Leu Pro Ile Leu Asn Lys Gln Ser Cys 530 535 540 Ser Ile Ser Asn Ile Glu Thr Val Ile Glu Phe Gln Gln Lys Asn Asn 545 550 555 560 Arg Leu Leu Glu Ile Thr Arg Glu Phe Ser Val Asn Ala Gly Val Thr 565 570 575 Thr Pro Val Ser Thr Tyr Met Leu Thr Asn Ser Glu Leu Leu Ser Leu 580 585 590 Ile Asn Asp Met Pro Ile Thr Asn Asp Gln Lys Lys Leu Met Ser Asn 595 600 605 Asn Val Gln Ile Val Arg Gln Gln Ser Tyr Ser Ile Met Cys Ile Ile 610 615 620 Lys Glu Glu Val Leu Ala Tyr Val Val Gln Leu Pro Leu Tyr Gly Val 625 630 635 640 Ile Asp Thr Pro Cys Trp Lys Leu His Thr Ser Pro Leu Cys Thr Thr 645 650 655 Asn Thr Lys Glu Gly Ser Asn Ile Cys Leu Thr Arg Thr Asp Arg Gly 660 665 670 Trp Tyr Cys Asp Asn Ala Gly Ser Val Ser Phe Phe Pro Gln Ala Glu 675 680 685 Thr Cys Lys Val Gln Ser Asn Arg Val Phe Cys Asp Thr Met Asn Ser 690 695 700 Arg Thr Leu Pro Ser Glu Val Asn Leu Cys Asn Val Asp Ile Phe Asn 705 710 715 720 Pro Lys Tyr Asp Cys Lys Ile Met Thr Ser Lys Thr Asp Val Ser Ser 725 730 735 Ser Val Ile Thr Ser Leu Gly Ala Ile Val Ser Cys Tyr Gly Lys Thr 740 745 750 Lys Cys Thr Ala Ser Asn Lys Asn Arg Gly Ile Ile Lys Thr Phe Ser 755 760 765 Asn Gly Cys Asp Tyr Val Ser Asn Lys Gly Val Asp Thr Val Ser Val 770 775 780 Gly Asn Thr Leu Tyr Cys Val Asn Lys Gln Glu Gly Lys Ser Leu Tyr 785 790 795 800 Val Lys Gly Glu Pro Ile Ile Asn Phe Tyr Asp Pro Leu Val Phe Pro 805 810 815 Ser Asp Glu Phe Asp Ala Ser Ile Ser Gln Val Asn Glu Lys Ile Asn 820 825 830 Gln Ser Leu Ala Phe Ile Arg Lys Ser Asp Glu Leu Leu Ser Ala Ile 835 840 845 Gly Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln Ala Tyr Val Arg 850 855 860 Lys Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu Gly Ala Ile Val 865 870 875 880 Ser Cys Tyr Gly Lys Thr Lys Cys Thr Ala Ser Asn Lys Asn Arg Gly 885 890 895 Ile Ile Lys Thr Phe Ser Asn Gly Cys Asp Tyr Val Ser Asn Lys Gly 900 905 910 Val Asp Thr Val Ser Val Gly Asn Thr Leu Tyr Cys Val Asn Lys Gln 915 920 925 Glu Gly Lys Ser Leu Tyr Val Lys Gly Glu Pro Ile Ile Asn Phe Tyr 930 935 940 Asp Pro Leu Val Phe Pro Ser Asp Glu Phe Asp Ala Ser Ile Ser Gln 945 950 955 960 Val Asn Glu Lys Ile Asn Gln Ser Leu Ala Phe Ile Arg Lys Ser Asp 965 970 975 Glu Leu Leu Ser Ala Ile Gly Gly Tyr Ile Pro Glu Ala Pro Arg Asp 980 985 990 Gly Gln Ala Tyr Val Arg Lys Asp Gly Glu Trp Val Leu Leu Ser Thr 995 1000 1005 Phe Leu 1010 <210> 52 <211> 480 <212> PRT <213> Artificial Sequence <220> <223> sc9-10 DS-Cav1 A149C Y458C (RSV-F) without signal peptide <220> <221> MISC_FEATURE <222> (454)..(480) <223> Foldon domain <400> 52 Gln Asn Ile Thr Glu Glu Phe Tyr Gln Ser Thr Cys Ser Ala Val Ser 1 5 10 15 Lys Gly Tyr Leu Ser Ala Leu Arg Thr Gly Trp Tyr Thr Ser Val Ile 20 25 30 Thr Ile Glu Leu Ser Asn Ile Lys Glu Asn Lys Cys Asn Gly Thr Asp 35 40 45 Ala Lys Val Lys Leu Ile Lys Gln Glu Leu Asp Lys Tyr Lys Asn Ala 50 55 60 Val Thr Glu Leu Gln Leu Leu Met Gln Ser Thr Pro Ala Thr Gly Ser 65 70 75 80 Gly Ser Ala Ile Cys Ser Gly Val Ala Val Cys Lys Val Leu His Leu 85 90 95 Glu Gly Glu Val Asn Lys Ile Lys Ser Ala Leu Leu Ser Thr Asn Lys 100 105 110 Ala Val Val Ser Leu Ser Asn Gly Val Ser Val Leu Thr Phe Lys Val 115 120 125 Leu Asp Leu Lys Asn Tyr Ile Asp Lys Gln Leu Leu Pro Ile Leu Asn 130 135 140 Lys Gln Ser Cys Ser Ile Ser Asn Ile Glu Thr Val Ile Glu Phe Gln 145 150 155 160 Gln Lys Asn Asn Arg Leu Leu Glu Ile Thr Arg Glu Phe Ser Val Asn 165 170 175 Ala Gly Val Thr Thr Pro Val Ser Thr Tyr Met Leu Thr Asn Ser Glu 180 185 190 Leu Leu Ser Leu Ile Asn Asp Met Pro Ile Thr Asn Asp Gln Lys Lys 195 200 205 Leu Met Ser Asn Asn Val Gln Ile Val Arg Gln Gln Ser Tyr Ser Ile 210 215 220 Met Cys Ile Ile Lys Glu Glu Val Leu Ala Tyr Val Val Gln Leu Pro 225 230 235 240 Leu Tyr Gly Val Ile Asp Thr Pro Cys Trp Lys Leu His Thr Ser Pro 245 250 255 Leu Cys Thr Thr Asn Thr Lys Glu Gly Ser Asn Ile Cys Leu Thr Arg 260 265 270 Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala Gly Ser Val Ser Phe Phe 275 280 285 Pro Gln Ala Glu Thr Cys Lys Val Gln Ser Asn Arg Val Phe Cys Asp 290 295 300 Thr Met Asn Ser Arg Thr Leu Pro Ser Glu Val Asn Leu Cys Asn Val 305 310 315 320 Asp Ile Phe Asn Pro Lys Tyr Asp Cys Lys Ile Met Thr Ser Lys Thr 325 330 335 Asp Val Ser Ser Ser Val Ile Thr Ser Leu Gly Ala Ile Val Ser Cys 340 345 350 Tyr Gly Lys Thr Lys Cys Thr Ala Ser Asn Lys Asn Arg Gly Ile Ile 355 360 365 Lys Thr Phe Ser Asn Gly Cys Asp Tyr Val Ser Asn Lys Gly Val Asp 370 375 380 Thr Val Ser Val Gly Asn Thr Leu Tyr Cys Val Asn Lys Gln Glu Gly 385 390 395 400 Lys Ser Leu Tyr Val Lys Gly Glu Pro Ile Ile Asn Phe Tyr Asp Pro 405 410 415 Leu Val Phe Pro Ser Asp Glu Phe Asp Ala Ser Ile Ser Gln Val Asn 420 425 430 Glu Lys Ile Asn Gln Ser Leu Ala Phe Ile Arg Lys Ser Asp Glu Leu 435 440 445 Leu Ser Ala Ile Gly Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln 450 455 460 Ala Tyr Val Arg Lys Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu 465 470 475 480 <210> 53 <211> 1704 <212> DNA <213> Artificial Sequence <220> <223> RSV-F DS-Cav1-SpyTag-C tag <220> <221> misc_feature <222> (1)..(75) <223> signal peptide <220> <221> misc_feature <222> (1552) (1632) <223> Foldon structural domain <220> <221> misc_feature <222> (1633) (1650) <223> (GSG)2 connector <220> <221> misc_feature <222> (1651)...(1689) <223> Spytag <220> <221> misc_feature <222> (1690) (1701) <223> C tag <220> <221> misc_feature <222> (1702)...(1704) <223> stop codon <400> 53 atggaactgc tgatcctgaa ggccaacgcc atcaccacca tcctgaccgc cgtgaccttc 60 tgcttcgcca gcggccagaa catcaccgag gaattctacc agagcacctg cagcgccgtg 120 agcaagggct acctgagcgc cctgcggacc ggctggtaca ccagcgtgat caccatcgag 180 ctgtccaaca tcaagaaaa caagtgcaac ggcaccgacg ccaaagtgaa gctgatcaag 240 caggaactgg acagtacaa gaacgccgtg accgagctgc agctctctgat gcagagcacc 300 cccgccacca aaacagagc cagaagagag ctgccccggt tcatgaacta caccctgaac 360 aacgccaaga aaaccaacgt gaccctgagc aagaagagaa agaagagatt cctgggcttc 420 ctgctgggcg tgggcagcgc cattgccagc ggcgtggccg tgtgtaaagt gctgcacctg 480 gaaggcgaag tgaaaagat gaagtccgcc ctgctgtcca ccaacaaggc cgtggtgtcc 540 ctgagcaacg gcgtgagcgt gctgaccttc aaggtgctgg atctgaagaa ctacatcgac 600 aagcagctgc tgcccatcct gaaagcag agctgcagca tcagcaacat cgagacagtg 660 720 gccggagtga ccaccccgt gtccacctac atgctgacca acagcgagct gctgtccctg 780 atcaatgaca tgcccatcac caacgaccag aaaaagctga tgagcaacaa cgtgcagatc 840 gtgcggcagc agagctactc catcatgtgc atcatcaaag aagaggtgct ggcctacgtg 900 gtgcagctgc ccctgtacgg cgtgatcgac acccccttgct ggaagctgca caccagcccc 960 ctgtgcacaa ccaacaccaa agagggcagc aacatctgcc tgacccggac cgaccggggc 1020 tggtactgcg aaacgccgg cagcgtgtcc ttctttccac aggccgagac atgcaaggtg 1080 cagagcaacc gggtgttctg cgacaccatg aacagcctga ccctgccctc cgaagtgaac 1140 ctgtgcaacg tggacatctt caaccccaag tacgactgca agatcatgac ctccaagacc 1200 gacgtgtcca gctccgtgat cacctccctg ggcgccatcg tgtcctgcta cggcaagacc 1260 aagtgcaccg ccagcaacaa gaacagaggc atcatcaaga ccttcagcaa cggctgcgac 1320 tacgtgtcca ataagggcgt ggacaccgtg tccgtgggca acacactgta ctacgtgaat 1380 aaggaag gcaagagcct gtacgtgaag ggcgagccca tcatcaactt ctacgacccc 1440 ctggtgttcc ccagcgacga gttcgacgcc agcatcagcc aggtgaacga gaagatcaac 1500 cagagcctgg ccttcatcag aaagagcgac gaactgctgt ccgccatcgg cggctacatc 1560 cccgaggccc ccagagatgg ccaggcctac gtgcggaagg acggcgagtg ggtgctgctg 1620 tctacatttc tgggaagcgg aggctctggt gcccatatcg tgatggtgga cgcctacaag 1680 cctaccaaag agcccgaggc ctaa 1704 <210> 54 <211> 1632 <212> DNA <213> Artificial Sequence <220> <223> RSV-F DS-Cav1 <220> <221> misc_feature <222> (1)..(75) <223> Signal peptide <220> <221> misc_feature <222> (1552)..(1632) <223> Foldon domain <400> 54 atggaactgc tgatcctgaa ggccaacgcc atcaccacca tcctgaccgc cgtgaccttc 60 tgcttcgcca gcggccagaa catcaccgag gaattctacc agagcacctg cagcgccgtg 120 agcaagggct acctgagcgc cctgcggacc ggctggtaca ccagcgtgat caccatcgag 180 ctgtccaaca tcaagaaaa caagtgcaac ggcaccgacg ccaaagtgaa gctgatcaag 240 caggaactgg acagtacaa gaacgccgtg accgagctgc agctctctgat gcagagcacc 300 cccgccacca aaacagagc cagaagagag ctgccccggt tcatgaacta caccctgaac 360 aacgccaaga aaaccaacgt gaccctgagc aagaagagaa agaagagatt cctgggcttc 420 ctgctgggcg tgggcagcgc cattgccagc ggcgtggccg tgtgtaaagt gctgcacctg 480 gaaggcgaag tgaaaagat gaagtccgcc ctgctgtcca ccaacaaggc cgtggtgtcc 540 ctgagcaacg gcgtgagcgt gctgaccttc aaggtgctgg atctgaagaa ctacatcgac 600 aagcagctgc tgcccatcct gaaagcag agctgcagca tcagcaacat cgagacagtg 660 720 gccggagtga ccaccccgt gtccacctac atgctgacca acagcgagct gctgtccctg 780 atcaatgaca tgcccatcac caacgaccag aaaaagctga tgagcaacaa cgtgcagatc 840 gtgcggcagc agagctactc catcatgtgc atcatcaaag aagaggtgct ggcctacgtg 900 gtgcagctgc ccctgtacgg cgtgatcgac accccctgct ggaagctgca caccagcccc 960 ctgtgcacaa ccaacaccaa agagggcagc aacatctgcc tgacccggac cgaccggggc 1020 tggtactgcg acaacgccgg cagcgtgtcc ttctttccac aggccgagac atgcaaggtg 1080 cagagcaacc gggtgttctg cgacaccatg aacagcctga ccctgccctc cgaagtgaac 1140 ctgtgcaacg tggacatctt caaccccaag tacgactgca agatcatgac ctccaagacc 1200 gacgtgtcca gctccgtgat cacctccctg ggcgccatcg tgtcctgcta cggcaagacc 1260 aagtgcaccg ccagcaacaa gaacagaggc atcatcaaga ccttcagcaa cggctgcgac 1320 tacgtgtcca ataagggcgt ggacaccgtg tccgtgggca acacactgta ctacgtgaat 1380 aagcaggaag gcaagagcct gtacgtgaag ggcgagccca tcatcaactt ctacgacccc 1440 ctggtgttcc ccagcgacga gttcgacgcc agcatcagcc aggtgaacga gaagatcaac 1500 cagagcctgg ccttcatcag aaagagcgac gaactgctgt ccgccatcgg cggctacatc 1560 cccgaggccc ccagagatgg ccaggcctac gtgcggaagg acggcgagtg ggtgctgctg 1620 tctacatttc tg 1632 <210> 55 <211> 1557 <212> DNA <213> Artificial Sequence <220> <223> RSV-F DS-Cav1 without signal peptide <220> <221> misc_feature <222> (1477)..(1557) <223> Foldon domain <400> 55 cagaacatca ccgaggaatt ctaccagagc acctgcagcg ccgtgagcaa gggctacctg 60 agcgccctgc ggaccggctg gtacaccagc gtgatcacca tcgagctgtc caacatcaaa 120 gaaaacaagt gcaacggcac cgacgccaaa gtgaagctga tcaagcagga actggacaag 180 tacaagaacg ccgtgaccga gctgcagctg ctgatgcaga gcacccccgc caccaacaac 240 agagccagaa gagagctgcc ccggttcatg aactacaccc tgaacaacgc caagaaaacc 300 aacgtgaccc tgagcaagaa gagaaagaga agattcctgg gcttcctgct gggcgtgggc 360 agcgccattg ccagcggcgt ggccgtgtgt aaagtgctgc acctggaagg cgaagtgaac 420 aagatcaagt ccgccctgct gtccaccaac aaggccgtgg tgtccctgag caacggcgtg 480 agcgtgctga ccttcaaggt gctggatctg aagaactaca tcgacaagca gctgctgccc 540 atcctgaaca agcagagctg cagcatcagc aacatcgaga cagtgatcga gttccagcag 600 aagaacaacc ggctgctgga aatcacccgg gagttcagcg tgaacgccgg agtgaccacc 660 cccgtgtcca cctacatgct gaccaacagc gagctgctgt ccctgatcaa tgacatgccc 720 atcaccaacg accagaaaaa gctgatgagc aacaacgtgc agatcgtgcg gcagcagagc 780 tactccatca tgtgcatcat caaagaagag gtgctggcct acgtggtgca gctgcccctg 840 tacggcgtga tcgacacccc ctgctggaag ctgcacacca gccccctgtg cacaaccaac 900 accaaagagg gcagcaacat ctgcctgacc cggaccgacc ggggctggta ctgcgacaac 960 gccggcagcg tgtccttctt tccacaggcc gagacatgca aggtgcagag caaccgggtg 1020 ttctgcgaca ccatgaacag cctgaccctg ccctccgaag tgaacctgtg caacgtggac 1080 atcttcaacc ccaagtacga ctgcaagatc atgacctcca agaccgacgt gtccagctcc 1140 gtgatcacct ccctgggcgc catcgtgtcc tgctacggca agaccaagtg caccgccagc 1200 aacaagaaca gaggcatcat caagaccttc agcaacggct gcgactacgt gtccaataag 1260 ggcgtggaca ccgtgtccgt gggcaacaca ctgtactacg tgaataagca ggaaggcaag 1320 agcctgtacg tgaagggcga gcccatcatc aacttctacg accccctggt gttccccagc 1380 gacgagttcg acgccagcat cagccaggtg aacgagaaga tcaaccagag cctggccttc 1440 atcagaaaga gcgacgaact gctgtccgcc atcggcggct acatccccga ggcccccaga 1500 gatggccagg cctacgtgcg gaaggacggc gagtgggtgc tgctgtctac atttctg 1557 <210> 56 <211> 567 <212> PRT <213> Artificial Sequence <220> [[ID=​​​​​​​​​​​​​​​​​​​​​​​​​​​<221> MISC_FEATURE <222> (551)..(563) <223> Spytag <220> <221> MISC_FEATURE <222> (564)..(567) <223> C label <400> 56 Met Glu Leu Leu Ile Leu Lys Ala Asn Ala Ile Thr Thr Ile Leu Thr 1 5 10 15<00***n Ile Lys 65 70 75 80 Gln Glu Leu Asp Lys Tyr Lys Asn Ala Val Thr Glu Leu Gln Leu Leu 85 90 95 Met Gln Ser Thr Pro Ala Thr Asn Asn Arg Ala Arg Arg Glu Leu Pro 100 105 110 Arg Phe Met Asn Tyr Thr Leu Asn Asn Ala Lys Lys Thr Asn Val Thr 115 120 125 ​​​​​​​​​​​​Leu Ser Lys Lys Arg Lys Arg Arg Phe Leu Gly Phe Leu Leu Gly Val 130 135 140 Gly Ser Ala Ile Ala Ser Gly Val Ala Val Cys Lys Val Leu His Leu 145 150 155 160 Glu Gly Glu Val Asn Lys Ile Lys Ser Ala Leu Leu Ser Thr Asn Lys 165 170 175 Ala Val Val Ser Leu Ser Asn Gly Val Ser Val Leu Thr Phe Lys Val 180 185 190 Leu Asp Leu Lys Asn Tyr Ile Asp Lys Gln Leu Leu Pro Ile Leu Asn 195 200 205 Lys Gln Ser Cys Ser Ile Ser Asn Ile Glu Thr Val Ile Glu Phe Gln 210 215 220 Gln Lys Asn Asn Arg Leu Leu Glu Ile Thr Arg Glu Phe Ser Val Asn 225 230 235 240 Ala Gly Val Thr Thr Pro Val Ser Thr Tyr Met Leu Thr Asn Ser Glu 245 250 255 Leu Leu Ser Leu Ile Asn Asp Met Pro Ile Thr Asn Asp Gln Lys Lys 260 265 270 Leo Met Ser Asn Asn Val Gln Ile Val Arg Gln Gln Ser Tyr Ser Ile 275 280 285 Met Cys Ile Ile Lys Glu Glu Val Leu Ala Tyr Val Val Gln Leu Pro 290 295 300 Leu Tyr Gly Val Ile Asp Thr Pro Cys Trp Lys Leu His Thr Ser Pro 305 310 315 320 Leu Cys Thr Thr Asn Thr Lys Glu Gly Ser Asn Ile Cys Leu Thr Arg 325 330 335 Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala Gly Ser Val Ser Phe Phe 340 345 350 Pro Gln Ala Glu Thr Cys Lys Val Gln Ser Asn Arg Val Phe Cys Asp 355 360 365 Thr Met Asn Ser Leu Thr Leu Pro Ser Glu Val Asn Leu Cys Asn Val 370 375 380 Asp Ile Phe Asn Pro Lys Tyr Asp Cys Lys Ile Met Thr Ser Lys Thr 385 390 395 400 Asp Val Ser Ser Ser Val Ile Thr Ser Leu Gly Ala Ile Val Ser Cys 405 410 415 Tyr Gly Lys Thr Lys Cys Thr Ala Ser Asn Lys Asn Arg Gly Ile Ile 420 425 430 Lys Thr Phe Ser Asn Gly Cys Asp Tyr Val Ser Asn Lys Gly Val Asp 435 440 445 Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr Val Asn Lys Gln Glu Gly 450 455 460 Lys Ser Leu Tyr Val Lys Gly Glu Pro Ile Ile Asn Phe Tyr Asp Pro 465 470 475 480 Leu Val Phe Pro Ser Asp Glu Phe Asp Ala Ser Ile Ser Gln Val Asn 485 490 495 Glu Lys Ile Asn Gln Ser Leu Ala Phe Ile Arg Lys Ser Asp Glu Leu 500 505 510 Leu Ser Ala Ile Gly Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln 515 520 525 Ala Tyr Val Arg Lys Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu 530 535 540 Gly Ser Gly Gly Ser Gly Ala His Ile Val Met Val Asp Ala Tyr Lys 545 550 555 560 Pro Thr Lys Glu Pro Glu Ala 565 <210> 57 <211> 544 <212> PRT <213> Artificial Sequence <220> <223> RSV-F DS-Cav1 <220> <221> MISC_FEATURE <222> (1)..(25) <223> Signal peptide <220> <221> MISC_FEATURE <222> (518)..(544) <223> Foldon domain <400> 57 Met Glu Leu Leu Ile Leu Lys Ala Asn Ala Ile Thr Thr Ile Leu Thr 1 5 10 15 Ala Val Thr Phe Cys Phe Ala Ser Gly Gln Asn Ile Thr Glu Glu Phe 20 25 30 Tyr Gln Ser Thr Cys Ser Ala Val Ser Lys Gly Tyr Leu Ser Ala Leu 35 40 45 Arg Thr Gly Trp Tyr Thr Ser Val Ile Thr Ile Glu Leu Ser Asn Ile 50 55 60 Lys Glu Asn Lys Cys Asn Gly Thr Asp Ala Lys Val Lys Leu Ile Lys 65 70 75 80 Gln Glu Leu Asp Lys Tyr Lys Asn Ala Val Thr Glu Leu Gln Leu Leu 85 90 95 Met Gln Ser Thr Pro Ala Thr Asn Asn Arg Ala Arg Arg Glu Leu Pro 100 105 110 Arg Phe Met Asn Tyr Thr Leu Asn Asn Ala Lys Lys Thr Asn Val Thr 115 120 125 Leu Ser Lys Lys Arg Lys Arg Arg Phe Leu Gly Phe Leu Leu Gly Val 130 135 140 Gly Ser Ala Ile Ala Ser Gly Val Ala Val Cys Lys Val Leu His Leu 145 150 155 160 Glu Gly Glu Val Asn Lys Ile Lys Ser Ala Leu Leu Ser Thr Asn Lys 165 170 175 Ala Val Val Ser Leu Ser Asn Gly Val Ser Val Leu Thr Phe Lys Val 180 185 190 Leu Asp Leu Lys Asn Tyr Ile Asp Lys Gln Leu Leu Pro Ile Leu Asn 195 200 205 Lys Gln Ser Cys Ser Ile Ser Asn Ile Glu Thr Val Ile Glu Phe Gln 210 215 220 Gln Lys Asn Asn Arg Leu Leu Glu Ile Thr Arg Glu Phe Ser Val Asn 225 230 235 240 Ala Gly Val Thr Thr Pro Val Ser Thr Tyr Met Leu Thr Asn Ser Glu 245 250 255 Leu Leu Ser Leu Ile Asn Asp Met Pro Ile Thr Asn Asp Gln Lys Lys 260 265 270 Leo Met Ser Asn Asn Val Gln Ile Val Arg Gln Gln Ser Tyr Ser Ile 275 280 285 Met Cys Ile Ile Lys Glu Glu Val Leu Ala Tyr Val Val Gln Leu Pro 290 295 300 Leu Tyr Gly Val Ile Asp Thr Pro Cys Trp Lys Leu His Thr Ser Pro 305 310 315 320 Leu Cys Thr Thr Asn Thr Lys Glu Gly Ser Asn Ile Cys Leu Thr Arg 325 330 335 Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala Gly Ser Val Ser Phe Phe 340 345 350 Pro Gln Ala Glu Thr Cys Lys Val Gln Ser Asn Arg Val Phe Cys Asp 355 360 365 Thr Met Asn Ser Leu Thr Leu Pro Ser Glu Val Asn Leu Cys Asn Val 370 375 380 Asp Ile Phe Asn Pro Lys Tyr Asp Cys Lys Ile Met Thr Ser Lys Thr 385 390 395 400 Asp Val Ser Ser Ser Val Ile Thr Ser Leu Gly Ala Ile Val Ser Cys 405 410 415 Tyr Gly Lys Thr Lys Cys Thr Ala Ser Asn Lys Asn Arg Gly Ile Ile 420 425 430 Lys Thr Phe Ser Asn Gly Cys Asp Tyr Val Ser Asn Lys Gly Val Asp 435 440 445 Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr Val Asn Lys Gln Glu Gly 450 455 460 Lys Ser Leu Tyr Val Lys Gly Glu Pro Ile Ile Asn Phe Tyr Asp Pro 465 470 475 480 Leu Val Phe Pro Ser Asp Glu Phe Asp Ala Ser Ile Ser Gln Val Asn 485 490 495 Glu Lys Ile Asn Gln Ser Leu Ala Phe Ile Arg Lys Ser Asp Glu Leu 500 505 510 Leu Ser Ala Ile Gly Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln 515 520 525 Ala Tyr Val Arg Lys Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu 530 535 540 <210> 58 <211> 519 <212> PRT <213> Artificial Sequence <220> <223> RSV-F DS-Cav1 without signal peptide <220> <221> MISC_FEATURE <222> (493) (519) <223> Foldon structural domain <400> 58 Gln Asn Ile Thr Glu Glu Phe Tyr Gln Ser Thr Cys Ser Ala Val Ser 1 5 10 15 Lys Gly Tyr Leu Ser Ala Leu Arg Thr Gly Trp Tyr Thr Ser Val Ile 20 25 30 Thr Ile Glu Leu Ser Asn Ile Lys Glu Asn Lys Cys Asn Gly Thr Asp 35 40 45 Ala Lys Val Lys Leu Ile Lys Gln Glu Leu Asp Lys Tyr Lys Asn Ala 50 55 60 Val Thr Glu Leu Gln Leu Leu Met Gln Ser Thr Pro Ala Thr Asn Asn 65 70 75 80 Arg Ala Arg Arg Glu Leu Pro Arg Phe Met Asn Tyr Thr Leu Asn Asn 85 90 95 Ala Lys Lys Thr Asn Val Thr Leu Ser Lys Lys Arg Lys Arg Arg Phe 100 105 110 Leu Gly Phe Leu Leu Gly Val Gly Ser Ala Ile Ala Ser Gly Val Ala 115 120 125 Val Cys Lys Val Leu His Leu Glu Gly Glu Val Asn Lys Ile Lys Ser 130 135 140 Ala Leu Leu Ser Thr Asn Lys Ala Val Val Ser Leu Ser Asn Gly Val 145 150 155 160 Ser Val Leu Thr Phe Lys Val Leu Asp Leu Lys Asn Tyr Ile Asp Lys 165 170 175 Gln Leu Leu Pro Ile Leu Asn Lys Gln Ser Cys Ser Ile Ser Asn Ile 180 185 190 Glu Thr Val Ile Glu Phe Gln Gln Lys Asn Asn Arg Leu Leu Glu Ile 195 200 205 Thr Arg Glu Phe Ser Val Asn Ala Gly Val Thr Thr Pro Val Ser Thr 210 215 220 Tyr Met Leu Thr Asn Ser Glu Leu Leu Ser Leu Ile Asn Asp Met Pro 225 230 235 240 Ile Thr Asn Asp Gln Lys Lys Leu Met Ser Asn Asn Val Gln Ile Val 245 250 255 Arg Gln Gln Ser Tyr Ser Ile Met Cys Ile Ile Lys Glu Glu Val Leu 260 265 270 Ala Tyr Val Val Gln Leu Pro Leu Tyr Gly Val Ile Asp Thr Pro Cys 275 280 285 Trp Lys Leu His Thr Ser Pro Leu Cys Thr Thr Asn Thr Lys Glu Gly 290 295 300 Ser Asn Ile Cys Leu Thr Arg Thr Asp Arg Gly Trp Tyr Cys Asp Asn 305 310 315 320 Ala Gly Ser Val Ser Phe Phe Pro Gln Ala Glu Thr Cys Lys Val Gln 325 330 335 Ser Asn Arg Val Phe Cys Asp Thr Met Asn Ser Leu Thr Leu Pro Ser 340 345 350 Glu Val Asn Leu Cys Asn Val Asp Ile Phe Asn Pro Lys Tyr Asp Cys 355 360 365 Lys Ile Met Thr Ser Lys Thr Asp Val Ser Ser Ser Val Ile Thr Ser 370 375 380 Leu Gly Ala Ile Val Ser Cys Tyr Gly Lys Thr Lys Cys Thr Ala Ser 385 390 395 400 Asn Lys Asn Arg Gly Ile Ile Lys Thr Phe Ser Asn Gly Cys Asp Tyr 405 410 415 Val Ser Asn Lys Gly Val Asp Thr Val Ser Val Gly Asn Thr Leu Tyr 420 425 430 Tyr Val Asn Lys Gln Glu Gly Lys Ser Leu Tyr Val Lys Gly Glu Pro 435 440 445 Ile Ile Asn Phe Tyr Asp Pro Leu Val Phe Pro Ser Asp Glu Phe Asp 450 455 460 Ala Ser Ile Ser Gln Val Asn Glu Lys Ile Asn Gln Ser Leu Ala Phe 465 470 475 480 Ile Arg Lys Ser Asp Glu Leu Leu Ser Ala Ile Gly Gly Tyr Ile Pro 485 490 495 Glu Ala Pro Arg Asp Gly Gln Ala Tyr Val Arg Lys Asp Gly Glu Trp 500 505 510 Val Leu Leu Ser Thr Phe Leu 515 <210> 59 <211> 2088 <212> DNA <213> Artificial Sequence <220> <223> gH without signal peptide <220> <221> misc_feature <222> (1077)..(1077) <223> The C>A mutation occurs at position 1077. <220> <221> misc_feature <222> (1)..(2082) <223> extracellular domain <220> <221> misc_feature <222> (2083)...(2088) <223> Transmembrane domain (truncated) <400> 59 cgatatggcg cagaagccgt atccgaaccg ctggacaaag cgtttcacct actgctcaac 60 acctacggga gacccatccg cttcctgcgt gaaaatacca cccagtgtac ctacaacagc 120 agcctccgta acagcacggt cgtcagggaa aacgccatca gtttcaactt tttccaaagc 180 tataatcaat actatgtatt ccatatgcct cgatgtcttt ttgcgggtcc tctggcggag 240 cagtttctga accaggtaga tctgaccgaa accctggaaa gataccaaca gagacttaac 300 acttacgcgc tggtatccaa agacctggcc agctaccgat ctttttcgca gcagctaaag 360 gcacaagaca gcctaggtga acagcccacc actgtgccac cgcccattga cctgtcaata 420 cctcacgttt ggatgccacc gcaaaccact ccacacggct ggacagaatc acataccacc 480 tcaggactac accgaccaca ctttaaccag acctgtatcc tctttgatgg acacgatcta 540 ctattcagca ccgtcacacc ttgtttgcac caaggctttt acctcatcga cgaactacgt 600 tacgttaaaa taacactgac cgaggacttc ttcgtagtta cggtgtccat agacgacgac 660 acacccatgc tgcttatctt cggccatctt ccacgcgtac ttttcaaagc gccctatcaa 720 cgcgacaact ttatactacg acaaactgaa aaacacgagc tcctggtgct agttaagaaa 780 gatcaactga accgtcactc ttatctcaaa gacccggact ttcttgacgc cgcacttgac 840 ttcaactacc tagacctcag cgcactacta cgtaacagct ttcaccgtta cgccgtggat 900 gtactcaaga gcggtcgatg tcagatgctg gaccgccgca cggtagaaat ggccttcgcc 960 tacgcattag cactgttcgc agcagcccga caagaagagg ccggcgccca agtctccgtc 1020 ccacgggccc tagaccgcca ggccgcactc ttacaaatac aagaatttat gatcacatgc 1080 ctctcacaaa caccaccacg caccacgttg ctgctgtatc ccacggccgt ggacctggcc 1140 aaacgagccc tttggacacc gaatcagatc accgacatca ccagcctcgt acgcctggtc 1200 tacatactct ctaaacagaa tcagcaacat ctcatccccc aatgggcact acgacagatc 1260 gccgactttg ccctaaaact acacaaaacg cacctggcct cttttctttc agccttcgca 1320 cgccaagaac tctacctcat gggcagcctc gtccactcca tgctggtaca tacgacggag 1380 agacgcgaaa tcttcatcgt agaaacgggc ctctgttcat tggccgagct atcacacttt 1440 acgcagttgt tagctcatcc acaccacgaa tacctcagcg acctgtacac accctgttcc 1500 agtagcgggc gacgcgatca ctcgctcgaa cgcctcacgc gtctcttccc cgatgccacc 1560 gtccccgcta ccgttcccgc cgccctctcc atcctatcta ccatgcaacc aagcacgctg 1620 gaaaccttcc ccgacctgtt ttgcttgccg ctcggcgaat ccttctccgc gctgaccgtc 1680 tccgaacacg tcagttatat cgtaacaaac cagtacctga tcaaaggtat ctcctaccct 1740 gtctccacca ccgtcgtagg ccagagcctc atcatcaccc agacggacag tcaaactaaa 1800 tgcgaactga cgcgcaacat gcataccaca cacagcatca cagtggcgct caacatttcg 1860 ctagaaaact gcgccttttg ccaaagcgcc ctgctagaat acgacgacac gcaaggcgtc 1920 atcaacatca tgtacatgca cgactcggac gacgtccttt tcgccctgga tccctacaac 1980 gaagtggtgg tctcatctcc gcgaactcac tacctcatgc ttttgaaaaa cggtacggta 2040 ctagaagtaa ctgacgtcgt cgtggacgcc accgacagtc gtctcctc 2088 <210> 60 <211> 696 <212> PRT <213> Artificial Sequence <220> <223> gH without signal peptide <220> <221> MISC_FEATURE <222> (1)..(694) <223> extracellular domain <220> <221> MISC_FEATURE <222> (694) (696) <223> Transmembrane domain (truncated) <400> 60 Arg Tyr Gly Ala Glu Ala Val Ser Glu Pro Leu Asp Lys Ala Phe His 1 5 10 15 Leu Leu Leu Asn Thr Tyr Gly Arg Pro Ile Arg Phe Leu Arg Glu Asn 20 25 30 Thr Thr Gln Cys Thr Tyr Asn Ser Ser Leu Arg Asn Ser Thr Val Val 35 40 45 Arg Glu Asn Ala Ile Ser Phe Asn Phe Phe Gln Ser Tyr Asn Gln Tyr 50 55 60 Tyr Val Phe His Met Pro Arg Cys Leu Phe Ala Gly Pro Leu Ala Glu 65 70 75 80 Gln Phe Leu Asn Gln Val Asp Leu Thr Glu Thr Leu Glu Arg Tyr Gln 85 90 95 Gln Arg Leu Asn Thr Tyr Ala Leu Val Ser Lys Asp Leu Ala Ser Tyr 100 105 110 Arg Ser Phe Ser Gln Gln Leu Lys Ala Gln Asp Ser Leu Gly Glu Gln 115 120 125 Pro Thr Thr Val Pro Pro Pro Ile Asp Leu Ser Ile Pro His Val Trp 130 135 140 Met Pro Pro Gln Thr Thr Pro His Gly Trp Thr Glu Ser His Thr Thr 145 150 155 160 Ser Gly Leu His Arg Pro His Phe Asn Gln Thr Cys Ile Leu Phe Asp 165 170 175 Gly His Asp Leu Leu Phe Ser Thr Val Thr Pro Cys Leu His Gln Gly 180 185 190 Phe Tyr Leu Ile Asp Glu Leu Arg Tyr Val Lys Ile Thr Leu Thr Glu 195 200 205 Asp Phe Phe Val Val Thr Val Ser Ile Asp Asp Asp Thr Pro Met Leu 210 215 220 Leu Ile Phe Gly His Leu Pro Arg Val Leu Phe Lys Ala Pro Tyr Gln 225 230 235 240 Arg Asp Asn Phe Ile Leu Arg Gln Thr Glu Lys His Glu Leu Leu Val 245 250 255 Leu Val Lys Lys Asp Gln Leu Asn Arg His Ser Tyr Leu Lys Asp Pro 260 265 270 Asp Phe Leu Asp Ala Ala Leu Asp Phe Asn Tyr Leu Asp Leu Ser Ala 275 280 285 Leu Leu Arg Asn Ser Phe His Arg Tyr Ala Val Asp Val Leu Lys Ser 290 295 300 Gly Arg Cys Gln Met Leu Asp Arg Arg Thr Val Glu Met Ala Phe Ala 305 310 315 320 Tyr Ala Leu Ala Leu Phe Ala Ala Ala Arg Gln Glu Glu Ala Gly Ala 325 330 335 Gln Val Ser Val Pro Arg Ala Leu Asp Arg Gln Ala Ala Leu Leu Gln 340 345 350 Ile Gln Glu Phe Met Ile Thr Cys Leu Ser Gln Thr Pro Pro Arg Thr 355 360 365 Thr Leu Leu Leu Tyr Pro Thr Ala Val Asp Leu Ala Lys Arg Ala Leu 370 375 380 Trp Thr Pro Asn Gln Ile Thr Asp Ile Thr Ser Leu Val Arg Leu Val 385 390 395 400 Tyr Ile Leu Ser Lys Gln Asn Gln Gln His Leu Ile Pro Gln Trp Ala 405 410 415 Leu Arg Gln Ile Ala Asp Phe Ala Leu Lys Leu His Lys Thr His Leu 420 425 430 Ala Ser Phe Leu Ser Ala Phe Ala Arg Gln Glu Leu Tyr Leu Met Gly 435 440 445 Ser Leu Val His Ser Met Leu Val His Thr Thr Glu Arg Arg Glu Ile 450 455 460 Phe Ile Val Glu Thr Gly Leu Cys Ser Leu Ala Glu Leu Ser His Phe 465 470 475 480 Thr Gln Leu Leu Ala His Pro His His Glu Tyr Leu Ser Asp Leu Tyr 485 490 495 Thr Pro Cys Ser Ser Ser Gly Arg Arg Asp His Ser Leu Glu Arg Leu 500 505 510 Thr Arg Leu Phe Pro Asp Ala Thr Val Pro Ala Thr Val Pro Ala Ala 515 520 525 Leu Ser Ile Leu Ser Thr Met Gln Pro Ser Thr Leu Glu Thr Phe Pro 530 535 540 Asp Leu Phe Cys Leu Pro Leu Gly Glu Ser Phe Ser Ala Leu Thr Val 545 550 555 560 Ser Glu His Val Ser Tyr Ile Val Thr Asn Gln Tyr Leu Ile Lys Gly 565 570 575 Ile Ser Tyr Pro Val Ser Thr Thr Val Val Gly Gln Ser Leu Ile Ile 580 585 590 Thr Gln Thr Asp Ser Gln Thr Lys Cys Glu Leu Thr Arg Asn Met His 595 600 605 Thr Thr His Ser Ile Thr Val Ala Leu Asn Ile Ser Leu Glu Asn Cys 610 615 620 Ala Phe Cys Gln Ser Ala Leu Leu Glu Tyr Asp Asp Thr Gln Gly Val 625 630 635 640 Ile Asn Ile Met Tyr Met His Asp Ser Asp Asp Val Leu Phe Ala Leu 645 650 655 Asp Pro Tyr Asn Glu Val Val Val Ser Ser Pro Arg Thr His Tyr Leu 660 665 670 Met Leu Leu Lys Asn Gly Thr Val Leu Glu Val Thr Asp Val Val Val 675 680 685 Asp Ala Thr Asp Ser Arg Leu Leu 690 695

Claims

1. A composition comprising a virus-like particle displaying an antigen component, wherein the composition comprises: i) an antigenic component comprising a first peptide tag, wherein the antigenic component exceeds 50 kDa and is a multimer, and is an immunogenic component of an HCMV pentamer or an immunogenic component of a prefusion RSV-F protein; and ii) a portion comprising a second peptide tag, wherein said portion is a surface antigen of hepatitis B virus (HBsAg), wherein the antigen component and the portion are linked by an isopeptide bond between the first peptide tag and the second peptide tag, and wherein the first peptide tag and the second peptide tag are a SpyTag / SpyCatcher pair.

2. The composition of claim 1, wherein the immunogenic component of the HCMV pentamer comprises each of the gH, gL, pUL128, pUL130, and pUL131 subunits.

3. The composition of claim 2, wherein the immunogenic component of the HCMV pentamer comprises a gH subunit having a truncated transmembrane domain.

4. The composition according to claim 2 or 3, wherein the gH, gL, pUL128, pUL130 and pUL131 subunits are amino acid sequences listed in SEQ ID NOs: 28, 31, 35, 33 and 36, respectively.

5. The composition of claim 2 or 3, wherein the first peptide tag is attached to the gH subunit. The composition of claim 5 , wherein the first peptide tag is attached to the C-terminus of the gH subunit.

7. The composition of claim 1, wherein the immunogenic component of the prefusion RSV-F protein is a stabilized prefusion F protein.

8. The composition of claim 1, wherein the immunogenic component of the prefusion RSV-F protein comprises a trimer of an F1 subunit and an F2 subunit.

9. A composition according to any one of claims 7 or 8, wherein the RSV-F protein is an amino acid sequence listed in any one of SEQ ID NO: 50 to 52 or 56 to 58.

10. The composition of claim 1, wherein the first peptide tag is attached to the C-terminus of the prefusion RSV-F protein.

11. The composition of any one of claims 1-3, 6-8 and 10, wherein the first peptide tag is a SpyTag.

12. The composition of claim 11, wherein the SpyTag is an amino acid sequence listed in SEQ ID NO:

30.

13. The composition of claim 11, wherein the SpyTag is attached via a linker.

14. The composition of claim 13, wherein the linker is an amino acid sequence listed in SEQ ID NO:

29.

15. The composition of any one of claims 1-3, 6-8, 10 and 12-14, wherein the second peptide tag is SpyCatcher.

16. The composition of claim 15, wherein the SpyCatcher is the amino acid sequence listed in SEQ ID NO:

38.

17. The composition of claim 15, wherein the moiety is attached to the SpyCatcher via a linker.

18. The composition of claim 17, wherein the linker is a flexible linker.

19. The composition of claim 18, wherein the linker is an amino acid sequence listed in SEQ ID NO:

39.

20. The composition of any one of claims 1-3, 6-8, 10, 12-14 and 16-19, wherein the composition is an immunogenic composition or a vaccine composition.

21. A vaccine comprising the composition according to any one of claims 1 to 20, for preventing and / or treating a disease.

22. A method of producing a composition according to any one of claims 1 to 20 or a vaccine according to claim 21, the method comprising: - introducing a first nucleic acid encoding a first genetic fusion of a first protein and a first peptide tag into a first host cell; - incubating the first host cell under conditions where the first genetic fusion is expressed; - introducing a second nucleic acid encoding a second genetic fusion of a second protein and a second peptide tag into a second host cell; - incubating the second host cell under conditions where the second genetic fusion is expressed; - optionally purifying the expressed components; - incubating the expressed component under conditions where an isopeptide bond is formed between the first peptide tag and the second peptide tag; and optionally purifying the resulting composition, wherein the first protein comprises an antigenic component and the second protein comprises a moiety.

23. A kit comprising a composition comprising a first immunogenic composition and optionally one or more boosting compositions comprising a second immunogenic composition, wherein the first immunogenic composition and / or the second immunogenic composition comprises a composition according to any one of claims 1 to claim 20 or a vaccine according to claim 21.

24. A vaccine for preventing and / or treating HCMV infection, comprising the composition according to any one of claims 2 to 6.

25. A vaccine for preventing and / or treating RSV infection, comprising the composition according to any one of claims 7 to claim 10.

26. A pharmaceutical composition comprising the vaccine according to any one of claims 21, 24 or 25, and a pharmaceutically acceptable buffer, excipient, carrier, adjuvant or a combination thereof.

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