Vaccine composition

Abstract

To provide a vaccine composition usable for an antigen of giant / a large number of constituent elements.SOLUTION: Provided is a vaccine composition, most markedly a vaccine composition which has a large antigenic constituent element, for example, exceeding 50kDa or being a multimer, that is, constituted of a subunit. Such an antigenic constituent element is particularly an object of interest because it is currently equivalent to a pathogen-derived antigenic constituent element to which vaccination is impossible. Provided is a composition including particles which display an antigenic constituent element, includes an antigenic constituent element including a first peptide tag and a portion including a second peptide tag, where the antigenic constituent element and the portion are connected through isopeptide bond between the first and second peptide tags, and the antigenic constituent element exceeds 50kDa or multimeric.SELECTED DRAWING: Figure 4

Description

[Background technology]

[0001] Vaccines are a safe and effective way to fight and eradicate infectious diseases. While vaccine development has been very successful, there remains a list of disease challenges for which no vaccine currently exists, including many serious pathogens that constitute formidable immunological disorders. Generally, an effective vaccine is considered to need to be delivered to the lymph nodes and remain there for a sufficient time to produce an immune response.

[0002] Vaccine development has shifted from using attenuated or dead pathogens to using smaller antigenic components of these pathogens, with the aim of generating the necessary protective immune response while avoiding the inherent risks associated with the use of such attenuated strains. Efforts have focused on attempts to express immunogenic sites of pathogen components (e.g., components necessary for a pathogen to infect cells), and these components have been limited to short / small peptides and proteins, simply put, due to the technical challenges of expressing large or numerous component antigens. However, a potential problem associated with using very short or small peptides is the risk of antigenically variable pathogens that evade the immune response induced by vaccination through changes in specific parts of their antigens.

[0003] The expression of large / multiple-component antigens offers immunological advantages, including the ability to generate antibodies against multiple neutralizing epitopes of a single pathogen. However, the expression of one or more large antigens in a way that allows for the maintenance of appropriate antigenic epitopes and the formation of complexes that are presented for the production of an effective antibody response remains extremely difficult. Therefore, there is still a need for improved methods for expressing large antigens and / or multiple-component antigens in a manner that can produce a clinically significant immune response.

[0004] Previous recombinant vaccines designed to elicit an immune response to multiple antigenic components either relied on each component being expressed and packaged separately in separate particles, for example, in the anti-HPV vaccines Cervarix and Gardasil, the recombinant major capsid L1 protein of a specific HPV strain is expressed separately and assembled as virus-like particles (VLPs), and then different types of VLPs are combined into the vaccine formulation. Alternatively, a number of short epitopes are selected and combined into a single recombinant vaccine (e.g., the multimer 001 influenza vaccine), but precisely because of their nature, these epitopes are short, linear epitopes, selected to avoid the manufacturing complexities involved in three-dimensional structure or refolding, and therefore not attempted to represent natural pathogens as presented to the immune system in active infection.

[0005] For example, β-herpes human cytomegalovirus (HCMV, also known as human herpesvirus-5 (HHV-5)) is a leading cause of neonatal developmental disorders. This ubiquitous virus infects over 60% of the general population, and initial infections are usually mild or asymptomatic. After infection, the virus remains latent in the body but can cause serious illness in immunocompromised individuals (i.e., HIV patients, transplant recipients, and those undergoing chemotherapy) or the elderly. HCMV is a leading infectious cause of birth defects in developing countries. Up to 4 out of 200 babies are born with HCMV due to congenital infection, and up to 10% of these suffer long-term consequences. HCMV infection has also been associated with hypertension and atherosclerosis in adults (Cheng et al. (2) (May 2009) Frueh K (ed.), "Cytomegalovirus infection causes an increase of arterial blood pressure". PLoS Pathog. 5(5):e1000427). HCMV is therefore a public health priority. However, despite intensive efforts, success with an HCMV vaccine has not yet been achieved.

[0006] Respiratory syncytial virus (RSV) is another ubiquitous virus that causes little harm to the health of infected healthy adults and older children. However, it is the second leading cause of death in infants under one year of age worldwide, and second only to malaria. The virus is responsible for an estimated 160,000 deaths worldwide annually. The virus causes serious respiratory infections, and complications include pneumonia and bronchiolitis. High-risk groups include infants under one year of age, immunocompromised patients, the elderly, and those with cardiac and pulmonary conditions. Again, despite many years of active research and development, there is currently no approved vaccine for RSV.

[0007] For diseases such as those caused by RSV and HCMV, for which no vaccines are currently available, current approaches to vaccine production have generally not demonstrated the desired efficacy. This indicates a great unmet need to provide alternative types of vaccines to address diseases with such catastrophic outcomes.

[0008] In recent years, several genetically encoded systems have been described to enable spontaneous or assisted amide bond formation. For example, SpyTag is a peptide engineered to form spontaneous and irreversible isopeptide bonds to its protein partner, SpyCatcher, when its two components are mixed. The positions of SpyTag and SpyCatcher components within the protein chain can be designed to be diverse, and they react under a wide range of pH, buffering, and temperature conditions. SpyTag / SpyCatcher pairs, as well as their variants and derivatives, have been used in vaccine development, but to date, they have only been used for simple antigen presentation. Other genetically encoded systems that enable spontaneous amide bond formation include SnoopTag / SnoopTagJr and SnoopCatcher; RrgATag / RrgATag2 / DogTag and RrgACatcher, IsopepTag / IsopepTag-N and Pilin-C or Pilin-N, PsCsTag and PsCsCatcher; as well as SnoopTagJr and DogTag (mediated by SnoopLigase), and variants of all of these systems.

[0009] The inventors have demonstrated that the use of large / multiple component antigens in vaccine compositions is possible using a genetically encoded system for enabling amide bond formation, which can improve the reaction to large / multiple component antigens. [Prior art documents] [Non-patent literature]

[0010] [Non-Patent Document 1] Cheng et al. (May 2009), supervised by Frueh K. "Cytomegalovirus infection causes an increase of arterial blood pressure". PLoS Pathog. 5(5):e1000427 [Overview of the project]

[0011] In a first aspect of the present invention, a composition comprising particles for displaying protein components: i) a protein component containing the first peptide tag, and ii) The portion containing the second peptide tag Includes, Protein components and parts are linked through isopeptide bonds between the first and second peptide tags, and the protein components exceed 50 kDa The above composition is provided.

[0012] In another aspect of the present invention, a composition comprising particles for displaying protein components: i) Protein components containing the first peptide tag, and ii) The portion containing the second peptide tag Includes, Protein components and parts are linked through isopeptide bonds between the first and second peptide tags, and the protein components are polymeric. The above composition is provided.

[0013] Protein components may have any function, for example, they may be enzymes or have enzymatic properties. Protein components may be full-length proteins, or they may be parts of full-length proteins, segments, domains, or truncations. Protein components may be antigens or immunogens. Protein components may also be referred to as antigenic components.

[0014] In another aspect of the present invention, a composition comprising particles that display antigenic components: iii) an antigenic component containing the first peptide tag, and iv) The portion containing the second peptide tag Includes, The antigenic component and the moiety are linked through an isopeptide bond between the first and second peptide tags described above, and the antigenic component exceeds approximately 50 kDa Provide the composition.

[0015] In some embodiments of any aspect of the present invention, the protein component or antigenic component is 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 larger, for example larger than 200 kDa, larger than 300 kDa, or larger than 400 kDa.

[0016] The multimer may contain any number of subunits and may or may not be covalently bonded in the protein or antigenic component. The multimer may contain 2 to 20 subunits, or 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more subunits. Alternatively, the multimer may be a dimer, trimer, tetramer, pentamer, hexamer, heptamer, octamer, nonamer or decamer. The multimer can be formed from any suitable pathogen, preferably a viral multimer.

[0017] Examples of large protein components, i.e., those exceeding 50 kDa or as described above, include the pentameric complex (PC) and gB glycoprotein from human cytomegalovirus (HCMV), G and F glycoproteins from RSV, hemagglutinin (HA) and neuraminidase (NA) antigens from influenza A virus, Plasmodium falciparum Pfs230, Plasmodium falciparum CSP, human HER2 receptor, PCSK9, VAR2CSA, Plasmodium falciparum R IPR, varicella-zoster virus (VZV) glycoprotein E, rabies virus glycoprotein and Epstein-Barr virus (EBV) gH / gL complex are included.

[0018] In some embodiments of any aspect of the present invention, the protein component or antigenic component can also be a monomer or multimer, such as a dimer, trimer, tetramer or pentamer. In some embodiments of any aspect of the present invention, the protein component or antigenic component can also be a protein or peptide complex.

[0019] Non-limiting examples of multimeric antigenic components include pentameric complexes (PC) and gB trimers from human cytomegalovirus (HCMV), G and F glycoproteins from RSV, hemagglutinin (HA) and neuraminidase (NA) antigens from influenza A virus, some of which are described herein. Other examples include components of disease pathogens such as viruses, bacteria, fungal pathogens, parasites or other disease vectors. Suitable multimeric antigenic components include, for example, those derived from viruses such as influenza (e.g., influenza hemagglutinin (HA) (e.g., influenza trimer)), respiratory syncytial virus (RSV).

[0020] It is also possible to attach the protein component to a first peptide tag by gene fusion and express it recombinantly in a suitable cell. For components that include post-translational modifications, such as glycosylation, it may also be preferable to express the recombinant protein in a eukaryotic or mammalian cell line.

[0021] In one embodiment, the “part” is a component on which protein components or antigenic components are displayed, which may also be available to the immune system. In one embodiment, the part multimerizes to form the particle. Suitablely, such part may be a virus, a bacterium, a multimerized scaffold for vaccination, or a protein component that multimerizes to form VLPs (virus-like particles). Suitablely, the part may be a component of a bacteriophage, tobacco mosaic virus particle, adeno-associated virus-like particle (AAVLP), Escherichia coli (E. coli), etc. In one embodiment, the part is itself a component of a virus, bacterium, etc., such that its multimerization (self-assembly) forms a particle for displaying protein components or antigenic components. In one embodiment, the part may be a viral structural protein, such as a viral envelope or capsid protein, or a surface antigen. Examples of structural proteins include hemagglutinin-neuraminidases derived from a variety of viruses, including the matrix M1 protein and viral envelope M2 protein from influenza virus, HBsAg from hepatitis B virus, Escherichia coli bacteriophage AP205 viral coat protein (CP3), and mumps. Suitable viral structural proteins are known to those skilled in the art. In other embodiments, the portion may also be a protein or peptide, e.g., a multimerizing domain, e.g., IMX313 forming nanoparticles, or a computer-derived particle, e.g., MI3. In further embodiments, the portion may also be synthetic nanoparticles or synthetic VLPs, e.g., gold, lipopeptides, or poly(lactic acid-glycolic acid copolymer) (PLGA) nanoparticles. Other suitable portions may also include liposomes or outer membrane vesicles. Preferably, the portion containing the second peptide tag is the portion to which the second peptide tag is attached.

[0022] The use of a virus-derived structural surface antigen may also be preferable. Therefore, a second peptide tag is attached to the structural surface antigen to enable the formation of virus-like particles (VLPs), either by attaching the second peptide tag to the particles or by allowing the particles to display the second peptide tag. VLPs are non-infectious, self-assembling nanoparticles, and their repeatable, molecularly defined construction is attractive for manipulating plurivalentity, particularly for vaccination. LPs have been produced from components of a very diverse viral family, including hepatitis B virus (including hepatitis B small molecule surface antigen (HBsAg)), parvoviridae (e.g., adeno-associated viruses), retroviridae (e.g., HIV), flaviviridae (e.g., hepatitis C virus), and bacteriophages (e.g., Qβ, AP205). Any of these may be suitable for use as part of the present invention.

[0023] A second peptide tag can also be attached to a portion of the sequence via gene fusion. This gene fusion is not limited to the terminal, but can be at any suitable point in the sequence. Those skilled in the art will recognize that the fusion protein can be recombinantly expressed in suitable cells.

[0024] The second peptide tag may also be displayed or attached to a portion by chemical conjugation. This would require, for example, the presence of a reactive amine group so that conjugation is feasible.

[0025] Therefore, in one embodiment, the portion is hepatitis B virus surface antigen (HBsAg). Preferably, HBsAg has the amino acid sequence (or a functional equivalent thereof) shown in Sequence ID No. 41, as described herein.

[0026] In one aspect of any part of the present invention, the protein component or antigenic component is an immunogenic component of the HCMV pentamer. Preferably, the antigenic or immunogenic component is capable of eliciting an immune response, such as an antibody response, when introduced into a subject, e.g., a patient. Thus, the “immunogenic component of the HCMV pentamer” is, for example, a component capable of eliciting an anti-HCMV antibody response in a subject. Preferably, 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 aspects, the immunogenic component comprises one or more of these “pUL” or “UL” components. In other aspects, the immunogenic component comprises one or more of these gH or gL components. In one embodiment, the immunogenic component comprises a combination of one or more "UL" components and one or more components selected from gH or gL components. In another embodiment of the present invention, the immunogenic component of the HCMV pentamer is an HCMV pentamer comprising all of the gH / gL / pUL128 / pUL130 / pUL131 subunits. Preferably, the gH / gL / pUL128 / pUL130 / pUL131 subunits have amino acid sequences or functional equivalents thereof that correspond to those derived from any known HCMV strain (including both laboratory strains and / or clinical isolates), including Towne (GI:239909366), AD169 (GI:219879600), Toledo (GI:290564358), and Merlin (GI:155573956). Functional equivalents refer to amino acid sequences that share a certain degree of homology and differ only in a few amino acids, but retain the functional properties that enable them to form antigenic subunits or pentamers that provide protective antibodies. Suitable variants of the components gH / gL / pUL128 / pUL130 / pUL131A are described, for example, in WO2014 / 005959 (see pages 4-10), which is incorporated herein.Preferably, in a vaccine approach, using HCMV pentameric subunits allows for immunogenic protection against infection from a wide range of HCMV virus strains because there is a high degree of homology between strains at the pentameric amino acid sequence level.

[0027] In some embodiments, the antigenic component may also correspond to a component of a disease pathogen, or a vector or part thereof. The antigenic component may, for example, be easily manufactured. To achieve this, the transmembrane domain may be absent. Preferably, in an HCMV pentamer, for example, the immunogenic component of the HCMV pentamer includes a gH subunit containing a partially excised transmembrane domain (partially excised by deleting one or more amino acids from this region) so that the subunit is secreted into the cell supernatant during protein production in host cells to facilitate purification.

[0028] In one embodiment, the gH / gL / pUL128 / pUL130 / pUL131A subunits have the amino acid sequences (or their functional equivalents) shown in SEQ ID NOs. 28, 31, 35, 33, and 36, respectively (with or without the signal peptides shown). By functional equivalent, we mean amino acid sequences that share some degree of homology and differ only in a few amino acids, but retain functional properties that enable the formation of, for example, an antigenic subunit or pentamer that provides a protective antibody. In some embodiments, the functional equivalents may also share 70%, 80%, 90%, or higher 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 shown in SEQ ID NOs. 13, 16, 20, 18, and 21, or codon-optimized versions thereof (with or without the coding sequence of the signal peptide). In some embodiments, any one of the gH / gL / pUL128 / pUL130 / pUL131A subunits may contain a signal peptide, e.g., a signal peptide present on the strain's native protein, a functional equivalent of the signal peptide, or a signal peptide derived from a different strain of HCMV. In some embodiments, any one of the gH / gL / pUL128 / pUL130 / pUL131A subunits may contain a signal peptide derived from a heterologous protein. The selection of the signal peptide may be determined to target the expressed protein to a specific cellular (or extracellular) location or to confer other functionality. After expression of the subunit(s), the signal peptide may be enzymatically cleaved (e.g., by a signal peptidase) by either the native cellular mechanisms of the expression system used or in vitro. In some embodiments, any one of the gH / gL / pUL128 / pUL130 / pUL131A subunits may be expressed without a signal peptide. In some embodiments, it is also possible to use native sequences containing introns, if these can result in higher expression levels. Appropriately, the native nucleic acid sequence of UL128 contains two introns.In another embodiment, the nucleic acid sequence of UL131A contains one intron. In some embodiments, the intron may be removed. In some embodiments, the native sequence may also be codon-optimized for a suitable expression system.

[0029] In one aspect of the present invention, the protein component or antigenic component is an immunogenic component of the RSV virus, such as an adherent glycoprotein (G protein) or a fusion glycoprotein (F protein), both of which control the early stages of infection. G is a highly glycosylated 90 kDa type II membrane-endogenous protein and is capable of mediating viral attachment to the host cell membrane through either interaction with heparan sulfate on proteoglycans, and is an excellent candidate for a protein component.

[0030] The F protein is a membrane-bound protein composed of three F0 monomers, which are processed during assembly into F1 and F2 subunits and covalently linked by two disulfide bonds. The F protein is highly conserved among RSV isolates derived from both A and B subgroups, and its amino acid sequence exhibits 90% or higher identity. F is a 574-amino acid class I fusion protein consisting of a 50-kilodalton (kDa) carboxy-terminal F1 fragment and a 20-kDa amino-terminal F2 fragment; it constitutes a heterodimer trimer. It is distinguished by two furin cleavage sites, which release a 27-amino acid glycopeptide and expose a hydrophobic fusion peptide at the F1 amino terminus. F1 has two N-linked glycosylation sites, while F2 has only one. After removing the 25-amino acid signal peptide and 27-amino acid glycopeptide between F2 and F1, the remaining external domain of F consists of 472 amino acids. Only 25 amino acids differ between subtypes A and B in the external domain of F.

[0031] To develop antigenic compositions from the RSV-F protein, several studies have focused on creating variants of the trimer pre-fusion protein. These variants have been produced by single-strand gene fusion of the two subunits of the mature pre-F protein. F2, fused to F1, and DS-Cav1 variants containing deletions of both the fusion peptide and the pep27 region have been created. Differences in the linkers between the F2 and F1 subunits appear to affect immunogenicity, and therefore, variants may employ different linker selections. The native RSV-F protein sequence can be found under deposit number P03420.1.

[0032] Several types of pre-fusion F proteins have been studied, developed, and subsequently published. All of these pre-fusion trimers may be suitable for use in the present invention. The DS-Cav1 stabilized fusion glycoprotein is derived from a native protein. EP2222710, incorporated herein, 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 the trimerization domain. A highly stable pre-fusion RSV-F protein is described by Krarup et al., Nat. Commun. 2015; 6: 8143, which is also incorporated herein.

[0033] WO2014 / 160463, incorporated herein, describes isolated recombinant RSV-F proteins stabilized by prefusion conformations, as well as nucleic acid molecules encoding recombinant RSV-F proteins.

[0034] WO2017 / 172890, incorporated herein, describes the substitution-modified pre-fusion RSV-F protein and the nucleic acid encoding the protein. Further explanation is also incorporated herein, 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-Yu Chuang, Aliaksandr Druz, Wing-Pui Kong, Yen-Ting Lai, Emily J. Rundlet, Yaroslav Tsybovsky, Yongping Yang, Ivelin S. Georgiev, Provided to Miklos Guttman, Christopher R. Lees, Marie Pancera, Mallika Sastry, Cinque Soto, Guillaume BE Stewart-Jones, Paul V. Thomas, Joseph G. Van Galen, Ulrich Baxa, Kelly K. Lee, John R. Mascola, Barney S. Graham, and Peter D. Kwong.

[0035] Exemplary nucleic acid sequences encoding recombinant F2-F1 external domain prototypes linked to the T4 fibrintin trimer are available under deposit numbers LP884611.1, LP884610.1, LP884609.1, and LP884608.1.

[0036] In some embodiments, the protein or antigenic component is a component of the disease pathogen. Alternatively, it may correspond to the vector or a part thereof. The antigenic component may lack a transmembrane domain, for example, to facilitate manufacturing. Appropriately, in the RSV-F protein or its pre-fusion conformation, for example, the immunogenic component of the F protein includes an F2-F1 subunit containing a partially excised transmembrane domain (partially excised by deleting one or more amino acids from this region) so that the subunit is secreted into the cell supernatant during protein production in the host cell, for ease of purification. Thus, the RSV-F protein lacks a functional TM domain. Alternatively, a gene fusion containing the first peptide tag may, in fact, prevent the F protein from being in the membrane, regardless of the presence of a functional transmembrane domain.

[0037] In one embodiment, each pre-fusion-stabilized subunit has the amino acid sequence (or its functional equivalent) shown in SEQ ID NOs. 50-58. By functional equivalent, we mean an amino acid sequence that shares some degree of homology and differs by only a few amino acids, but retains the functional properties necessary to form an antigenic subunit that provides, for example, a protective antibody. In some embodiments, the functional equivalent may also share 70%, 80%, 90%, or higher homology with the relevant amino acid sequence. In one embodiment, the pre-fusion-stabilized RSV-F trimer may not contain a hetero-trimerization domain.

[0038] The protein component includes a first peptide tag. This first peptide tag may be attached to the protein component by expressing a recombinant fusion protein. Those skilled in the art know the techniques for gene fusion of peptide sequences to express recombinant proteins in suitable cell lines. With respect to post-translational modifications, such as glycosylation, it is preferable to express the recombinant protein in eukaryotic or mammalian cell lines.

[0039] Preferably, using a first peptide tag and a second peptide tag forming an isopeptide bond, such as the SpyTag-SpyCatcher system described herein, makes it possible to "decorate" the portion displaying the large antigen, e.g., VLP, with large and / or multimeric antigens in the correct organization and orientation so that the antigen is presented to the immune system in a manner that can generate anti-antigen (e.g., anti-HCMV) antibodies that can provide protective / neutralizing / immunogenic effects. The traditional approach of vaccination using soluble antigens (even large antigens, e.g., multimeric / pentamers) may be less effective in producing protective / neutralizing / immunogenic effects. Preferably, the display of antigens (e.g., multimeric antigens) on particles, e.g., VLPs or nanoparticles, results in the presentation of a geometrically repeating array of the same antigen that can robustly induce an immune response, in contrast to soluble antigens. Larger VLPs or other suitable particles compared to "free" antigens may also have a higher immunogenic effect. Furthermore, the orientation of the display of a multimeric antigen, such as an HCMV pentamer, can be important for immunogenicity. The use of pair-forming tags, such as the SpyTag-SpyCatcher system described herein, for attachment to the multimeric antigen on the particle allows the antigen to attach to the particle in a specific preferred orientation. For example, in the case of HCMV, the gH / gL subunit may be less likely to have a neutralizing epitope than the "UL" subunit. Therefore, preferably, the present invention allows the orientation of the display of the HCMV pentamer on the particle to be determined by the appropriate placement of the first peptide tag, such that, for example, the "UL" subunit is displayed facing outward on the particle and thus more readily available to the individual's immune system. Alternatively / furthermore, the placement of the first peptide tag on the antigen may also be determined to produce an orientation of the antigen similar to that on a natural virus, thereby presenting the immune system with a particle that displays the antigen in an orientation more likely to induce an immune response against an invading live virus.

[0040] In contrast, traditional approaches to presenting proteins on VLPs may involve chemical linking, which means these chemical reactions may be more random, however However, this approach has the disadvantage that the correct (e.g., immunologically favorable) orientation of the antigen may not be reliably obtained, and may only account for a small percentage of the resulting ligation reactions. Furthermore, the processes involved in chemical conjugate may reduce the likelihood that the 3D structure necessary for proper antigen presentation can be maintained. Some of the drawbacks of the traditional approach 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.

[0041] Similarly, gene fusion of antigens to viral coat proteins has proven difficult and time-consuming due to problems associated with misfolding and difficulties in determining optimal expression conditions for both components. Furthermore, gene fusion is likely unsuitable for the expression of large antigens or multi-component antigens because achieving effective expression in the correct conformation is extremely difficult.

[0042] The location of the first peptide tag must be carefully designed so that the innate protein conformation is maintained and, optionally, any post-translational modifications are preserved, in order to present the protein or antigenic component in an immunogenic manner. For some antigens, the preservation of glycosylation does not affect the way the epitope is presented, but for others, either its preservation or removal improves efficacy. For protein components that are transmembrane proteins, the transmembrane portion of the protein component provides an excellent target for placing the first peptide tag, as this portion does not participate in the conformation of the protein component for which this sequence is antigenic and serves a role that is no longer required, for example, in vaccines. If the protein component does not contain a transmembrane protein, it may be beneficial to fuse the first peptide tag to the C or N terminus of the component or its subunits (in the case of a multimer), although the first peptide tag can also be contained within any part of the sequence. Alternatively, the first peptide tag may be placed within a loop on the protein or antigenic component.

[0043] To present immunogenic components, such as the immunogenic component of the HCMV pentamer, the position of the first peptide tag must be carefully designed so as to maintain the native protein conformation. With respect to HCMV, in one embodiment, attachment is via the gH subunit, preferably via the C-terminus of the gH subunit, or via 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 toward the outside of the particle, as discussed above. In this specification, the target region is a portion of a protein known to produce antibodies with a neutralizing effect, and may also be referred to as the immunogenic portion.

[0044] To present immunogenic components, such as the RSV-F pre-fusion F protein, the position of the first peptide tag must be carefully designed so as to maintain the native protein conformation. With respect to the RSV-F pre-fusion protein, in one embodiment, the attachment is appropriately through the C-terminus of the F pre-fusion and through the 3' end of the nucleic acid encoding the fusion. In addition to maintaining the conformation of the pre-fusion F protein (or its components), this rational design also presents the most neutralizable epitope of the pre-fusion F protein toward the outside of the particle, as discussed above. The same considerations would apply to any other variation of the F protein. The inclusion of the first peptide tag at the C-terminus works well, keeping the immunogenic protein components correctly folded. This has been proven by enlightened scholars.

[0045] In one embodiment, the first and second peptide tags are parts of a peptide tag / binding partner pair capable of forming an isopeptide bond. This isopeptide bond may be spontaneous, i.e., without assistance, or it may require assistance, i.e., a ligase or other helper. Preferably, the first and second peptide tags are SpyTag / SpyCatcher pairs. Preferably, 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, or modifications of all these systems.

[0046] Appropriately, the first peptide tag is a peptide tag derived from a peptide tag / binding partner pair, e.g., SpyTag, and the second peptide tag is a binding partner, e.g., SpyCatcher. In another embodiment, the first peptide tag is a binding partner, e.g., SpyCatcher, and the second peptide tag is a peptide tag component derived from a peptide tag / binding partner pair, e.g., SpyTag.

[0047] Appropriately, the first peptide tag is a peptide tag derived from a peptide tag / binding partner pair, e.g., SnoopTag, and the second peptide tag is the binding partner, e.g., SnoopCatcher. In another embodiment, the first peptide tag is the binding partner, e.g., SnoopCatcher, and the second peptide tag is a peptide tag component derived from a peptide tag / binding partner pair, e.g., SnoopTag. Thus, it can be seen that the first peptide tag may be either a “tag” or a “catcher”; and the second peptide tag may be the partner of this pair, either a “catcher” or a “tag.” Appropriate 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.

[0048] In one embodiment, a protein or antigenic component is attached as a first peptide tag to any one of SpyTag, SnoopTag, RrgATag, RrgATag2, DogTag, IsopepTag, IsopepTag-N, PsCsTag, and SnoopTagJr.

[0049] The first peptide tag may also be attached via a linker that may be rigid or flexible, if necessary. Those skilled in the art will recognize which linker is appropriate.

[0050] In another embodiment, the portion attaches to one of the following as a second peptide tag: SpyCatcher, SnoopCatcher, RrgACatcher, Pilin-C, Pilin-N, PsCsCatcher, and DogTag (mediated by SnoopLigase).

[0051] The part can be any suitable part, including a synthetic polymerization platform, as previously discussed. It is also possible to attach the second peptide tag to any suitable position within the portion, without affecting its ability to fold and form the appropriate conformation. Gene fusion may be preferable. It may also be preferable to include a second peptide tag at the C or N terminus of the portion, although the second peptide tag may also be included in any part of the sequence. Alternatively, the second peptide tag may be placed within the loop of the portion. For example, gene fusion of SpyCatcher to the N terminus of the viral coat protein (CP3) of RNA bacteriophage AP205 is described in Brune et al., Scientific Reports volume 6, paper number: 19234 (2016). An alternative fusion using a self-assembling synthetic protein as a multimerization platform is discussed in Brune et al., ACS Nano, 2018, 12(9), pp 8855-8866. Alternatively, the second peptide tag may be attached via chemical conjugate.

[0052] The second peptide tag may also be attached via a linker that may be rigid or flexible, if necessary. Those skilled in the art will recognize which linker is appropriate.

[0053] In one embodiment, an antigenic component, such as an HCMV pentamer or its immunogenic component, is attached to the SpyTag. A suitable SpyTag has the amino acid sequence shown in SEQ ID NO: 30.

[0054] SpayTag can also be attached via a linker. Suitable linkers include those having the amino acid sequence shown in SEQ ID NO: 29. In another embodiment, the portion attaches to a SpyCatcher binding partner (second peptide tag). The portion may also be HBsAg, appropriately. A suitable SpyCatcher has the amino acid sequence shown in SEQ ID NO: 38. In one embodiment, the SpyCatcher attaches via a linker. The linker may be a rigid linker or a flexible linker, and may have the amino acid sequence shown in SEQ ID NO: 39.

[0055] In another embodiment, the protein composition or antigenic composition further comprises a protein containing another, preferably different, first peptide tag, according to any aspect or aspect of the present invention.

[0056] In another embodiment, a composition according to any aspect or aspect of the present invention further comprises another, preferably different, antigen containing a first peptide tag, for example, another HCMV antigen. Preferably, the other HCMV antigen is glycoprotein B. Preferably, the glycoprotein B sequence is described, for example, in WO2014 / 005959; see SEQ ID NOs. 21, 22, 23, or 36. In one embodiment, the composition comprises particles (e.g., VLPs) displaying both an HCMV pentamer and the other HCMV antigen.

[0057] In one embodiment, the composition is an immunogenic composition or a vaccine composition. Preferably, the immunogenic or vaccine composition is capable of inducing an immune response, such as an antibody response, upon administration to an individual. Appropriately, the immune response may also be a protective immune response. A suitable immunogenic composition may further include further components, including adjuvants, immunostimulants, and / or pharmaceutically acceptable excipients.

[0058] Suitable adjuvants can be based on, for example, aluminum, peptides, squalene, liposomes, oil-in-water emulsions, and saponins, and may include Alhydrogel®, MF59, AS01, Matrix M, Muramyl dipeptide, and Quil A. Water-in-oil adjuvants are also suitable, such as squalene-oil-in-water emulsions, e.g., Addavax. TM That is appropriate.

[0059] Accordingly, in another aspect or embodiment of the present invention, an immunogenic or vaccine composition comprising a composition according to the present invention is provided. Preferably, the vaccine composition comprises a vaccine dose which is a certain amount of the composition according to the present invention that provides immunogenicity, preferably an immune protective effect against infectious pathogens / vectors, e.g., a neutralizing effect from HCMV infection. Preferably, the vaccine composition comprises a vaccine dose which is a certain amount of the composition according to the present invention that provides a neutralizing effect against infectious pathogens / vectors, e.g., a neutralizing effect from RSV infection. Antibodies generated against the immunogenic composition, preferably neutralizing antibodies, can also be detected and measured by methods well known to those skilled in the art, including, for example, a standardized ELISA assay or a microneutralization assay as described herein.

[0060] According to another aspect: i) The portion containing the first peptide tag ii) Proteins containing a second peptide tag Includes, The first peptide tag and the second peptide tag form an isopeptide bond. A VLP is provided. In some embodiments, the portion is HBsAg. However, as previously stated, it is also possible to use any suitable portion.

[0061] Appropriately, the first peptide tag is a peptide tag derived from a peptide tag / binding partner pair, e.g., SpyTag, and the second peptide tag is a binding partner, e.g., SpyCatcher. In another embodiment, the first peptide tag is a binding partner, e.g., SpyCatcher, and the second peptide tag is a peptide tag derived from a peptide tag / binding partner pair, e.g., SpyTag. Other suitable peptide tag / binding partner pairs are described herein and will be known to those skilled in the art. Appropriately, 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, or modifications of all these systems.

[0062] Appropriately, 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 also be a protein or peptide complex of 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 greater, for example, greater than 200 kDa, greater than 300 kDa, or greater than 400 kDa.

[0063] 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. The multimeric antigen may appropriately be an HCMV pentamer as described herein. The protein may appropriately be an RSV-F protein or a derivative thereof (e.g., a pre-fusion F protein). In one embodiment, the protein containing the second peptide tag is an immunogenic component of an HCMV pentamer. An HCMV pentamer (gH / gL / pUL128 / pUL130 / pUL131) as described herein, and containing appropriate linkers and tags, has a molecular weight greater than 160 kDa. It has. Other suitable large or multimeric proteins or antigens include antigens derived from other infectious pathogens, such as viruses including influenza virus and RSV.

[0064] Preferably, in this method, using HBsAg as a carrier (VLP) may also produce an anti-HepB boost, also referred to as an anti-Hepatitis B virus (HBV) reaction.

[0065] In another aspect: i) Protein containing the first peptide tag ii) The portion containing the second peptide tag Includes, The first peptide tag and the second peptide tag form an isopeptide bond. A VLP is provided. In some embodiments, the portion is HBsAg. However, as previously stated, it is also possible to use any suitable portion.

[0066] Appropriately, the first peptide tag is a peptide tag derived from a peptide tag / binding partner pair, e.g., SpyTag, and the second peptide tag is a binding partner, e.g., SpyCatcher. In another embodiment, the first peptide tag is a binding partner, e.g., SpyCatcher, and the second peptide tag is a peptide tag component derived from a peptide tag / binding partner pair, e.g., SpyTag. Other suitable peptide tag / binding partner pairs are described herein and will be known to those skilled in the art. Appropriately, 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, or modifications of all these systems.

[0067] Suitablely, the protein containing the first peptide tag is a protein or peptide complex greater than 50 kDa. The protein containing the first peptide tag may also be a protein or peptide complex of 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 greater than 400 kDa. 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. Suitablely, the multimeric antigen may be an HCMV pentamer as described herein. Suitablely, the protein may also be an RSV-F protein or a derivative thereof (e.g., a pre-fusion F protein). In one embodiment, the protein containing the first peptide tag is an immunogenic component of an HCMV pentamer, as described herein, and an HCMV pentamer (gH / gL / pUL128 / pUL130 / pUL131A) containing a suitable linker and tag having a molecular weight greater than 160 kDa. Other suitable large or multimeric proteins or antigens include antigens derived from other infectious pathogens, including viruses such as influenza virus and RSV.

[0068] Preferably, in this method, using HBsAg as a carrier (VLP) may also result in an anti-HBV boost. In another aspect of the present invention, H linked to SpyTag as described herein We provide CMV pentamers.

[0069] In accordance with another aspect of the present invention, a method for producing a composition or VLP according to the present invention: -Introduce a first nucleic acid encoding a first gene fusion of a first protein to a first peptide tag into a first host cell; -The first host cells are incubated under conditions for expressing the first gene fusion; optionally, the expressed components are purified; -Introduce a second nucleic acid encoding a second gene fusion of a second protein to a second peptide tag into a second host cell; - The second host cells are incubated under conditions for expressing the second gene fusion; optionally, the expressed components are purified; - Incubate the expressed components under conditions for the formation of an isopeptide bond between the first peptide tag and the second peptide tag; optionally, purify the resulting composition. The present invention provides the method, including the steps involved.

[0070] Ideally, the expressed components are incubated together to form isopeptide bonds. Isopeptide bond formation may also require co-incubation with a ligase or similar substance.

[0071] Suitablely, a method for producing a composition or VLP according to the present invention may also be for producing a composition comprising antigenic components to be displayed on a VLP. In some embodiments, when the “immunogenic components of the HCMV pentamer” include the entire HCMV pentamer, the recombinant production of the HCMV pentamer components requires that each subunit be expressed in the correct stoichiometric proportions so that the pentamer is formed and correctly folded for assembly. In these embodiments, it is necessary to exclude complexes consisting of only the required pentameric parts (e.g., gH / gL dimer and tetramer, or a tetramer lacking any one of the five subunits) from the final product. Preferably, the present invention overcomes the problems that would otherwise be associated by expressing all vaccine components (i.e., HBsAg and the five subunits of the HCMV pentamer) in a single system by providing a simple approach of separately fabricating the components and then conjugating them. Thus, in one embodiment, the purified tag is incorporated onto UL130 (Hofmann et al., DOI 10.1002 / bit 25670). Similar principles would be applicable to other immunogenic components.

[0072] In some embodiments, if the “immunogenic components of the RSV-F protein” include the entire F protein or its derivatives, the recombinant production of the components of the F protein or its derivatives requires that they be correctly folded for assembly together with the derivatives containing the pre-fusion F protein trimer.

[0073] Preferably, the method is for producing a composition comprising an HCMV pentamer displayed on an HBsAg VLP. Preferably, the method is for producing a composition comprising an RSV-F prefusion F protein trimer displayed on an HBsAg VLP.

[0074] In another aspect of the present invention, a vaccine is provided for use in the prevention and / or treatment of a disease. Preferably, the vaccine comprises a composition or VLP according to any aspect or embodiment of the present invention. In one embodiment, the disease is HCMV infection. In another aspect, a method for the prevention of HCMV is provided. Preferably, the vaccine is for use in humans. Preferably, the vaccine is for use in adults, e.g., women of reproductive age or pregnant women. For use in the present invention. In another aspect, the present invention provides a method for inducing an immunogenic response to HCMV, such as a protective immune response, in an individual, comprising the step of administering a composition according to any aspect or embodiment of the present invention.

[0075] In another aspect of the present invention, a composition is provided that conforms to any aspect of the present invention for use as a pharmaceutical agent. In a further aspect of the present invention, a composition according to any aspect of the present invention is provided for use as a vaccine, preferably a vaccine for use in the prevention and / or treatment of HCMV infection. According to the present invention, a composition for use as a drug or vaccine may also be administered to adults, for example, women of reproductive age or pregnant women.

[0076] In another aspect, the present invention provides nucleic acid molecules for use in a method according to the present invention. In one embodiment, the nucleic acid molecule according to the present invention comprises a nucleic acid sequence encoding an amino acid sequence such as that shown in any of SEQ ID NOs: 27-41. In one embodiment, the nucleic acid molecule according to the present invention comprises a nucleic acid sequence such as that shown in any of SEQ ID NOs: 12-26 or 42-46.

[0077] In another aspect, the present invention provides a plurality of nucleic acid molecules, including nucleic acid molecules encoding amino acid sequences shown in SEQ ID NOs: 27-41. In one embodiment, the nucleic acid molecules of the present invention include those having sequences such as those shown in SEQ ID NOs: 12-26 or 42-46.

[0078] In another aspect, the present invention provides nucleic acid molecules for use in a method according to the present invention. In one embodiment, the nucleic acid molecule according to the present invention comprises a nucleic acid sequence encoding an amino acid sequence such as that shown in any of SEQ ID NOs. 50 to 58. In one embodiment, the nucleic acid molecule according to the present invention comprises a nucleic acid sequence such as that shown in any of SEQ ID NOs. 47 to 55.

[0079] In another aspect, the present invention provides a plurality of nucleic acid molecules, including nucleic acid molecules encoding amino acid sequences such as those shown in SEQ ID NOs: 50 to 58. In one embodiment, the nucleic acid molecules of the present invention include those having sequences such as those shown in any of SEQ ID NOs: 47 to 55.

[0080] In another aspect, the present invention provides a vector comprising one or more nucleic acid molecules according to the present invention. Preferably, the vector is an expression vector for expressing the amino acid sequence of any component of a composition according to the present invention.

[0081] In another aspect, the present invention provides host cells for expressing components of compositions according to the present invention. Suitable host cells may also be for transient or stable expression of these components. Methods and host cells for expressing CMV proteins are described, for example, in WO2014 / 005959 and WO2016 / 067239, both of which are incorporated herein by reference. In some embodiments, the components may also be glycosylated.

[0082] In another aspect of the present invention, a kit comprising a composition according to the present invention for use in a prime-boost vaccination regimen is provided. Preferably, the kit may also comprise a prime composition comprising a first immunogenic composition according to the present invention, and a boost composition comprising a second immunogenic composition according to the present invention. Alternatively, a kit may be provided that provides a single-dose or multi-dose vaccination regimen, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 doses. Thus, in another aspect, Therefore, the present invention provides a medication measure comprising a dose applied at approximately three-week intervals. [Brief explanation of the drawing]

[0083] [Figure 1] SDS-PAGE and Western blot analysis of purified pentameric SpyTag under non-reducing and reducing conditions. Lane 1: ColorPlus pre-stained broad protein ladder showing size in kDa; Lane 2: Non-reducing sample; Lane 3: Reducing sample. A) SDS-PAGE and Coomassi staining analysis, showing the location of HCMV pentameric components on the left of the gel for non-reducing and on the right for reduced. B) Western blot analysis using anti-HCMV pentameric antibody. [Figure 2] SDS-PAGE and Western blot analysis of purified SpyCatcher-HBsAg under non-reducing (NR) and reducing (R) conditions. A) SDS-PAGE and Coomassi staining analysis. B) Western blot analysis using anti-HBsAg monoclonal antibody. [Figure 3] HPLC analysis using an s200increase3.2 / 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 as a single major peak at the void volume of the column. [Figure 4] SDS-PAGE and Western blot analysis of conjugated pentamer-SpyTag and SpyCatcher-HBsAg under reducing conditions. 1: ColorPlus pre-stained broad protein ladder with size shown in kDa; 2: Conjugated; 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. [Figure 5]HPLC analysis was performed using an S200increase3.2 / 300 column. 30 μl of conjugated pentamer-SpyTag-SpyCatcher-HBsAg was loaded, and this eluted as a single major peak at the void volume of the column. [Figure 6] Immunogenicity of Addavax-adjuvanted HCMV pentamer-HBsAg vaccine versus pentamer protein vaccine after single immunization. BALB / c mice were immunized with 1 μg or 0.1 μg of HCMV pentamer-SpyTag, either as a soluble protein or as a pentamer-HBsAg VLP. Titer was measured from mouse serum by standardized ELISA. The line represents the mean, and the error bars represent the standard deviation (n=10). Mice immunized with HCMV pentamer-HBsAg VLP showed a substantially stronger serum IgG antibody response compared to mice immunized with HCMV pentamer protein alone, even at a 10x lower VLP dose for equivalent pentamer content. [Figure 7] Neutralizing activity in serum of mice immunized with HCMV pentamer-HBsAg vaccine compared to pentamer protein vaccines. Vaccines were adjuvanted with Addavax, and responses are shown after one (prime) or two (boost) immunizations. NT50 was measured on ARPE-19 cells infected with the AD169wt131 strain (displaying a functional pentamer). Neutralizing titers for Cytogam and a commercially available neutralizing anti-gH mAb (HCMV16(51C1) from Bio-Rad Antibodies) in the same assay are shown. [Figure 8]Immunogenicity of unadjuvanted HCMV pentamer-HBsAg vaccine versus pentamer protein vaccine after one or two immunizations. BALB / c mice were immunized with HCMV pentamer-SpyTag (pentamer-HBsAg) conjugated to 1 μg or 0.1 μg of SpyCatcher-HBsAg, or with 1 μg of pentamer-SpyTag protein. Titer was measured by standardized ELISA using mouse serum. The line represents the mean, and the error bars represent the standard deviation (n=10). Mice immunized with HCMV pentamer-HBsAg VLP showed a substantially stronger serum IgG antibody response compared to mice immunized with HCMV pentamer protein alone, even at a 10x lower VLP dose equivalent to that of the pentamer. [Figure 9] Neutralizing activity in serum of mice immunized with HCMV pentamer-HBsAg vaccine compared to pentamer protein vaccine. The vaccine was not adjuvanted and responses are shown after one (prime-post) or two (boost-post) immunizations. NT50 was measured on ARPE-19 cells infected with the AD169wt131 strain (displaying a functional pentamer). Neutralizing titers for Cytogam and a commercially available neutralizing anti-gH mAb (HCMV16(51C1) from Bio-Rad Antibodies) in the same assay are shown. [Figure 10] SDS-PAGE and Western blot analysis of purified RSV-F-SpyTag under non-reducing and reducing conditions. A) SDS-PAGE and Coomassi staining analysis, lane 1: ColorPlus pre-stained broad protein ladder; lane 2: non-reducing sample; lane 3: reduced sample. B) Western blot analysis using anti-RSV-F monoclonal antibody, lane 1: ColorPlus pre-stained broad protein ladder; lane 2: non-reducing sample; lane 3: reduced sample. [Figure 11]SDS-PAGE and Western blot analysis of SpyCatcher-HBsAg and conjugated RSV-F-SpyTag under reducing conditions. 1: ColorPlus pre-stained broad protein ladder; 2: RSV-F-SpyTag-SpyCatcher-HBsAg conjugate; 3: RSV-F-SpyTag; 4: SpyCatcher-HBsAg. A) SDS-PAGE and Coomassi staining analysis. B) Western blot with anti-HBsAg monoclonal antibody. C) Western blot with anti-RSV-F monoclonal antibody. [Figure 12] Immunogenicity of conjugated RSV-F-SpyTag--SpyCatcher-HBsAg ("Sc9-10-HBsAg") versus unconjugated RSV-F-SpyTag ("Sc9-10"). BALB / c mice were immunized with either 1 μg of SpyCatcher-HBsAg conjugated RSV-F-SpyTag (RSV-F VLP) or 1 μg of RSV-F-SpyTag protein, either unadjuvanted or adjuvanted with Addavax™. [Modes for carrying out the invention]

[0084] Virus-like particles Traditionally, vaccine approaches have used attenuated or dead whole pathogens, but this has been replaced by the use of recombinant subunits containing appropriate pathogen-derived proteins. More recently, approaches using virus-like particles (VLPs) have been developed. VLPs are particles that resemble viruses in size (approximately 20-200 nm), shape, and repeating protein arrangement, but lack any pathogen-derived genetic material. Due to their size, VLPs are easily excreted by lymph nodes and are ideal for uptake and presentation by antigen-presenting cells. Furthermore, their repeating structure facilitates complement fixation and B cell receptor crosslinking (Kushnir et al. Vaccine 2012; Vol 31(1):58-83). However, these mechanisms of action are not limited to theory.

[0085] HCMV Human cytomegalovirus (HCMV, also known as human herpesvirus-5 (HHV-5)) is a virus that most adults have been exposed to at some point, and the initial infection is usually only 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 congenital abnormalities in developing countries. Up to 4 out of 200 babies are born with HCMV due to congenital infection, and up to 10% of these suffer long-term consequences. HCMV infection has been associated with hypertension and atherosclerosis in adults (Cheng et al. (May 2009), supervised by Frueh K, "Cytomegalovirus infection causes an increase of arterial blood pressure"). PLoS Pathog. 5(5):e1000427).

[0086] The HCMV pentamer complex, containing the viral proteins gH / gL / pUL128 / pUL130 / pUL131A, has been identified as a potentially useful vaccine target for HCMV based on the observation that antibodies against this complex can neutralize viral entry into epithelial cells and reduce the risk of perinatal HCMV transmission. However, despite intensive efforts, a successful HCMV vaccine has not yet been developed.

[0087] HCMV pentamer HCMV strains, including clinical isolates and laboratory strains, have different genomic sequences. HCMV strains include Merlin (GI:155573956), Towne (GI239909366), AD169 (GI:219879600), Toledo (GI290564358), and TB40 / E. HCMV contains numerous membrane proteins and protein complexes. The pentameric protein gH / gL / pUL128 / pUL130 / pUL131A is important for HCMV infection of epithelial and endothelial cells and is thought to be via the endocytosis pathway. Other combinations of components of this complex have been shown to be important for infection of fibroblasts, for example. The "pUL" subunit / component is also referred to as "UL"; "pUL131" is also referred to as "pUL131A" and "pUL131a," or "UL131A."

[0088] A diverse range of HCMV strains have been deposited in ATCC and can be found as Merlin (VR-1590), Towne (VR-977), and AD169 (VR-538). Genome sequences can be referenced through deposit numbers: Merlin (AY446894.2), Towne (GO121041.1), AD169 (FJ527563.1), Toledo (GU37742.2), and TB40 / E (KF297339.1).

[0089] RSV Respiratory syncytial virus (RSV) is the leading cause of serious respiratory illness in children worldwide. An estimated 3.4 million children under the age of five are hospitalized annually with severe RSV lower respiratory tract infections, with the highest incidence occurring in infants under six months of age. The majority of deaths occur in infants under one year of age and in developing countries. Currently, options for prevention and control are limited.

[0090] RSV-F pre-fusion trimer The F glycoprotein is a type I virus fusion protein. The RSV F precursor (F0) is thought to be cleaved at two sites by a furin-like protease, producing three fragments. The shorter N-terminal fragment (F2) covalently attaches to the larger C-terminal fragment (F1) via two disulfide bonds. The 27-amino acid intervening fragment dissociates after cleavage and is not found in the mature protein.

[0091] As discussed earlier, many stabilized pre-fusion F trimers are available. In the examples presented herein, exemplary sequences encoding these pre-fusion trimers are found in SEQ ID NOs: 48, 48, 54, and 55. Sequences containing fusion with SpyTag are included as SEQ ID NOs: 47 and 53. The amino acid sequences for the pre-fusion trimers are shown as SEQ ID NOs: 51, 52, 57, and 58, and those containing SpyTag are SEQ ID NOs: 40 and 56. Other exemplary sequences are mentioned herein.

[0092] Protein tag / binding partner pair Proteins capable of spontaneous isopeptide bond formation (so-called "isopeptide proteins") Proteins capable of spontaneous isopeptide bond formation can also be expressed as separate fragments providing a peptide tag and a polypeptide-binding partner to the peptide tag, where the two fragments are covalently bonded to each other and provide irreversible interactions (i.e., two-part linkers) (see, for example, WO2018 / 189517 and WO2018 / 197854, both of which are incorporated herein, along with WO2011 / 098772 and WO2016 / 193746). In this regard, proteins capable of spontaneous isopeptide bond formation can also be expressed as separate fragments providing a peptide tag and a polypeptide-binding partner to the peptide tag, where the two fragments are covalently reconstituted by isopeptide bond formation, thereby linking the peptide tag and the molecule or component fused to its polypeptide-binding partner. The isopeptide bond formed by the peptide tag and its polypeptide binding partner is stable under conditions where non-covalent interactions would rapidly dissociate, for example, over long periods (e.g., several weeks), at high temperatures (up to at least 95°C), high pressure, or under harsh chemical treatments (e.g., pH 2–11, organic solvents, surfactants, or denaturants).

[0093] An isopeptide bond is an amide bond formed between a carboxyl / carboxamide and an amino group, where at least one of the carboxyl or amino group is outside the protein backbone (protein skeleton). These bonds are chemically irreversible under typical biological conditions, and they are resistant to most proteases. Because isopeptide bonds are inherently covalent, they give rise to some of the strongest protein-protein interactions.

[0094] In short, a two-part linker, i.e., a peptide tag and its polypeptide binding partner (a so-called peptide tag / binding partner pair), can also be derived from a protein (isopeptide protein) capable of spontaneously forming isopeptide bonds, where a protein domain is separately expressed to produce a peptide "tag" containing one of the residues involved in isopeptide bonding (e.g., aspartic acid or asparagine, or lysine), and a peptide or polypeptide binding partner (or "catcher") containing the other residue involved in isopeptide bonding (e.g., lysine, or aspartic acid or asparagine) and at least one other residue necessary for forming 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 separately incorporating the peptide tag and binding partner into different molecules or components, such as a protein, it is possible to covalently bond the said molecules or components through the isopeptide bond formed between the peptide tag and the binding partner, i.e., to form a linker between the molecule or component incorporating the peptide tag and binding partner.

[0095] Spontaneous formation of isopeptide bonds can occur in isolation, and it may not require the addition of any other entities. For some peptide tag and tag partner pairs, the presence of a helper entity, such as a ligase, may be necessary to generate the isopeptide bond.

[0096] The peptide tag / binding partner pair (2-part linker) called SpyTag / SpyCatcher is used with the FbaB protein of Streptococcus pyogenes (Zakeri et al., 2012, Proc Natl Academia). Derived from the CnaB2 domain (Sci USA 109, E690-697), it has been 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, 3 0).

[0097] Appropriately, the first and second peptide tags form a peptide tag / binding pair referred to as SpyTag / SpyCatcher. Appropriately, the SpyCatcher component is a delta-N1 (ΔN1) SpyCatcher, which has 23 amino acid partial excisions at its N-terminus compared to "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)) (SEQ ID NO: 38).

[0098] In other embodiments, the first and second peptide tags form peptide tag / binding pairs that are mutants of SpyTag / SpyCatcher exhibiting increased reaction rates for isopeptide bond formation, such as those described in concurrently pending application GB1706430.4. In some embodiments, these mutants may be useful for the attachment of large proteins (e.g., >50 kDa or >100 kDa, or >160 kDa HCMV pentameric proteins as described herein) and / or when slow reactions or steric hindrance may be problematic.

[0099] In another embodiment, the isopeptide protein may include, for example, SnoopTag / SnoopCatcher as described in WO 2016 / 193746.

[0100] In some embodiments, one or both isopeptide proteins can retain the reactivity of the isopeptide bond while having partial N-terminal excision.

[0101] Exemplary first and second peptide tag pairs (peptide tag / binding partner pairs; reactive pairs) are described, for example, in WO2011 / 098722, WO2016 / 193746, GB1706430.4, GB1705750.6 or Li, L. et al., J. Mol. Biol. 426, 309-317 (2014), and are listed in the table below.

[0102] [Table 1]

[0103] Mutants, derivatives, and modifications of binding pairs may be prepared by any suitable means. Mutants, derivatives, and functionally effective modifications may also involve amino acid additions, substitutions, alterations, or deletions that retain the same function with respect to the ability to form isopeptide bonds with suitable binding partners.

[0104] For some binding pairs, mediation by a third entity, such as an enzyme, is necessary. For example, SnoopLigase facilitates the binding of SnoopTagJr and DogTag. It can be used to mediate pair formation. Therefore, pair formation may require the assistance of enzymes such as ligases.

[0105] HBsAg "HBsAg" refers to the surface antigen (HBsAg) or a portion thereof derived from the hepatitis B virus. In one embodiment, HBsAg can also refer to the HBsAg sequence, such as the one shown in Sequence ID No. 41, which includes the N-terminus of HBsAg, for example, the 226 amino acids of the S protein of the hepatitis B virus (adw serotype). Appropriately, HBsAg can refer to Valenzuela et al., (1979) 'Nucleotide sequence of the gene coding for the major protein of hepatitis B virus surface antigen'. It contains a four-amino acid sequence, Pro Val Thr Asn, which corresponds to the four carboxyl terminal residues of the hepatitis B virus (adw serotype) pre-S2 protein, as described in Nature 280:815-819. VLPs formed from HBsAg have been approved for clinical use against hepatitis B, 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) (Kushnir et al. Vaccine 2012; Vol 31(1):58-83). HBsAg has also completed Phase III clinical trials and is currently used as the basis for RTS,S, 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).

[0106] Linker details The distance between proteins (e.g., VLPs and decorative antigens) may affect the availability of antigenic epitopes within the protein, the stability of one or more proteins, and also the accessibility of either isopeptide binding partner (e.g., SpyTag / SpyCatcher), thus affecting the conjugation efficiency. Therefore, it is possible to select a linker with appropriate properties to optimize availability, stability, and / or accessibility. Linkers can be broadly subdivided into flexible and rigid subtypes.

[0107] Flexibility Linker If the domains to be linked require movement, flexible linkers may be used. These typically consist of small, nonpolar (e.g., Gly) or polar (e.g., Ser, Thr) amino acids, where 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 also aid in maintaining stability in solution, and length adjustment can affect the proper folding of the protein (Chen et al., 2013). Any suitable flexible linker with properties and length appropriate to the relevant entity may be used. Ideally, flexible linkers may include combinations of 2 to 70 amino acids of this type.

[0108] example:

[0109] [Table 2]

[0110] Rigid linker In some cases, rigid linkers may be preferable because they can assist in providing protein separation. Rigid linkers have a secondary structure. One of the most common rigid linkers employs an α-helical structure (EAAAK). n(wherein n is the number of iterations) (Arai et al., (2001) Protein Eng. Aug;14(8):529-32). Other rigid linkers include (XP) n In the formula, X is any amino acid, but preferably Ala(A), Lys(K), or Glu(E), which can also be included, with proline providing conformational constraints (Chen et al., 2013).

[0111] 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 having properties and length appropriate to the relevant entity may be used. Appropriately, the rigid linker may include combinations of 2 to 70 amino acids of this type.

[0112] example:

[0113] [Table 3]

[0114] Host cells and expression vectors Host cells for the expression of nucleic acids that produce proteins and compositions according to the present invention will be known to those skilled in the art.

[0115] In one embodiment, the host cell would be suitable for transient expression. In another embodiment, The host cell would be a cell capable of forming a stable cell line. Appropriately, the coding sequences encoding antigenic components, such as the HCMV pentamer and RSV-F protein, including those containing isopeptide-forming peptide tags, would be incorporated into a single host cell. In one embodiment, each nucleic acid sequence encoding a multimeric subunit, such as a pentamer, would be contained in a different plasmid / vector such that transfection with, for example, all five plasmids / vectors of the host cell, when cultured under appropriate conditions, would produce the pentamer produced by the host cell. In another embodiment, the plasmid / vector may contain a combination of one or more coding sequences so that at least one, two, three, four, or five plasmids can be introduced. Alternatively, the entire fusion peptide coding sequence may be provided in a single vector so that the entire protein component and the first peptide tag are encoded on the same vector.

[0116] In one embodiment, these vectors are used for the stable integration of coding sequences into the host cell genome. Host cells suitable for stable expression include mammalian cells, such as rodent cells including HEK cells (human embryonic kidney 293 cells) or CHO (Chinese hamster ovary) cells. Mammalian cells and vectors suitable for the expression of protein components of compositions according to the present invention are known to those skilled in the art and are described, for example, in WO2016 / 067239, pp. 15-16, and Hofmann et al., (2015) Biotech and Bioeng, 112(12):2505-2515. Exemplary stable construct sequences for the expression of components according to the present invention may be found in Example 3 below.

[0117] Affinity Purification In some embodiments, expression constructs for use in the expression of components of compositions according to the present invention may also include one or more “tag” sequences to facilitate 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 producing system. Those skilled in the art of recombinant protein production know of systems such as His tags and Strep tags, which may be included for purification purposes. Such tags dramatically assist in protein purification and have little 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 proteins (MBPs), glutathione-S-transferase (GST), and FLAG tags.

[0118] Both protein components and / or parts may contain affinity purification tags. For ease of use, these are typically gene-fused to the C or N terminus of the protein.

[0119] Therefore, in some embodiments, for example, the gH, gL, pUL128, pUL130, pUL131A (or fragment thereof) subunits of HCMV, the RSV pre-fusion F protein, or the HBsAg peptide / protein may contain additional amino acid residues at the N or C terminus to facilitate purification. Such additional amino acid residues may include tags such as His-tags or C-tags. In some embodiments, C-tags can 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 tags may be ligated to the amino acid sequence in a manner that allows for cleavage after purification, for example, by using a cleavable linker. In other embodiments, non-affinity purification methods may be used.

[0120] In other embodiments, the RSV pre-fusion F protein may also contain additional amino acid residues at its C or N terminus to facilitate purification. As illustrated herein, the RSV pre-F protein has a C-tag for affinity purification.

[0121] Conjugation of the first and second peptide tag pairs The conjugate of the first and second peptide tags / binding partners / reactive pairs can be carried out overnight at 4°C. Alternatively, the conjugate reaction can be carried out at room temperature for 3-4 hours, as the coupling rate is expected to increase at room temperature. The optimal ratio of the first and second binding partners for a given coupling reaction depends on the size of each binding partner. For example, a 1:1.5 molar ratio of VLP monomer to antigen may be sufficient for smaller antigens (~20 kDa), while a 1:1 mass ratio may be sufficient for larger antigens (>100 kDa) combined with the same VLP monomer. However, both ratios will result in excess antigen (smaller binding partners). Any excess antigen can be removed, for example, by 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 antigens are conjugated and therefore downstream purification is unnecessary. Lower concentrations can reduce the reaction rate; therefore, an appropriate final protein concentration of approximately 1 mg / ml is optimal for conjugation reactions. A wide range of buffers with near-neutral pH are suitable for coupling / conjugation. A standard choice for conjugation buffer is TBS (20 mM Tris and 150 mM NaCl, pH 7.4). In some situations, the addition of a 10x stock of citrate buffer (40 mM Na2HPO4, 200 mM sodium citrate, pH 6.2) can also be used, as described by Brune et al. Sci Rep. (2016).

[0122] Pharmaceutical composition and use The composition of the present invention can also be incorporated into a vaccine or immunogenic composition. Preferably, the vaccine or immunogenic composition would contain the particles of the present invention in an immunogenic dose.

[0123] The pharmaceutical composition may also comprise the particles or immunogen compositions described in the present invention, provided 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, an excipient, or a carrier. The pharmaceutical composition may also comprise suitable excipients and formulations for maintaining the stability of the composition. The formulation may also comprise an adjuvant. In one embodiment, the formulation comprises AddaVax TM Alternatively, an oil-in-water emulsion based on a similar squalene may be included with a formulation similar to MF59®. Other suitable adjuvants include liposome-based adjuvants, e.g., Matrix M and AS01. Other suitable adjuvants include aluminum-based formulations, e.g., Alhydrogel®. In one embodiment, the formulation may also contain EDTA at a concentration of, for example, 5 mM. Suitable excipients or formulations may depend on the characteristics of the particle or immunogenic composition; for example, the choice of expression system may affect the stability, glycosylation, or folding of the protein in the composition, which in turn may affect the optimal formulation of the composition. Methods for determining suitable excipients, formulations, or adjuvants will be known to those skilled in the art.

[0124] Various further aspects and embodiments of the present invention will be apparent to those skilled in the art in consideration of this disclosure. All documents referenced herein are incorporated herein by reference in their entirety.

[0125] "and / or" in this specification means two specified terms, including or excluding the other. Each of the features or components shall be construed as a specific disclosure. For example, “A and / or B” shall be construed as each of the specific disclosures of (i) A, (ii) B, and (iii) A and B, as each is individually shown herein.

[0126] Unless otherwise indicated in the background, the descriptions and definitions of the features set forth above are not limited to any particular aspect or embodiment of the present invention and apply equally to all aspects and embodiments described herein.

[0127] While the present invention has been described by reference to several embodiments, it will be further recognized by those skilled in the art that other embodiments can be constructed without being limited to the embodiments disclosed and without departing from the scope of the invention as defined in the accompanying claims.

[0128] In this specification, "recombinant" means a polynucleotide of genomic, cDNA, semi-synthetic, or synthetic origin, which by its origin or manipulation: (1) does not associate with all or some of the naturally associated polynucleotides, and / or (2) is linked to polynucleotides other than those naturally linked. In this specification, the term "recombinant" means, with respect to proteins or polypeptides, a polypeptide produced by the expression of a recombinant polynucleotide.

[0129] Unless otherwise specified, the processes, including the steps, may be carried out in any suitable order. Therefore, the steps may be carried out in any suitable order. Sequence identity between polypeptide sequences is preferably determined by a pair-sorting algorithm using the Needleman-Wunsch broad sorting algorithm (Needleman and Wunsch 1970), with default parameters (e.g., using the EBLOSUM62 scoring matrix with a gap opening penalty of 10.0 and a gap elongation penalty of 0.5). This algorithm is preferably implemented in the needle tool in the EMBOSS package (Rice, Longden and Bleasby 2000). Sequence identity should be calculated over the entire length of the polypeptide sequences of the present invention.

[0130] Any homologue of the components referred to herein is typically a functional homologue and typically at least 40% homologous to the appropriate region of the protein. Homology can also be measured using known methods. For example, the UWGCG Package provides a BESTFIT program that can be used to calculate homology (e.g., using the default settings) (Devereux et al. (1984) Nucleic Acids Research 12, 387-395). Using the PILEUP and BLAST algorithms, e.g., Altschul SF (1993) J Mol Evol 36:290-300; Altschul, S, F et al. (1990) J As described in Mol Biol 215:403-10, it is also possible to calculate homology or align sequences (typically with default settings). Software for performing BLAST analysis is available from the National Center for It is publicly available through Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ).

[0131] The BLAST algorithm performs a statistical analysis of the similarity between two sequences; for example, Karlin and Altschul (1993) Proc. Natl. Acad. See Sci. USA 90: 5873-5787. One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indicator of the probability that a match between two nucleotide or amino acid sequences occurs by chance. For example, if the second sequence matches the first A sequence is considered similar to another sequence if the minimum sum probability when comparing the sequences 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.

[0132] The mutant polypeptide contains (or consists of) a sequence having at least 40% identity with the native protein. In a preferred embodiment, the mutant sequence may be at least 55%, 65%, 70%, 75%, 80%, 85%, 90%, and more preferably at least 95%, 97%, or 99% homology with a particular region of the native protein at least 20, preferably at least 30, for example, at least 40, 60, 100, 200, 300, 400, or more adjacent amino acids, or even across the entire mutant sequence. Alternatively, the mutant sequence may be at least 55%, 65%, 70%, 75%, 80%, 85%, 90%, and more preferably at least 95%, 97%, or 99% homology with the full-length native protein. Typically, the mutant sequence differs from a suitable region of the native protein by at least 2, 5, 10, 20, 40, 50, or 60 mutations (each of which may be a substitution, insertion, or deletion), or fewer. The mutant sequence of the present invention may have percentage identity with a specific region of the full-length native protein, which is the same as any of the specified percentage homology values ​​over any length of the sequence described above (i.e., at least 40%, 55%, 80%, or 90%, and more preferably at least 95%, 97%, or 99% identity).

[0133] Protein variants also include partial excisions. Any partial excision may be used as long as the variant remains functional. Partial excisions will typically be constructed to remove sequences that are non-essential to activity / function, particularly to the formation of isopeptide bonds, and / or do not affect the conformation of the folded protein, especially the folding of any immunogenic sites. Partial excisions may also be selected to improve the ease of production of components. Appropriate partial excisions can be routinely identified by systematic partial excisions of sequences of varying lengths from the N or C-terminus.

[0134] Mutants of native proteins further include mutants that have one or more amino acid insertions, substitutions, or deletions—e.g., 2, 3, 4, 5-10, 10-20, 20-40, or more—with respect to a specific region of the native protein. Deletions and insertions are preferably made outside the antigenic region. Insertions are typically made at the N- or C-terminus of sequences derived from the native protein, for example, for recombinant expression. Substitutions are also typically made in regions that are non-essential to activity / function and / or do not affect the conformation of the folded protein. Such substitutions may be made to improve the solubility or other properties of the protein. Substitutions may be made to increase the stability of the protein.

[0135] Substitutions preferably introduce one or more conservative changes, in which an amino acid is replaced by another amino acid having a similar chemical structure, similar chemical properties, or similar side-chain volume. The introduced amino acids may have similar polarity, hydrophilicity, hydrophobicity, basicity, acidity, neutralizing properties, or charge to the amino acid they substitute for. Alternatively, the conservative change may introduce another amino acid that is aromatic or aliphatic in place of a pre-existing aromatic or aliphatic amino acid. Conservative amino acid changes are well known in the art.

[0136] A derivative is an entity that arises from or is created from a parent entity by the substitution of a part of the parent entity. [Examples]

[0137] [Example 1] Formation of exemplary polymer-VLP composition (HCMV pentamer-HBsAg VLP) ExpiFectamine TM HCMV pentamers were transiently expressed in Expi293F cells using the 293 transfection reagent (ThermoFisher Scientific) and five separate plasmids encoding the following sequences. The HCMV pentamers described below are approximately 162 kDa in size and do not contain glycosylation (they include the tag and linker, but not the signal peptide).

[0138] Nucleotide sequence The HCMV pentamer sequence expressed the native sequence (including introns) derived from the Merlin strain (GenBank:AY446894.2; a low-passage (i.e., attenuated) HCMV strain), with the exception of two introduced mutations (one in gH and one in UL128) described in the appropriate sections below.

[0139] gH-SpyTag-His nucleotide sequence (SEQ ID NO: 12) In this sequence (SEQ ID NO: 12), a silent mutation C>A was introduced at position 1146 for GeneArt® synthesis, because the natural sequence CACCTGC surrounding this nucleotide was warned to be potentially problematic. The construct includes: a signal peptide (nt 1-69), an external domain (nt 70-2151), a partially excised transmembrane domain (nt 2152-2157) (the signal peptide, external domain, and partially excised transmembrane domain are all shown in SEQ ID NO: 13), a linker (nt 2158-2175; SEQ ID NO: 14), a SpyTag (nt 2176-2214; SEQ ID NO: 15), a 6xHis tag (nt 2215-2232), and a stop codon (nt 2233-2235). Nucleotides 1-2157 (SEQ ID NO: 13) correspond to the gH coding sequence.

[0140] gL nucleotide sequence (SEQ ID NO: 16) This sequence contains: signal peptide (nt 1-90), external domain (nt 91-834), and stop codon (nt 835-837).

[0141] UL130-C-tagged nucleotide sequence (SEQ ID NO: 17) This sequence contains: signal peptide (nt 1-75), external domain (nt 76-642), linker (nt 643-687), C-tag (nt 688-699), and stop codon (nt 700-702).

[0142] UL128 nucleotide sequence (SEQ ID NO: 20) (includes two introns present in the natural sequence) This sequence contains: signal peptide (nt 1~81), introns: nt 165~287, nt 423~542, outer domain exons (nt 82~164, nt 288~422, nt 543~756), and stop codons (nt 757~759).

[0143] A T>C mutation was introduced at nucleotide 634. The T634 nucleotide is mentioned in the GenBank file as causing immature termination of UL128 in the Merlin strain, and we therefore used annotations from a different strain (GenBank:GQ396662.1, strain HAN38) that provide information on which base to substitute in order to restore full-length protein expression.

[0144] UL131A nucleotide sequence (SEQ ID NO: 21) (including introns present in the natural sequence) In this sequence: signal peptide (nt 1~54), intron (nt 237~34 4) External domain exons (nt 55~236, nt 345~495), stop codons (nt 496~498).

[0145] SpyCatcher-HBsAg nucleotide sequence (SEQ ID NO: 22) This sequence contains: SpyCatcher delta N1 (nt 1~276), flexibility linker (nt 277~303), PVTN linker (nt 304~315), HBsAg (nt 316~993), C-tag (nt 994~1005), and stop codon (nt 1006~1008).

[0146] amino acid sequence The expression of the above nucleotide sequence is predicted to result in the following amino acid sequence. gH-SpyTag-His amino acid sequence (SEQ ID NO: 27) Predicted molecular weight: 81.852 kDa (without signal peptide), 84.364 kDa (with signal peptide).

[0147] This sequence contains: signal peptide (aa 1-23), extracellular domain (aa 24-717), partially excised transmembrane domain (aa 787-719) (the signal peptide, extracellular domain, and partially excised transmembrane domain are all shown in SEQ ID NO: 28), linker (aa 720-725; SEQ ID NO: 29), SpyTag (aa 726-738; SEQ ID NO: 30), and 6xHis tag (aa 739-744). Amino acid residues 1-719 correspond to the amino acid sequence of the natural Merlin strain gH, including the partially excised TM domain (SEQ ID NO: 28).

[0148] gL amino acid sequence (SEQ ID NO: 31) Predicted molecular weight: 27.522 kDa (without signal peptide), 30.815 kDa (with signal peptide).

[0149] This sequence contains a signal peptide (aa 1-30) and an external domain (aa 31-278). Amino acid residues 1-278 correspond to the amino acid sequence of the natural Merlin strain gL. UL130-C-tagged amino acid sequence (SEQ ID NO: 32) Predicted molecular weight: 23.167 kDa (without signal peptide), 26.081 kDa (with signal peptide).

[0150] This sequence contains: signal peptide (aa 1-25), external domain (aa 26-214) (both the signal peptide and external domain are shown in SEQ ID NO: 33), linker (aa 215-229; SEQ ID NO: 34), and C-tag (aa 230-233). Amino acid residues 1-214 correspond to the amino acid sequence of the natural Merlin strain UL130.

[0151] UL128 amino acid sequence (SEQ ID NO: 35) Predicted molecular weight: 16.659 kDa (without signal peptide), 19.717 kDa (with signal peptide).

[0152] This sequence contains: signal peptide (aa 1-27), and external domain (aa 28-171). Amino acid residues 1-171 correspond to the amino acid sequence of the natural Merlin strain UL128. UL131A amino acid sequence (SEQ ID NO: 36) Predicted molecular weight: 12.985 kDa (without signal peptide), 14.989 kDa (with signal peptide).

[0153] In the above sequence: signal peptide (aa 1-18), external domain (aa 19-129). Amino acid residues 1-129 correspond to the amino acid sequence of the natural Merlin strain UL131A. .

[0154] SpyCatcher-HBsAg amino acid sequence (SEQ ID NO: 37) Predicted molecular weight including tags and linkers: 36.824 kDa. This sequence contains: SpyCatcher delta N1 (aa 1~92; SEQ ID NO: 38), flexibility linker (aa 93~101; SEQ ID NO: 39), PVTN linker (aa 102~105; SEQ ID NO: 40), HBsAg (aa 106~331; SEQ ID NO: 41), and C-tag (aa 332~335).

[0155] Purification of the pentamer The pentamer-SpyTag was expressed in EXPI293F cells and secreted into the supernatant (due to the deletion of (part of) the TM domain from the gH subunit). The first attempt to purify the HCMV pentamer using affinity purification relied on the expression of the gH subunit containing the C-tag, which resulted in the isolation of gH / gL heterohomodimers and pentamers. In another strategy, the C-tag was added to the UL130 subunit (SEQ ID NO: 17 (nucleotide) and SEQ ID NO: 32 (amino acid)), enabling the purification of the pentamer from the supernatant using C-tag affinity purification (ThermoFisher) and size exclusion chromatography. The pentamer appeared as expected under non-reducible and reducible conditions when analyzed by SDS-PAGE (Figure 1A), and reacted with an anti-HCMV pentamer antibody (Native Antigen Company (AbCMV2450)) (Figure 1B), with only small amounts of contamination observed at ~14 kDa.

[0156] Purification of HBsAg VLP monomers SpyCatcher-HBsAg was expressed in Pichia pastoris and purified from cell homogenates. Under reducing conditions on an SDS-PAGE gel, the main protein bands corresponded to the expected monomer size (approximately 37 kDa), while larger bands indicated the presence of oligomeric species and demonstrated excellent particle crosslinking (Figure 2A, lane "R"). Under non-reducing conditions (lane "NR"), the substance remained mainly at the top of the gel with some smearing, indicating that VLP particles were well formed and therefore too large to migrate completely within the gel (Figure 2A). Both non-reduced and reduced SpyCatcher-HBsAg reacted strongly with a mouse anti-HBsAg monoclonal antibody (obtained from Bio-Rad (MCA4658)) (Figure 2B), demonstrating that the presence of SpyCatcher did not negatively affect the reactive epitope. Both HCMV pentamer-SpyTag and SpyCatcher-HBsAg eluted as single peaks when evaluated by HPLC size exclusion analysis on an s200increase 3.2 / 300 column (Figures 3A-B). HCMV pentamer-SpyTag eluted at ~400 kDa (Figure 3A), which is larger than expected. However, this can be explained by the fact that the pentameric structure is not spherical, and in such cases, it is known that the protein retention time is altered during size exclusion chromatography. SpyCatcher-HBsAg eluted into the void volume of the column, indicating that particles were properly formed and monomers were undetectable in solution (Figure 3B).

[0157] Antigen-VLP conjugation HCMV pentamer-SpyTag was conjugated to SpyCatcher-HBsAg overnight at 4°C to produce HCMV-pentamer-coated HBsAg VLPs. A buffer containing Tris-buffered saline (TBS; 20mM Tris and 150mM NaCl, pH 7.4) supplemented with 5mM EDTA was used for conjugation. Conjugation was monitored using SDS-PAGE, Western blotting, and HPLC. Compared to conjugation reactions using either pentamer-SpyTag or SpyCatcher-HBsAg alone, the conjugation reaction yielded ~130kDa under reducing conditions. A novel band was observed (Figure 4A, lane 2), which was reactive with both monoclonal anti-HBsAg (Figure 4B) and polyclonal anti-HCMV pentamer (Figure 4C) antibodies, indicating that the band contained at least conjugated HBsAg-gH. Analysis by HPLC size exclusion chromatography revealed that 97% eluted within the main peak corresponding to the predicted size of the conjugated HCMV pentamer-HBsAg monomer (Figure 5).

[0158] [Example 2] In vivo testing of HCMV-Spy-Tag-SpyCatcher-HBsAg VLP (adjuvant-treated). Conjugated HCMV pentamer-HBsAg VLPs and unconjugated HCMV pentamer-SpyTags were used in an immunization schedule using BALB / c mice to (i) confirm the immunogenicity of the produced HCMV pentamer-SpyTags and (ii) compare the immunogenicity of unconjugated HCMV pentamer-SpyTags versus conjugated HCMV pentamer-HBsAg VLPs.

[0159] The following prime-boost-boost schedule was used with 3-week intervals: Day 0: Immunization (Prime); Day 20: Tail blood sampling; Day 21: Immunization (Boost 1); Day 41: Tail blood sampling; Day 42: Immunization (Boost 2); Day 63: Cardiac blood sampling.

[0160] The immune groups were as follows. For each group, n=10: 1) AddaVax TM (Invivogen) 1 μg of HCMV pentamer - SpyTag 2) AddaVax TM 1 μg of HCMV pentamer - SpyTag--SpyCatcher - HBsAg VLP (equivalent of 1 μg of pentamer) in 3) AddaVax TM SpyCatcher - HBsAg VLP in (standardized against the amount of SpyCatcher - HBsAg in Group 2) 4) AddaVax TM 0.1 μg of HCMV pentamer in 5) AddaVax TM 0.1 μg of HCMV pentamer - SpyTag--SpyCatcher - HBsAg VLP (equivalent of 0.1 μg of pentamer) in 6) TBS (20 mM Tris and 150 mM NaCl, pH 7.4) AddaVax TM is a squalene - based oil - in - water nanoemulsion containing a formulation similar to MF59 (registered trademark), which is approved in Europe for adjuvanted influenza vaccines. Squalene oil - in - water emulsions are known to induce both cellular (Th1) and humoral (Th2) immune responses. Other suitable adjuvants will be known to those skilled in the art.

[0161] Immunogenicity was evaluated using ELISA. Standardized ELISA against HCMV pentamer was used to determine the titer of the antisera generated in each group. The plates were coated overnight at 5 μg / ml of pentamer (without SpyTag), 50 μL / well; washed; blocked with milk for 1 hour; washed; mouse sera (approximate dilutions in PBS) were applied for 1 hour; washed; goat anti - mouse - alkaline phosphatase antibody (1:10,000) was applied for 1 hour; washed; and developed.

[0162] Including both unconjugated (groups 1 and 4) and conjugated HCMV pentamer-HBsAg (groups 2 and 5) at different doses, this allows for a comparison of immunogenicity between the conjugated HCMV pentamer-HBsAg VLP vaccine and the unconjugated HCMV pentamer-SpyTag, thereby enabling estimations for other HCMV pentamer vaccines (e.g., soluble pentamers). Groups 3 and 6 serve as negative controls.

[0163] At each time point, the OD value of the sample was read at the appropriate dilution, and the ELISA units were determined using standard curves prepared on each plate. Figure 6 shows the data for the post-priming results of groups 1, 2, 4, and 5. HCMV pentamer-HBsAg immunized mice showed a substantially stronger serum IgG antibody response using both 1 μg and 0.1 μg doses compared to mice immunized with unconjugated HCMV pentamer at doses of 1 μg or 0.1 μg. Again, as shown in Figure 6, the ELISA units for groups 3 and 6 provided a baseline for this assay.

[0164] The functional activity of the resulting antibodies was investigated using a microneutralization assay based on Wang et al. (Vaccine 33(2015) 7254-7261; DOI: 10.1016 / j.vaccine.2015.10.110). The neutralization titers for groups 1, 2, 4, and 5 are shown in Figure 7. Serum from mice immunized with pentamer-HBsAg VLP was substantially more neutralizing than serum from mice immunized with pentamer-SpyTag protein alone.

[0165] [Example 3] Stable structure array Two stable constructs (adopted from Hofmann et al., (2015) Biotech and Bioeng, 112(12):2505-2515) were optimized for CHO expression of HCMV pentamer-SpyTag components. Introns were removed from the HCMV pentamer sequence, but the signal sequence was retained.

[0166] HCMV gH-SpyTag / gL stable expression construct The stable vector construct HCMV-gH-(GSG)2-SpyTag-His-IRES-gL was designed to include the gH-SpyTag-His component (sequence number 42) and the gL component (sequence number 43) upstream and downstream of EV71 IRES, respectively. The coding sequence used in this construct is described below.

[0167] Nucleotide sequence gH-(GSG)2-SpyTag-His (without introns) (SEQ ID NO: 42) inserted upstream of EV71 IRES This sequence contains: signal peptide (nt 1~69), external domain (nt 70~2151), partially excised transmembrane domain (nt 2152~2157), (GSG)2 linker (nt 2158~2175), SpyTag (nt 2176~2214), His-tag (nt 2215~2232), and stop codon (nt 2233~2235).

[0168] gL (non-intron) inserted downstream of EV71 IRES (SEQ ID NO: 43) This sequence contains: signal peptide (nt 1-90), external domain (nt 91-834), and stop codon (nt 835-837). HCMV UL128 / UL130 / UL131A stable expression construct The stable construct HCMV-UL128-IRES-UL130-(G4S)3-C-tag-IRES-UL131A was designed to contain the UL128 component (SEQ ID NO: 44), the UL130 component (SEQ ID NO: 45), and the UL131A component (SEQ ID NO: 46). The UL130 component was inserted after the first EV71 IRES in the plasmid, and the UL131A component was inserted after the second EV71 IRES. The coding sequences used in this construct are listed below.

[0169] Nucleotide sequence UL128 (intron-free) (SEQ ID NO: 44) In this sequence: signal peptide (nt 1~81), outer domain (nt 82~51 3) Stop codon (nt 514~516).

[0170] UL130-(G4S)3-C-tag (intron-free) (SEQ ID NO: 45) This sequence contains: signal peptide (nt 1-75), external domain (nt 76-642), (G4S)3 linker (nt 643-687), C tag (nt 688-699), and stop codon (nt 700-702).

[0171] UL131A (intron-free) (SEQ ID NO: 46) This sequence contains: signal peptide (nt 1-54), external domain (nt 55-387), and stop codon (nt 388-390).

[0172] [Example 4] In vivo testing of HCMV-SpyTag-SpyCatcher-HBsAg (non-adjuvanted) The immunogenicity of conjugated HCMV pentamer-HBsAg VLP versus unconjugated HCMV pentamer-SpyTag proteins was further investigated using an immunization schedule with BALB / c mice.

[0173] The following prime-boost-boost schedule was used with 3-week intervals: Day 0: Immunization (Prime); Day 20: Tail blood sampling; Day 21: Immunization (Boost 1); Day 41: Tail blood sampling; Day 42: Immunization (Boost 2); Day 63: Cardiac blood sampling.

[0174] The immune groups were as follows. For each group, n=10: 1) 1 μg non-adjuvanted HCMV pentamer - SpyTag 2) 1 μg non-adjuvanted HCMV pentamer-SpyTag--SpyCatcher-HBsAg VLP (equivalent to 1 μg of the pentamer) 3) 0.1 μg non-adjuvanted HCMV pentamer-SpyTag--SpyCatcher-HBsAg VLP (equivalent to 0.1 μg of the pentamer) Immunogenicity was evaluated using ELISA. The titer of the antiserum produced in each group was determined using a standardized ELISA against HCMV pentamers. Plates were coated overnight with 5 μg / ml pentamer (without SpyTag), 50 μL / well; washed; blocked with milk for 1 hour; washed; applied mouse serum (approximate dilution in PBS) for 1 hour; washed; applied goat anti-mouse alkaline phosphatase antibody (1:10,000) for 1 hour; washed; and developed.

[0175] At each time point, the OD value of the sample was read at the appropriate dilution, and the ELISA units were determined using standard curves prepared on each plate. Post-priming and post-boosting data are shown in Figure 8. HCMV pentamer-HBsAg immunized mice showed a substantially stronger serum IgG antibody response using both 1 μg and 0.1 μg doses compared to mice immunized with 1 μg of HCMV pentamer alone as a soluble protein.

[0176] The functional activity of the resulting antibodies was examined using a microneutralization assay based on Wang et al. (2015). Neutralization titers after priming and boosting are shown in Figure 9. Serum from mice immunized with non-adjuvanted pentamer-HBsAg VLP was substantially more neutralizing than serum from mice immunized with non-adjuvanted pentamer-SpyTag protein alone.

[0177] [Example 5] RSV-F-SpyTag expression and purification The sequence derived from the antigen RSV-F Sc9-10 DS-Cav1 A149C Y458C was fused to a SpyTag to generate an RSV-F-SpyTag, and then ExpiCHO TM Expression system kits and ExpiFectamine TM Using transfection reagent (ThermoFisher Scientific), the nucleotide sequence in plasmid pcDNA3.4, sequence number 47, was transfected with ExpiCHO TM The expression was achieved by transiently transfecting the cells.

[0178] RSV-F Sc9-10 DS-Cav1 A149C Y458C (National Institutes of Health) is identified by Joyce et al. (2016) (Iterative structure-based improvement of a respiratory syncytial virus fusion glycoprotein vaccine. Nat This variant is a pre-fusion RSV-F variant of the respiratory syncytial virus fusion protein (pre-fusion RSV-F), as described in Struct Mol Biol. 2016 Sep; 23(9): 811-820). This variant is a pre-fusion stabilized form of the fusion (F) glycoprotein containing a gene-linked F subunit, lacking the fusion peptide, containing a T4 fibrintin trimerizing motif (foldon domain), and its promoter-to-promoter movement is stabilized by an additional promoter-to-promoter disulfide bond (A149C Y458C).

[0179] Nucleotide sequence RSV-F-SpyTag-C tagged nucleotide sequence (SEQ ID NO: 47) The original sequence of Sc9-10 DS-Cav1 A149C Y458C was modified at 3' by deletions of the thrombin region, 6xHis-tag, and Strep-tag(registered trademark) II. These deletion domains were replaced with linker-SpyTag-C-tag sequences to produce a 1587nt cassette (SEQ ID NO: 47) containing Sc9-10 DS-Cav1 A149C Y458C (nt 1~1515, signal peptide (nt 1~75), and T4 fibrintin foldon domain (nt 1435~1515)), (GSG)2 linker (nt 1516~1533; SEQ ID NO: 14), SpyTag (nt 1534~1572; SEQ ID NO: 15), C-tag (nt 1573~1584), and stop codon (nt 1585~1587). Sequence ID 48 contains the Sc9-10 DS-Cav1 A149C Y458C nucleotide sequence, excluding the linker, SpyTag, C-tag, and stop codon. Sequence ID 49 contains the Sc9-10 DS-Cav1 A149C Y458C nucleotide acid sequence, excluding the signal peptide, linker, SpyTag, or C-tag.

[0180] amino acid sequence The expression of the nucleotide sequence, SEQ ID NO: 47, is in the following domains: Sc9-10 DS-Cav1 A149C Y458C ((aa 1~505, including signal peptide (aa 1~25) and foldon domain (aa 479~505)), linker (aa The RSV-F-SpyTag-C-tagged amino acid sequence (SEQ ID NO: 500) was expected to result, including amino acids 506-511 (SEQ ID NO: 29), a Spy tag (aa 512-524; SEQ ID NO: 30), and a C-tag (aa 525-528). The predicted molecular weight of the protein was 57.9 kDa with the signal peptide and 55.3 kDa without the signal peptide. The Sc9-10 DS-Cav1 A149C Y458C amino acid sequence without the linker, SpyTag, or C-tag is included in SEQ ID NO: 51. The Sc9-10 DS-Cav1 A149C Y458C amino acid sequence without the signal peptide, linker, SpyTag, or C-tag is included in SEQ ID NO: 52.

[0181] Purification of RSV-F-SpyTag The RSF-F-SpyTag antigen was secreted from cells and purified from the supernatant using C-tag affinity purification and size exclusion chromatography. The pyTag appeared as expected under both non-reducible and reducing conditions when analyzed by SDS-PAGE (Figure 10A), and reacted with the anti-RSV-F[2F7] monoclonal antibody (ab43812; Abcam) (Figure 10B).

[0182] Purification of HBsAg VLP monomers SpyCatcher-HBsAg (VLP monomer) was prepared and purified as described in Example 1 above. See also Figure 2.

[0183] Conjugation of RSV-F-SpyTag to SpyCatcher-HBsAg RSV-F-SpyTag was conjugated to SpyCatcher-HBsAg overnight at 4°C to produce HBsAg VLPs coated with the 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. Conjugation was monitored using SDS-PAGE and Western blot analysis (Figure 11). When the conjugation reaction was compared to either RSV-F-SpyTag or SpyCatcher-HBsAg alone, a novel band of ~105 kDa (lane 2) was observed under reducing conditions (Figure 11A), which was reactive with both the anti-HBsAg monoclonal antibody (MCA4658, Bio-Rad) (Figure 11B) and the anti-RSV-F[2F7] monoclonal antibody (ab43812; Abcam) (Figure 11C), indicating that this band contained conjugated RSV-F-SpyTag--SpyCatcher-HBsAg.

[0184] [Example 6] Immunogenicity of conjugated RSV-F-SpyTag-SpyCatcher-HBsAg An immunization schedule was designed using BALB / c mice to confirm the immunogenicity of the produced RSV-F antigen and to compare the immunogenicity of conjugated RSV-F-SpyTag--SpyCatcher-HBsAg VLP versus unconjugated RSV-F-SpyTag protein. Based on the amount of RSV-F-SpyTag in the sample, groups were administered, and a prime-boost schedule with 3-week intervals was selected, with the final time point being 2 weeks after boost immunization.

[0185] Mice immunized with RSV-F-SpyTag--SpyCatcher-HBsAg after priming showed either no adjuvant-treated vaccine (Figure 6) or Addavax TM Regardless of whether it is included in the formulation (Figure 6), mice immunized with RSV-F-SpyTag protein alone show a substantially stronger serum IgG antibody response.

[0186] Sequence List

[0187] [Table 4]

Claims

1. A composition comprising virus-like particles that display antigenic components: i) an antigenic component containing the first peptide tag, and ii) The portion containing the second peptide tag Includes, Antigenic components and parts are linked through isopeptide bonds between the first and second peptide tags, wherein the antigenic component exceeds 50 kDa, the antigenic component is a protein component, the part is the surface antigen of hepatitis B virus (HBsAg), and the first and second peptide tags are a SpyTag and SpyCatcher pair. The aforementioned composition.

2. The composition according to claim 1, wherein the antigenic component is greater than 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, 200 kDa, 300 kDa, or 400 kDa.

3. The composition according to claim 1 or claim 2, wherein the antigenic component is a monomer or a polymer, and the polymer may be a trimer, tetramer, pentamer, hexamer, heptamer, octamer, noumer or decaper.

4. The composition according to any one of claims 1 to 3, wherein the antigenic component comprises any one of the following immunogenic components: gB glycoprotein derived from human cytomegalovirus (HCMV), G glycoprotein derived from RSV, F glycoprotein derived from RSV, hemagglutinin (HA) antigen derived from influenza A virus, neuraminidase (NA) antigen derived from influenza A virus, Plasmodium falciparum Pfs230 protein, Plasmodium falciparum CSP protein, human HER2 receptor, PCSK9, VAR2CSA, Plasmodium falciparum RIPR protein, varicella-zoster virus (VZV) glycoprotein E, rabies virus glycoprotein, or Epstein-Barr virus (EBV) gH / gL complex.

5. The composition according to any one of claims 1 to 4, wherein the antigenic component comprises the immunogenic component of the RSV-F protein.

6. The composition according to claim 5, wherein the immunogenic component of the RSV-F protein is a pre-fusion F protein, and may also be a stabilized pre-fusion F protein.

7. The immunogenic component of the RSV prefusion F protein is F １ and F ２ The composition according to claim 6, comprising a trimer of a subunit.

8. The composition according to any one of claims 5 to 7, wherein the RSV-F protein has the amino acid sequence shown in any one of SEQ ID NOs. 50 to 52 or 56 to 58.

9. The composition according to any one of claims 6 to 8, wherein the first peptide tag is attached to the C-terminus of the pre-fusion F protein.

10. The composition according to any one of claims 1 to 9, wherein the first peptide tag is SpyTag.

11. The composition according to claim 10, wherein SpyTag has the amino acid sequence shown in SEQ ID NO:

30.

12. The composition according to claim 10 or 11, wherein SpyTag is attached via a linker.

13. The composition according to claim 12, wherein the linker has the amino acid sequence shown in SEQ ID NO:

29.

14. The antigenic component containing the first peptide tag, (i) containing the amino acid sequence shown in either SEQ ID NO: 50 or 56, (ii) Encoded by the nucleotide sequence shown in either SEQ ID NO: 47 or 53, The composition according to claim 1.

15. The composition according to any one of claims 1 to 14, wherein the second peptide tag is SpyCatcher.

16. The composition according to claim 15, wherein SpyCatcher has the amino acid sequence shown in SEQ ID NO:

38.

17. The composition according to claim 15 or 16, wherein a portion is attached to the SpyCatcher through a linker, preferably a flexible linker.

18. The composition according to claim 17, wherein the linker has the amino acid sequence shown in SEQ ID NO:

39.

19. The portion containing the second peptide tag is (i) containing the amino acid sequence shown in SEQ ID NO: 37, or (ii) Encoded by the nucleotide sequence shown in Sequence ID No. 22, The composition according to claim 1.

20. The composition according to any one of claims 1 to 19, which is an immunogenic composition or a vaccine composition.

21. A vaccine comprising the composition according to any one of claims 1 to 19, for use in the prevention and / or treatment of a disease.

22. A method for producing the composition according to any one of claims 1 to 19, or the vaccine according to claim 21: - The first nucleic acid encoding the first gene fusion of the first protein to the first peptide tag is introduced into the first host cell; - The first host cell is incubated under conditions for expressing the first gene fusion; - A second nucleic acid encoding a second gene fusion of a second protein to a second peptide tag is introduced into a second host cell; - The second host cell is incubated under conditions for expressing the second gene fusion; -Optionally, purify the expressed components; - Incubate the expressed components under conditions for the formation of an isopeptide bond between the first peptide tag and the second peptide tag; and optionally, purify the resulting composition. The process includes the above method, A method in which the first protein contains an antigenic component and the second protein contains a portion.

23. A kit comprising a composition comprising a first immunogenic composition and optionally one or more booster compositions comprising a second immunogenic composition, wherein the first and / or second immunogenic composition comprises the composition according to any one of claims 1 to 19 or the vaccine according to claim 21.

24. A vaccine for use in the prevention and / or treatment of RSV infection, comprising the composition according to any one of claims 5 to 9.

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

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