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 combat and eradicate infectious diseases. Although vaccine development has been very successful, there remains a list of disease challenges for which no vaccines currently exist, including many important pathogens that represent formidable immunological obstacles. It is generally considered that an effective vaccine must be transported to lymph nodes and remain there for a sufficient time to generate an immune response.

[0002] Vaccine development has been moving from the use of attenuated or killed pathogens to the use of smaller antigenic components of these pathogens, aiming to avoid the risks inherent in the use of these attenuated strains while generating the necessary protective immune response. Efforts have been focused on trying to express the immunogenic sites of pathogen components (e.g., components that are necessary for the pathogen to infect cells), and these components have been limited to short / small peptides and proteins, simply because of the technical problems involved in expressing antigens of large or multiple components. However, a potential problem with using very short or small peptides is the risk of pathogens being antigenically variable, which escape the immune response induced by vaccination through changes in certain parts of the antigen.

[0003] There are immunological advantages associated with the expression of large / multiple component antigens, including the ability to allow the generation of antibodies against multiple neutralizing epitopes of a single pathogen. However, the expression of one or more large antigens that can form complexes such that the appropriate antigenic epitopes are maintained and presented for the generation of an effective antibody response remains very challenging. Thus, there remains a need for improved methods for expressing large and / or multiple component antigens in a manner that can generate a clinically significant immune response.

[0004] Previous recombinant vaccines designed to induce immune responses against multiple antigenic components either rely on each component being expressed and packaged separately in separate particles, e.g., in the case of the anti-HPV vaccines, Cervarix and Gardasil, the recombinant major capsid L1 proteins of specific HPV strains are expressed separately and assembled as virus-like particles (VLPs), and then different types of VLPs are combined into a vaccine formulation. Alternatively, multiple short epitopes are selected and combined into a single recombinant vaccine (e.g., multimeric 001 influenza vaccine), but by their very nature, these epitopes are short linear epitopes, selected to avoid the manufacturing complexities involved in three-dimensional structure or refolding, and therefore do not attempt to represent the natural pathogen as presented to the immune system in active infection.

[0005] For example, the 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 infection is usually mild or asymptomatic. After infection, the virus remains latent in the body, but can cause serious disease in immunocompromised individuals (i.e., HIV patients, transplant patients, and those undergoing chemotherapy) or the elderly. HCMV is the leading infectious cause of congenital anomalies in developing countries. Up to 4 / 200 babies are born with HCMV due to congenital infection, and of these, up to 10% suffer from long-term consequences. HCMV infection has also been associated with hypertension and atherosclerosis in adults (Cheng et al. (2013)). (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, a successful HCMV vaccine has not been achieved to date.

[0006] Respiratory syncytial virus (RSV) is another ubiquitous virus that causes little health harm in infected healthy adults and older children. However, it is the second leading cause of death in infants under 1 year old worldwide, second only to malaria. The virus is responsible for an estimated 160,000 deaths per year worldwide. The virus causes serious respiratory infections, and complications include pneumonia and bronchiolitis. High-risk groups include infants under 1 year old and immunocompromised patients, elderly people, and those with heart and lung conditions. Again, despite years of active research and development, there is currently no licensed vaccine against RSV.

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

[0008] In recent years, several genetically encoded systems have been described to allow spontaneous or assisted amide bond formation. For example, SpyTag is a peptide that has been engineered to form a spontaneous and irreversible isopeptide bond to its protein partner SpyCatcher when the two components are mixed. The positions of the SpyTag and SpyCatcher components within the protein chain can be designed to be in diverse positions and react under a wide range of pH, buffer and temperature conditions. The SpyTag / SpyCatcher pair, as well as its variants and derivatives, have been used in vaccine development, but to date, only for simple antigen presentation. Other genetically encoded systems for allowing 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; and SnoopTagJr and DogTag (mediated by SnoopLigase), as well as variants of all of these systems.

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

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

[0011] In a first aspect of the invention there is provided a composition comprising a particle displaying a protein component: i) a protein component comprising a first peptide tag, and ii) a portion containing a second peptide tag Including, The protein components and moieties are linked through isopeptide bonds between the first and second peptide tags, and the protein components are greater than 50 kDa. The composition is provided.

[0012] In another aspect of the invention, there is provided a composition comprising a particle displaying a protein component: i) a protein component comprising a first peptide tag, and ii) a portion containing a second peptide tag Including, The protein components and moieties are linked through isopeptide bonds between the first and second peptide tags, and the protein components are multimeric. The composition is provided.

[0013] The protein component may have any function, for example it may be an enzyme or it may have enzymatic properties. The protein component may be a full length protein or it may be a part, segment, domain or truncation of a full length protein. The protein component may be an antigen or immunogen. The protein component may also be referred to as an antigenic component.

[0014] In another aspect of the invention, there is provided a composition comprising a particle displaying an antigenic entity: iii) an antigenic component comprising a first peptide tag, and iv) A portion containing a second peptide tag Including, The antigenic component and moiety are linked through an isopeptide bond between the first and second peptide tags, and the antigenic component is greater than approximately 50 kDa. The composition is provided.

[0015] In some embodiments of any aspect of the invention, the protein or antigenic component may be 60 kDa, 70 kDa, 80 kDa, 90 kDa, 100 kDa, 110 kDa, 120 kDa, 130 kDa, 140 kDa, 150 kDa, 160 kDa, 170 kDa, 180 kDa, 190 kDa or greater, such as greater than 200 kDa, greater than 300 kDa, or greater than 400 kDa.

[0016] Multimers may contain any number of subunits, which may or may not be covalently linked in protein or antigenic moiety. Multimers may contain 2-20 subunits, or 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more subunits. Alternatively, multimers may be dimers, trimers, tetramers, pentamers, hexamers, heptamers, octamers, nonamers or decamers. Multimers may be formed from any suitable pathogen, but are preferably viral multimers.

[0017] Non-limiting examples of large protein components, i.e., those greater than 50 kDa or as described above, include the pentameric complex (PC) and gB glycoprotein from human cytomegalovirus (HCMV), the G and F glycoproteins from RSV, the hemagglutinin (HA) and neuraminidase (NA) antigens from influenza A virus, Plasmodium falciparum Pfs230, Plasmodium falciparum CSP, human HER2 receptor, PCSK9, VAR2CSA, Plasmodium falciparum Rfs230, VAR2CSA ... IPRs include varicella zoster virus (VZV) glycoprotein E, rabies virus glycoprotein and the Epstein-Barr virus (EBV) gH / gL complex.

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

[0019] Non-limiting examples of multimeric antigenic components include the pentameric complex (PC) and gB trimer from human cytomegalovirus (HCMV), the G and F glycoproteins from RSV, and the 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 those derived from viruses, such as influenza (e.g., influenza hemagglutinin (HA) (e.g., influenza trimer)), respiratory syncytial virus (RSV), and the like.

[0020] Protein components can be attached to a first peptide tag by gene fusion and expressed recombinantly in a suitable cell. For components that include post-translational modifications, such as glycosylation, it may be preferable to express the recombinant protein in eukaryotic or mammalian cell lines.

[0021] In one embodiment, the "moiety" is a component on which the protein component or antigenic component is displayed, e.g., can be available to the immune system. In one embodiment, the moiety multimerizes to form said particle. Suitably, such moiety can be a virus, a bacterium, a multimerization scaffold for vaccination, or a protein component that multimerizes to form a VLP (virus-like particle). Suitably, the moiety can be a component of a bacteriophage, a tobacco mosaic virus particle, an adeno-associated virus-like particle (AAVLP), E. coli, etc. In one embodiment, the moiety is itself a component of a virus, a bacterium, etc., such that multimerization (self-assembly) of the moiety forms a particle for displaying the protein component or antigenic component. In one embodiment, the moiety can be a viral structural protein, e.g., a viral envelope or capsid protein, or a surface antigen. Examples of structural proteins include matrix M1 protein and viral envelope M2 protein from influenza virus, HBsAg from Hepatitis B virus, Escherichia coli bacteriophage AP205 viral coat protein (CP3), hemagglutinin-neuraminidase from various viruses including mumps, etc. Suitable viral structural proteins are known to those skilled in the art. In other embodiments, the moiety can be a protein or peptide, such as a multimerization domain, such as IMX313 forming nanoparticles, or a computer-derived particle, such as MI3. In further embodiments, the moiety can be a synthetic nanoparticle or synthetic VLP, such as gold, lipopeptide or poly(lactic-co-glycolic acid) (PLGA) nanoparticle. Other suitable moieties can include liposomes or outer membrane vesicles. Suitably, the moiety comprising the second peptide tag is the moiety to which the second peptide tag is attached.

[0022] The use of structural surface antigens from viruses may be preferred. Thus, a second peptide tag is attached to the structural surface antigen to allow the formation of a virus-like particle (VLP), which either attaches the second peptide tag to the particle or displays the second peptide tag. VLPs are non-infectious self-assembling nanoparticles, and their repetitive, molecularly defined architecture makes them attractive for engineering multivalency, particularly for vaccination. LPs have been produced from members of a wide variety of viral families, including Hepatitis B virus (including the Hepatitis B small surface antigen (HBsAg)), Parvoviridae (e.g., adeno-associated virus), Retroviridae (e.g., HIV), Flaviviridae (e.g., Hepatitis C virus) and bacteriophages (e.g., Qβ, AP205), any of which may be suitable for use as part of the present invention.

[0023] A second peptide tag can also be attached to the moiety through gene fusion. This gene fusion can be at any suitable point in the sequence, not just limited to the termini. One skilled in the art will recognize that the fusion protein can be recombinantly expressed in a suitable cell.

[0024] The second peptide tag can alternatively be displayed or attached to the moiety by chemical conjugation, which would require, for example, the presence of a reactive amine group for conjugation to be feasible.

[0025] Thus, in one embodiment, the moiety is Hepatitis B virus surface antigen (HBsAg). Suitably, the HBsAg has the amino acid sequence set forth in SEQ ID NO: 41 (or a functional equivalent thereof), as described herein.

[0026] In one embodiment of any aspect of the invention, the protein component or antigenic component is an immunogenic component of the HCMV pentamer. Suitably, the antigenic or immunogenic component is one that is capable of generating an immune response, such as an antibody response, against the component when introduced into a subject, such as a patient. Thus, an "immunogenic component of the HCMV pentamer" is a component that is capable of generating an anti-HCMV antibody response, for example, in a subject. Suitably, the immunogenic component comprises one or more (at least one) of HCMV pentamer subunit components selected from gH, gL, pUL128, pUL130 and pUL131 (also known as pUL131A). In some embodiments, the immunogenic component comprises one or more of these "pUL" or "UL" components. In other embodiments, 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 invention, the immunogenic component of the HCMV pentamer is an HCMV pentamer comprising all of the gH / gL / pUL128 / pUL130 / pUL131 subunits. Suitably, the gH / gL / pUL128 / pUL130 / pUL131 subunits have amino acid sequences corresponding to those 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), or a functional equivalent thereof. By functional equivalent is meant amino acid sequences that share a certain degree of homology and differ only in a few amino acids, but retain the functional property of being able to form antigenic subunits or pentamers that provide protective antibodies. Suitable variants of components gH / gL / pUL128 / pUL130 / pUL131A are described, for example, in WO2014 / 005959 (see pages 4-10), which is incorporated herein by reference.Advantageously, the use of HCMV pentameric subunits in vaccine approaches can provide immunogenic protection against infection from a broad range of HCMV viral strains due to the high degree of homology between strains at the pentameric amino acid sequence level.

[0027] In some embodiments, the antigenic entity can correspond to a component of a disease pathogen, or a vector or portion thereof. The antigenic entity can be, for example, a component that is easy to manufacture. Suitably, in an HCMV pentamer, for example, the immunogenic component of the HCMV pentamer comprises a gH subunit that includes a truncated transmembrane domain (truncated by deleting one or more amino acids from this region) such that the subunit is secreted into the cell supernatant during protein production in a host cell to facilitate purification.

[0028] In one embodiment, the gH / gL / pUL128 / pUL130 / pUL131A subunits have the amino acid sequences (or functional equivalents) shown in SEQ ID NOs: 28, 31, 35, 33, 36, respectively (with or without the signal peptides shown). By functional equivalents is meant amino acid sequences that share a degree of homology and differ only in a few amino acids, but retain the functional property of being able to form antigenic subunits or pentamers that provide, for example, protective antibodies. In some embodiments, functional equivalents may share 70%, 80%, 90% or more homology with the relevant amino acid sequence. 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, 21, or codon-optimized versions thereof (with or without the coding sequence for the signal peptide). In some embodiments, any one of the gH / gL / pUL128 / pUL130 / pUL131A subunits can have a signal peptide, such as a signal peptide present on the native protein of the strain, a functional equivalent of a signal peptide, or a signal peptide from a different strain of HCMV. In some embodiments, any one of the gH / gL / pUL128 / pUL130 / pUL131A subunits can have a signal peptide from a heterologous protein. The choice of signal peptide can 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 can be enzymatically cleaved (e.g., by a signal peptidase), either by the native cellular machinery of the expression system used or in vitro. In some embodiments, any one of the gH / gL / pUL128 / pUL130 / pUL131A subunits can be expressed without a signal peptide. In some embodiments, it is also possible to use native sequences containing introns, as these may result in higher expression levels.Suitably, the native nucleic acid sequence of UL128 contains two introns.In another embodiment, the nucleic acid sequence of UL131A includes one intron. In some embodiments, the intron may be removed. In some embodiments, the native sequence may be codon-optimized for a suitable expression system.

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

[0030] The F protein is an integral membrane protein, composed of three F0 monomers, which are processed during assembly into F1 and F2 subunits, covalently linked by two disulfide bonds. The F protein is highly conserved among RSV isolates from both the A and B subgroups, and the amino acid sequence shows 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 trimer of heterodimers. 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. F There are two N-linked glycosylation sites in F2 and only one in F1. After removing the 25 amino acid signal peptide and the 27 amino acid glycopeptide between F2 and F1, the remaining ectodomain of F consists of 472 amino acids. Only 25 amino acids differ between subtypes A and B in the F ectodomain.

[0031] To develop antigenic compositions from the RSV-F protein, some studies have focused on creating mutants of the trimer pre-fusion protein. Mutants have been produced by genetically fusing two subunits of mature pre-F into a single chain. A DS-Cav1 mutant has been created that contains F2 genetically fused to F1 and a deletion of both the fusion peptide and the pep27 region. Differences in the linker between the F2 and F1 subunits likely affected immunogenicity, and therefore mutants may use different linker choices. The native RSV-F protein sequence can be found in accession number P03420.1.

[0032] Several types of pre-fusion F proteins have been studied and developed, and have been published as a result. These pre-fusion trimers may all be suitable for use in the present invention. The DS-Cav1 stabilized fusion glycoprotein is derived from a natural protein. EP2222710, which is incorporated herein by reference, also discloses a recombinant RSV antigen comprising a soluble F protein polypeptide comprising the F2 and F1 domains of the RSV-F protein polypeptide, and a trimerization domain. Krarup et al., Nat. Commun. 2015; 6: 8143, describes a highly stable pre-fusion RSV-F protein, which is also incorporated herein by reference.

[0033] WO2014 / 160463, incorporated herein by reference, describes an isolated recombinant RSV-F protein that is stabilized in a pre-fusion conformation, as well as a nucleic acid molecule encoding the recombinant RSV-F protein.

[0034] WO2017 / 172890, incorporated herein by reference, describes substitution-modified pre-fusion RSV-F proteins and nucleic acids encoding the proteins. Further description can be found 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 ectodomain protomers linked to the T4 fibritin trimerization domain are available under the accession numbers: LP884611.1, LP884610.1, LP884609.1 and LP884608.1.

[0036] In some embodiments, the protein or antigenic component is a component of a disease pathogen. Alternatively, it may correspond to a vector or a portion thereof. The antigenic component may lack a transmembrane domain, for example, to facilitate production. Suitably, the immunogenic component of the RSV-F protein or, in its pre-fusion conformation, for example, the F protein, comprises an F2-F1 subunit that includes a truncated transmembrane domain (truncated 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 to facilitate purification. Thus, the RSV-F protein lacks a functional TM domain. Alternatively, a gene fusion that includes a first peptide tag may in fact prevent the F protein from being present in the membrane, regardless of the presence of a functional transmembrane domain.

[0037] In one embodiment, the pre-fusion stabilized subunits each have an amino acid sequence set forth in SEQ ID NOs: 50-58 (or a functional equivalent thereof). By functional equivalent is meant an amino acid sequence that shares some degree of homology and differs only in a few amino acids, but retains functional properties, such as being able to form an antigenic subunit that provides protective antibodies. In some embodiments, functional equivalents may share 70%, 80%, 90% or more homology with the relevant amino acid sequence. In one embodiment, the pre-fusion stabilized RSV-F trimer may not include a heterologous trimerization domain.

[0038] The protein component comprises 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 techniques for gene fusion of peptide sequences to express recombinant proteins in appropriate cell systems. For moieties that include post-translational modifications, such as glycosylation, it is preferred to express the recombinant protein in eukaryotic or mammalian cell lines.

[0039] Advantageously, the use of a first peptide tag and a second peptide tag forming an isopeptide bond, such as the SpyTag-SpyCatcher system as described herein, allows for "decorating" the large antigen, such as a VLP-displaying moiety, with large and / or multimeric antigens in the correct organization and orientation such that the antigen is presented to the immune system in such a way that it is capable of generating anti-antigen (e.g. anti-HCMV) antibodies that can provide a protective / neutralizing / immunogenic effect. Traditional approaches of vaccination with soluble antigens (even large antigens, e.g. multimers / pentamers) may be less effective in generating a protective / neutralizing / immunogenic effect. Advantageously, the display of antigens (e.g. multimeric antigens) on particles, such as VLPs or nanoparticles, results in the presentation of geometrically repeated arrays of identical antigens, which, in contrast to soluble antigens, can robustly induce an immune response. Compared to "free" antigens, the larger size of VLPs or other suitable particles may also have a higher immunogenic effect. Furthermore, the orientation of the display of multimeric antigens, such as HCMV pentamers, may be important for immunogenicity. The use of pairing tags, such as the SpyTag-SpyCatcher system as described herein, to attach multimeric antigens on particles allows the antigens to be attached to the particles in a particular preferred orientation. For example, in the case of HCMV, the gH / gL subunits may be less likely to have neutralizing epitopes than the "UL" subunits. Thus, advantageously, the present invention allows the orientation of the display of HCMV pentamers on particles to be determined by appropriate positioning of the first peptide tag, such that, for example, the "UL" subunits are displayed towards the outside of the particle and are therefore more readily available to the immune system of an individual. Alternatively / additionally, the positioning of the first peptide tag on the antigen can be determined to produce a similar orientation of the antigen to that on the native virus, thereby presenting to the immune system a particle that displays the antigen in an orientation more likely to induce an immune response against the invasive live virus.

[0040] In contrast, traditional approaches to display proteins on VLPs may involve chemical linkage, which may be more random in these chemical reactions, but Therefore, the disadvantage is that the correct (e.g., immunologically favorable) orientation of the antigen may not be reliably obtained and may represent only a small percentage of the resulting conjugation reactions. Furthermore, the process involved in chemical conjugation may reduce the likelihood that the 3D structure required for proper antigen presentation can be maintained. Some disadvantages 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 with misfolding and in determining optimal expression conditions for both components. Furthermore, gene fusion may not be suitable for the expression of large or multicomponent antigens, since efficient expression in the correct conformation is very difficult to achieve.

[0042] In order to present a protein or antigenic component in an immunogenic manner, the position of the first peptide tag needs to be carefully designed so that the native protein conformation is maintained, and optionally, if appropriate, any post-translational modifications are retained. For some antigens, the retention of glycosylation does not affect the way the epitope is presented, while for others, either its maintenance or removal improves efficacy. For protein components that are transmembrane proteins, the transmembrane part of the protein component provides an excellent target for placing the first peptide tag, since this sequence is not involved in the conformation of the antigenic protein component and serves a role that is no longer required, for example, in a vaccine. If the protein component does not include a transmembrane protein, it may be beneficial to fuse the first peptide tag to the C- or N-terminus of the component or its subunit (in the case of a multimer), although the first peptide tag can also be included in any part of the sequence. Alternatively, it may be possible to place the first peptide tag in a loop on the protein or antigenic component.

[0043] To present an immunogenic component, such as an immunogenic component of an HCMV pentamer, the position of the first peptide tag must be carefully designed so that the native protein conformation is maintained. For HCMV, in one embodiment, attachment is through the gH subunit, suitably through the C-terminus of the gH subunit, or through the transmembrane domain (or part thereof) of the gH subunit. In addition to maintaining the conformation of the pentamer (or the components of the pentamer), 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 the part of the protein known to generate antibodies with neutralizing effect, and may also be referred to as the immunogenic part.

[0044] To present an immunogenic component, such as the immunogenic component of the RSV pre-fusion F protein, the position of the first peptide tag must be carefully designed so that the native protein conformation is maintained. For RSV-F pre-fusion proteins, in one embodiment, the attachment is suitably 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 neutralizing epitope of the pre-fusion F protein toward the outside of the particle as discussed above. The same considerations would apply to any other variant of the F protein. The inclusion of the first peptide tag at the C-terminus works well while keeping the immunogenic protein component correctly folded, as described herein. It has been proven by the wise.

[0045] In one embodiment, the first and second peptide tags are part of a peptide tag / binding partner pair that can form an isopeptide bond. This isopeptide bond can be spontaneous, i.e. unassisted, or it can require assistance, i.e. ligase or other helpers. Suitably, the first and second peptide tags are a SpyTag / SpyCatcher pair. Suitably, the first and second peptide tags are selected from the list comprising 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), and mutants, derivatives or modifications of all these systems.

[0046] Suitably, the first peptide tag is a peptide tag 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 from a peptide tag / binding partner pair, e.g., SpyTag.

[0047] Suitably, the first peptide tag is a peptide tag from a peptide tag / binding partner pair, such as SnoopTag, and the second peptide tag is a binding partner, such as SnoopCatcher. In another embodiment, the first peptide tag is a binding partner, such as SnoopCatcher, and the second peptide tag is a peptide tag component from a peptide tag / binding partner pair, such as SnoopTag. It is therefore understood that the first peptide tag can be either a "tag" or a "catcher"; the second peptide tag can be the partner of this pair, respectively, a "catcher" or a "tag". Suitable peptide tag / binding partner pairs are described in detail in WO2011 / 09877, WO2016 / 193746, WO2018 / 18951 and WO2018 / 197854, which are incorporated herein by reference.

[0048] In one embodiment, the 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 can be attached through a linker, which can be rigid or flexible, if necessary. Those skilled in the art will recognize which linkers are appropriate.

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

[0051] The moiety can be any suitable moiety, including a synthetic multimerization platform, as previously discussed. The second peptide tag can be attached at any suitable location in the moiety that does not affect its ability to fold and form the proper conformation. Genetic fusion may be preferred. It may be preferred to include the second peptide tag at the C- or N-terminus of the moiety, but the second peptide tag may also be included in any part of the sequence. Alternatively, it may be possible to place the second peptide tag in the loop of the moiety. For example, genetic fusion of SpyCatcher to the N-terminus of the viral coat protein (CP3) of the RNA bacteriophage AP205 is described in Brune et al., Scientific Reports volume 6, article number: 19234 (2016). Alternative fusions using self-assembling synthetic proteins as multimerization platforms are discussed in Bruun et al., ACS Nano, 2018, 12(9), pp 8855-8866. Alternatively, the second peptide tag can be attached through chemical conjugation.

[0052] The second peptide tag can, if necessary, be attached through a linker, which can be strong or flexible. Those skilled in the art will recognize which linkers are appropriate.

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

[0054] The SpayTag may 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 moiety is attached to a SpyCatcher binding partner (second peptide tag). The moiety may suitably be HBsAg. A suitable SpyCatcher has the amino acid sequence shown in SEQ ID NO: 38. In one embodiment, the SpyCatcher is attached through a linker. The linker may be a rigid or flexible linker, suitably the linker has the amino acid sequence shown in SEQ ID NO: 39.

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

[0056] In another embodiment, the composition according to any aspect or embodiment of the present invention further comprises another, preferably different, antigen comprising the first peptide tag, such as another HCMV antigen. Suitably, the other HCMV antigen is glycoprotein B. Suitably, the glycoprotein B sequence is, for example, as described in WO2014 / 005959. See SEQ ID NO: 21, 22, 23 or 36. In one embodiment, the composition comprises a particle (e.g. a VLP) displaying both the HCMV pentamer and the other HCMV antigen.

[0057] In one embodiment, the composition is an immunogenic composition or a vaccine composition.Preferably, said immunogenic or vaccine composition is capable of inducing an immune response, such as an antibody response, upon administration to an individual.Suitably, the immune response may be a protective immune response.Suitable immunogenic compositions may further comprise additional components, including adjuvants, immunostimulants and / or pharma-ceutically acceptable excipients.

[0058] Suitable adjuvants may 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 QuilA. Water-in-oil adjuvants are also suitable. Squalene-oil-in-water emulsions, such as Addavax TM is appropriate.

[0059] Thus, in another aspect or embodiment of the present invention, there is provided an immunogenic or vaccine composition comprising a composition according to the present invention. Suitably, the vaccine composition comprises a vaccine dose, which is an amount of a composition according to the present invention that provides an immunogenic, preferably an immune protective effect against an infectious pathogen / vector, such as a neutralizing effect against HCMV infection. Suitably, the vaccine composition comprises a vaccine dose, which is an amount of a composition according to the present invention that provides a neutralizing effect against an infectious pathogen / vector, such as a neutralizing effect against RSV infection. Antibodies, preferably neutralizing antibodies, generated against the immunogenic composition can be detected and measured by methods well known to those skilled in the art, including, for example, standardized ELISA assays or microneutralization assays, as described herein.

[0060] According to another aspect: i) a portion containing a first peptide tag ii) a protein comprising a second peptide tag Including, The first peptide tag and the second peptide tag form an isopeptide bond. VLPs are provided. In some embodiments, the moiety is HBsAg. However, as described above, any suitable moiety can be used.

[0061] Suitably, the first peptide tag is a peptide tag 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 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. Suitably, the first and second peptide tags are selected from the list comprising 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] Suitably, the protein comprising the second peptide tag is a protein or peptide complex larger than 50 kDa. The protein comprising the second peptide tag can be a protein or peptide complex larger than 50 kDa, 60 kDa, 70 kDa, 80 kDa, 90 kDa, 100 kDa, 110 kDa, 120 kDa, 130 kDa, 140 kDa, 150 kDa or 160 kDa, 170 kDa, 180 kDa, 190 kDa or larger, for example, larger than 200 kDa, larger than 300 kDa, or larger than 400 kDa.

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

[0064] Advantageously, in this method, the use of HBsAg as a carrier (VLP) may also result in an anti-HepB boost, alternatively described as an anti-Hepatitis B virus (HBV) response.

[0065] In another aspect: i) a protein comprising a first peptide tag; ii) a portion containing a second peptide tag Including, The first peptide tag and the second peptide tag form an isopeptide bond. VLPs are provided. In some embodiments, the moiety is HBsAg. However, as described above, any suitable moiety can be used.

[0066] Suitably, the first peptide tag is a peptide tag 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 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. Suitably, the first and second peptide tags are selected from the list comprising 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] Suitably, the protein comprising the first peptide tag is a protein or peptide complex larger than 50 kDa. The protein comprising the first peptide tag can 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 larger, in particular a protein or peptide complex of 200 kDa, 300 kDa or even larger than 400 kDa. In one embodiment, the protein comprising the first peptide tag is a multimeric protein. In one embodiment, the protein comprising the second peptide tag is an antigen, preferably a multimeric antigen. Suitably, the multimeric antigen can be an HCMV pentamer as described herein. Suitably, the protein can be an RSV-F protein or a derivative thereof (e.g., a pre-fusion F protein). In one embodiment, the protein comprising the first peptide tag is an immunogenic component of the HCMV pentamer. As described herein, the HCMV pentamer (gH / gL / pUL128 / pUL130 / pUL131A) comprising the appropriate linker and tag has a molecular weight of more than 160 kDa. Other suitable large or multimeric proteins or antigens include antigens from other infectious pathogens, including viruses such as influenza virus, RSV, etc.

[0068] Advantageously, in this method, the use of HBsAg as a carrier (VLP) may also result in an anti-HBV boost. In another aspect of the invention, a H linked to a SpyTag as described herein is CMV pentamers are provided.

[0069] According to another aspect of the invention, there is provided a method for producing a composition or a VLP according to the invention, comprising: - introducing into a first host cell a first nucleic acid encoding a first genetic fusion of a first protein to a first peptide tag; - incubating said first host cell under conditions for expression of said first gene fusion; optionally, purifying the expressed component; - introducing into a second host cell a second nucleic acid encoding a second genetic fusion of a second protein to a second peptide tag; - incubating the second host cell under conditions for expressing the second gene fusion; optionally, purifying the expressed component; - incubating the expressed construct under conditions for the formation of an isopeptide bond between the first peptide tag and the second peptide tag; and optionally purifying the resulting composition. The method includes the steps of:

[0070] Suitably, the expressed components are incubated together to form isopeptide bonds. Formation of isopeptide bonds may require co-incubation with a ligase or the like.

[0071] Suitably, the method of producing a composition or VLP in accordance with the present invention may be for producing a composition comprising an antigenic component displayed on a VLP. In some embodiments, when the "immunogenic component of HCMV pentamer" comprises the entire HCMV pentamer, recombinant production of the component of HCMV pentamer requires that each subunit is expressed in the correct stoichiometry so that the pentamer is formed and folded correctly for assembly. In these embodiments, complexes of only the necessary pentamer parts (e.g. gH / gL dimer and tetramer, or tetramers lacking any one of the five subunits) need to be excluded from the final product. Advantageously, the present invention overcomes the problems that would otherwise be associated by expressing all of the vaccine components in one system (i.e. HBsAg and the five subunits of the HCMV pentamer) by providing a simple approach to make the components separately and then conjugate them. Thus, in one embodiment, a purification 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, when the "immunogenic component of the RSV-F protein" includes the entire F protein or a derivative thereof, recombinant production of the component F protein or a derivative thereof, together with the derivative including the pre-fusion F protein trimer, is required for assembly to be correctly folded.

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

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

[0075] In another aspect of the invention there is provided a composition according to any aspect of the invention for use as a medicament. In a further aspect of the invention there is provided a composition according to any aspect of the invention for use as a vaccine, preferably a vaccine for use in the prevention and / or treatment of HCMV infection. In accordance with the invention, a composition for use as a medicament or vaccine may be administered to adults, such as women of reproductive age or pregnant women.

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

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

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

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

[0081] In another aspect, the present invention provides a host cell for expressing the components of the composition according to the present invention.Suitable host cells can 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 can be glycosylated.

[0082] In another aspect of the invention there is provided a kit comprising a composition according to the invention for use in a prime-boost vaccination regime. Suitably the kit may comprise a prime composition comprising a first immunogenic composition according to the invention, and a boost composition comprising a second immunogenic composition according to the invention. Alternatively it is possible to provide a kit providing a single or multiple dose vaccination regime, i.e. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 doses. Thus in another aspect Thus, the present invention provides a dosing regimen comprising doses applied approximately three week apart. [Brief description of the drawings]

[0083] [Figure 1] SDS-PAGE and Western blot analysis of purified pentameric SpyTag under non-reducing and reducing conditions. Lane 1: ColorPlus prestained broad range protein ladder with sizes shown in kDa; Lane 2: non-reduced sample; Lane 3: reduced sample. A) SDS-PAGE and Coomassie staining analysis, non-reduced to the left and reduced to the right of the gel showing the position of the HCMV pentameric components. B) Western blot analysis with anti-HCMV pentamer antibody. [Diagram 2] SDS-PAGE and Western blot analysis of purified SpyCatcher-HBsAg under non-reducing (NR) and reducing (R) conditions. A) SDS-PAGE and Coomassie staining analysis. B) Western blot analysis using anti-HBsAg monoclonal antibody. [Diagram 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 prestained broad range protein ladder with sizes shown in kDa; 2: conjugated; 3: pentamer-SpyTag; 4: SpyCatcher-HBsAg. A) SDS-PAGE and Coomassie staining analysis. B) Western blot with anti-HBsAg monoclonal antibody. C) Western blot with anti-pentamer polyclonal antibody. [Diagram 5]HPLC analysis using a s200increase3.2 / 300 column. 30 μl of conjugated pentamer-SpyTag-SpyCatcher-HBsAg was loaded and eluted as a single major peak in the void volume of the column. [Figure 6] Immunogenicity of HCMV pentamer-HBsAg vaccine versus pentameric protein vaccine adjuvanted with Addavax, after a single immunization. BALB / c mice were immunized with 1 μg or 0.1 μg of HCMV pentamer-SpyTag, either as soluble protein or as pentamer-HBsAg VLP. Titers were measured from mouse sera by standardized ELISA. Lines indicate means and error bars indicate standard deviations (n=10). Mice immunized with HCMV pentamer-HBsAg VLP show substantially stronger serum IgG antibody responses compared to mice immunized with HCMV pentamer protein alone, even at a 10x lower dose of VLP equivalent to the pentamer. [Figure 7] Neutralizing activity in sera of mice immunized with HCMV pentamer-HBsAg vaccine compared to pentamer protein vaccine. Vaccines were adjuvanted with Addavax and responses after one (post-prime) or two immunizations (post-boost) are shown. NT50 was measured on ARPE-19 cells infected with AD169wt131 strain (displaying functional pentamers). 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 pentameric protein vaccine after one or two immunizations. BALB / c mice were immunized with 1 μg or 0.1 μg of HCMV pentamer-SpyTag conjugated to SpyCatcher-HBsAg (pentamer-HBsAg) or 1 μg of pentamer-SpyTag protein. Titers were measured by standardized ELISA from mouse sera. Lines indicate means and error bars indicate standard deviations (n=10). Mice immunized with HCMV pentamer-HBsAg VLPs exhibit substantially stronger serum IgG antibody responses compared to mice immunized with HCMV pentamer protein alone, even at a 10× lower dose of VLPs equivalent to the pentamer. [Figure 9] Neutralizing activity in sera of mice immunized with HCMV pentamer-HBsAg vaccine compared to pentamer protein vaccine. Vaccine was not adjuvanted and responses after one (post-prime) or two immunizations (post-boost) are shown. NT50 was measured on ARPE-19 cells infected with AD169wt131 strain (displaying functional pentamers). 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 Coomassie staining analysis, lane 1: ColorPlus prestained broad-range protein ladder; lane 2: non-reduced sample; lane 3: reduced sample. B) Western blot analysis with anti-RSV-F monoclonal antibody, lane 1: ColorPlus prestained broad-range protein ladder; lane 2: non-reduced sample; lane 3: reduced sample. [Figure 11]SDS-PAGE and Western blot analysis of RSV-F-SpyTag conjugated with SpyCatcher-HBsAg under reducing conditions. 1: ColorPlus prestained broad range protein ladder; 2: RSV-F-SpyTag--SpyCatcher-HBsAg conjugate, 3: RSV-F-SpyTag, 4: SpyCatcher-HBsAg. A) SDS-PAGE and Coomassie staining analysis. B) Western blot 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 1 μg of RSV-F-SpyTag (RSV-F VLP) conjugated to SpyCatcher-HBsAg or 1 μg of RSV-F-SpyTag protein, either unadjuvanted or adjuvanted with Addavax™. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

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

[0085] HCM V Human cytomegalovirus (HCMV, also known as human herpesvirus-5 (HHV-5)) is a virus to which most adults have been exposed, and initial infection is usually only mild or asymptomatic. After infection, the virus is latent in the body, but can cause serious disease in immunocompromised or elderly individuals. HCMV is also a major infectious cause of congenital anomalies in developing countries. Up to 4 / 200 babies are born with HCMV due to congenital infection, and of these, up to 10% suffer from long-term consequences. HCMV infection has been associated with hypertension and atherosclerosis in adults (Cheng et al. (May 2009) Frueh K ed. "Cytomegalovirus infection causes an increase of arterial blood pressure". PLoS Pathog. 5(5):e1000427).

[0086] The HCMV pentameric 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, no successful HCMV vaccine has been developed to date.

[0087] HCMV pentamer HCMV strains, including clinical isolates and laboratory strains, differ in genome sequence. HCMV strains include Merlin (GI:155573956), Towne (GI239909366) and 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 an endocytic pathway. Other combinations of components of this complex have been shown to be important, for example, for infection of fibroblasts. The "pUL" subunit / component is also referred to as "UL"; "pUL131" is also referred to as "pUL131A" and "pUL131a" or "UL131A".

[0088] Various HCMV strains have been deposited with the ATCC and can be found under the following accession numbers: Merlin (VR-1590), Towne (VR-977) and AD169 (VR-538). Genomic sequences can be referenced through the following accession numbers: Merlin (AY446894.2), Towne (GO121041.1), AD169 (FJ527563.1), Toledo (GU37742.2) and TB40 / E (KF297339.1).

[0089] RSV Respiratory syncytial virus is the leading cause of serious respiratory disease in children worldwide. An estimated 3.4 million children under 5 years of age are hospitalized annually with serious RSV lower respiratory tract infections, with the highest incidence in children under 6 months of age. Most deaths occur in infants under 1 year of age and in developing countries. Currently, options for prevention and control are limited.

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

[0091] As discussed above, 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 including fusions with SpyTag are included as SEQ ID NOs: 47 and 53. Amino acid sequences are shown as SEQ ID NOs: 51, 52, 57 and 58 for pre-fusion trimers, and SEQ ID NOs: 40 and 56 for those including SpyTag. Other exemplary sequences are mentioned herein.

[0092] Protein tag / binding partner pairs 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 that provide a peptide tag and a polypeptide binding partner of the peptide tag, where the two fragments can be covalently reassembled by isopeptide bond formation, thereby linking a molecule or component fused to the peptide tag and its polypeptide binding partner. The isopeptide bond formed by a peptide tag and its polypeptide binding partner is stable under conditions under which non-covalent interactions would rapidly dissociate, e.g., over extended periods of time (e.g., weeks), at high temperatures (up to at least 95°C), high pressure, or under harsh chemical treatments (e.g., pH 2-11, organic solvents, detergents or denaturants).

[0093] Isopeptide bonds are amide bonds formed between carboxyl / carboxamide and amino groups, where at least one of the carboxyl or amino groups is on the outside of the protein backbone (protein backbone). Such bonds are chemically irreversible under typical biological conditions, and they are resistant to most proteases. Because isopeptide bonds are covalent in nature, they produce some of the strongest measured protein-protein interactions.

[0094] Briefly, the two-part linker, i.e. the peptide tag and its polypeptide binding partner (so-called peptide tag / binding partner pair), can also be derived from a protein capable of spontaneously forming an isopeptide bond (isopeptide protein), where domains of the protein are expressed separately to produce a peptide "tag" that contains one of the residues involved in the isopeptide bond (e.g. aspartic acid or asparagine, or lysine), and a peptide or polypeptide binding partner (or "catcher") that contains the other residue involved in the isopeptide bond (e.g. lysine, or aspartic acid or asparagine) and at least one other residue required to form an isopeptide bond (e.g. glutamic acid). Mixing of the peptide tag and the binding partner results in the spontaneous formation of an isopeptide bond between the tag and the binding partner. Thus, by separately incorporating the peptide tag and the binding partner into different molecules or components, e.g. proteins, it is possible to covalently link 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 molecules or components incorporating the peptide tag and the binding partner.

[0095] The spontaneous formation of an isopeptide bond can be isolated and can occur without the need for the addition of any other entity. For some peptide tags and tag partner pairs, the presence of a helper entity, such as a ligase, can be required to generate the isopeptide bond.

[0096] A peptide tag / binding partner pair (two-part linker) called SpyTag / SpyCatcher was synthesized by binding to the Streptococcus pyogenes FbaB protein (Zakeri et al., 2012, Proc Natl Acad Sci (2012) 143:143–154). Sci USA 109, E690-697) and have 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] Suitably, the first and second peptide tags form a peptide tag / binding pair called SpyTag / SpyCatcher. Suitably, the SpyCatcher component is deltaN1 (ΔN1) SpyCatcher, which has a 23 amino acid truncation at the 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 a peptide tag / binding pair that is a mutant of SpyTag / SpyCatcher that exhibits increased reaction rates for isopeptide bond formation, such as those described in co-pending application GB1706430.4. In some embodiments, these mutants may be useful for attachment of large proteins (e.g., >50 kDa or >100 kDa, e.g., the >160 kDa HCMV pentameric protein as described herein) and / or where slow reactions or steric hindrance may be problematic.

[0099] In other embodiments, the isopeptide protein can include a SnoopTag / SnoopCatcher, for example, as described in WO 2016 / 193746.

[0100] In some embodiments, one or both of the isopeptide proteins can have an N- or C-terminal truncation and still retain the reactivity of the isopeptide bond.

[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 set forth in the table below.

[0102] [Table 1]

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

[0104] For some binding pairs, a third entity, such as an enzyme, must be mediated. For example, SnoopLigase mediates the bond formation between SnoopTagJr and DogTag. Thus, pairing may require the assistance of an enzyme such as a ligase.

[0105] HBsAg By "HBsAg" is meant the surface antigen (HBsAg) derived from the hepatitis B virus or a portion thereof. In one embodiment, HBsAg may refer to the N-terminus of HBsAg, e.g. the HBsAg sequence as shown in SEQ ID NO: 41, which includes the 226 amino acids of the S protein of hepatitis B virus (adw serotype). Suitably, HBsAg may include the HBsAg sequence as set forth in 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 carboxy-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 been used as the basis of the pre-erythrocytic malaria vaccine RTS,S, which has completed Phase III clinical trials and is the most advanced malaria vaccine to date (http: / / www.malariavaccine.org / sites / www.malariavaccine.org / files / content / page / files / RTSS%20FAQs_FINAL.pdf; Kaslow and Biernaux, Vaccine 2015, Vol. 33(52): 7425-7432).

[0106] Linker Details The distance between the proteins (e.g., the VLP and the decorated antigen) can have an impact on the availability of antigenic epitopes in the proteins, the stability of the protein or proteins, and can also have an impact on the conjugation efficiency due to the accessibility of any of the isopeptide binding partners (e.g., SpyTag / SpyCatcher). Therefore, it is also possible to select linkers with appropriate properties to optimize the availability, stability, and / or accessibility. Linkers can be broadly subclassified into flexible and rigid subtypes.

[0107] Flexible Linker Flexible linkers may be used when the domains to be linked require movement. These usually consist of small non-polar (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). Addition of Ser or Thr may also help maintain stability in solution, and length adjustment may affect proper folding of the protein (Chen et al., 2013). Any suitable flexible linker may be used, with properties and length appropriate for the relevant entity. Suitably, the flexible linker may include a combination of between 2 and 70 amino acids of these types.

[0108] example:

[0109] [Table 2]

[0110] Rigid Linker In some cases, rigid linkers may be preferred because they may help provide protein separation. Rigid linkers have secondary structure. One of the most common rigid linkers adopts an alpha helical structure (EAAAK). n(wherein n is the number of repeats) (Arai et al., (2001) Protein Eng. Aug;14(8):529-32). Other rigid linkers include (XP) n It is also possible to include: where X is any amino acid, but preferentially Ala (A), Lys (K) or Glu (E), where the proline provides the conformational constraint (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 may be used, having properties and length suitable for the relevant entity. Suitably, the rigid linker may include a combination of between 2 and 70 amino acids of these types.

[0112] example:

[0113] [Table 3]

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

[0115] In one embodiment, the host cell will be suitable for transient expression. In one embodiment, the host cell will be a cell capable of forming a stable cell line. Suitably, the coding sequences encoding the antigenic components, e.g., the HCMV pentamer and the RSV-F protein, including those containing isopeptide bond forming peptide tags, will be integrated into one host cell. In one embodiment, the nucleic acid sequences encoding the subunits of a multimer, such as a pentamer, will each be contained in a different plasmid / vector, such that transfection of a host cell, e.g., with all five plasmids / vectors, will result in the pentamer being produced by the host cell when cultured under suitable conditions. In other embodiments, the plasmid / vector may contain a combination of one or more coding sequences, such that at least one, two, three, four or five plasmids can be introduced. Alternatively, the entire fusion peptide coding sequence may be provided in one vector, such that all protein components and the first peptide tag are encoded on the same vector.

[0116] In one embodiment, these vectors are used for stable integration of coding sequences into the host cell genome. Suitable host cells 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 expression of protein components of the compositions according to the invention are known to the skilled artisan and are described, for example, in WO2016 / 067239, pages 15-16, and Hofmann et al., (2015) Biotech and Bioeng, 112(12):2505-2515. Exemplary stable construct sequences for expression of components according to the invention can be found in Example 3 below.

[0117] Affinity purification In some embodiments, expression constructs for use in expressing components of compositions according to the invention may 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 components and the first peptide tag from the system in which they are produced. Those skilled in the art of recombinant protein production are aware of systems such as His-tags and Strep-tags that may be included for purification purposes. Such tags dramatically aid 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 protein (MBP), glutathione-S-transferase (GST) and FLAG tags.

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

[0119] Thus, in some embodiments, for example, the gH, gL, pUL128, pUL130, pUL131A (or fragments 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, for example, a tag, such as a His-tag or a C-tag. In some embodiments, the C-tag can provide a cleaner purification. Other suitable tag sequences include maltose binding protein (MBP), Strep-tag, glutathione-S-transferase (GST) and FLAG tag. In some embodiments, the tag may be linked to the amino acid sequence in such a way that it can be cleaved 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 can contain additional amino acid residues at the C- or N-terminus to facilitate purification. As exemplified herein, the RSV pre-F protein has a C-tag for affinity purification.

[0121] Conjugation of the first and second peptide tag pairs Conjugation of the first and second peptide tags / binding partners / reactive pairs can be performed overnight at 4°C. Alternatively, the conjugation reaction can be performed at room temperature for 3-4 hours, as the coupling rate is expected to increase at room temperature. The optimal first and second binding partner ratio 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 partner). 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 not as efficient as SEC. Alternatively, the VLP / particle to antigen ratio can be optimized so that all antigen is conjugated and thus no downstream purification is required. A suitable final protein concentration of approximately 1 mg / ml is optimal for the conjugation reaction, as lower concentrations may decrease the reaction rate. A wide range of buffers with near neutral pH are compatible with coupling / conjugation. The standard choice of conjugation buffer is TBS (20 mM Tris and 150 mM NaCl, pH 7.4). In some circumstances, 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 in Brune et al. Sci Rep. (2016).

[0122] Pharmaceutical Compositions and Uses The compositions of the invention may also be incorporated into a vaccine or immunogenic composition. Suitably the vaccine or immunogenic composition will contain the particles of the invention in an immunogenic dose.

[0123] The pharmaceutical composition may comprise a particle or immunogenic composition according to the invention provided with a pharma- ceutically acceptable carrier. Suitable carriers are well known to those skilled in the art. In one embodiment, the pharmaceutical composition comprises a buffer, excipient or carrier. Suitably, the pharmaceutical composition may comprise excipients and formulations suitable for maintaining the stability of the composition. Suitably, the formulation may comprise an adjuvant. In one embodiment, the formulation comprises AddaVax. TM Or similar squalene-based oil-in-water emulsions can be included with formulations similar to MF59®. Other suitable adjuvants include liposome-based adjuvants, such as Matrix M and AS01. Other suitable adjuvants include aluminum-based formulations, such as Alhydrogel®. In one embodiment, the formulation can include EDTA, for example, at a concentration of 5 mM. The appropriate excipient or formulation can depend on the properties of the particle or immunogenic composition; for example, the choice of expression system can affect the stability, glycosylation, or folding of the proteins of the composition, which in turn can affect the optimal formulation of the composition. Those skilled in the art will know how to determine the appropriate excipient, formulation, or adjuvant.

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

[0125] "And / or" is used herein to refer to two specified terms, either with or without the other. For example, "A and / or B" is to be construed as a specific disclosure of (i) A, (ii) B, and (iii) each of A and B, just as if each were individually set forth herein.

[0126] Unless the context dictates otherwise, the feature descriptions and definitions provided above are not limited to any particular aspect or embodiment of the present invention and apply equally to all aspects and embodiments described.

[0127] While the present invention has been described by way of example with reference to certain embodiments, those skilled in the art will further recognize that the invention is not limited to the disclosed embodiments and that alternative embodiments may be constructed without departing from the scope of the present invention as defined in the appended claims.

[0128] "Recombinant," as used herein in describing a polynucleotide, means a polynucleotide of genomic, cDNA, semisynthetic, or synthetic origin that, by its origin or manipulation: (1) is not associated with all or a portion of a polynucleotide with which it is naturally associated, and / or (2) is linked to a polynucleotide other than that with which it is naturally linked. The term "recombinant," as used herein in reference to a protein or polypeptide, refers to a polypeptide produced by expression of a recombinant polynucleotide.

[0129] Unless otherwise specified, a process comprising steps may be performed in any suitable order. Thus, steps may be performed in any suitable order. Sequence identity between polypeptide sequences is preferably determined by a pairwise alignment algorithm using the Needleman-Wunsch global alignment algorithm (Needleman and Wunsch 1970) using default parameters (e.g., with the EBLOSUM62 scoring matrix, with a gap opening penalty=10.0, and a gap extension penalty=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 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 be measured using known methods. For example, the UWGCG Package provides the BESTFIT program that can be used to calculate homology (e.g., used with default settings) (Devereux et al. (1984) Nucleic Acids Research 12, 387-395). The PILEUP and BLAST algorithms can be used to find homology, for example, as described in Altschul SF (1993) J Mol Evol 36:290-300; Altschul, S, F et al. (1990) J Mol Evol 36:290-300. It is also possible to calculate homology or align sequences (typically with default settings) as described in Mol Biol 215:403-10. Software for performing BLAST analyses 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; see, e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 5873-5787. One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability that a match between two nucleotide or amino acid sequences will occur by chance. For example, if a first sequence is found in a second sequence, the probability that a match between the first and second sequences will occur by chance is 0.01. A sequence is considered to be similar to another sequence if the smallest sum probability in 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] A variant polypeptide comprises (or consists of) a sequence having at least 40% identity to the native protein. In preferred embodiments, the variant sequence may be at least 55%, 65%, 70%, 75%, 80%, 85%, 90%, and more preferably at least 95%, 97% or 99% homologous to a particular region of the native protein over at least 20, preferably at least 30, such as at least 40, 60, 100, 200, 300, 400 or more contiguous amino acids, or even over the entire sequence of the variant. Alternatively, the variant sequence may be at least 55%, 65%, 70%, 75%, 80%, 85%, 90%, and more preferably at least 95%, 97% or 99% homologous to the full-length native protein. Typically, the variant sequence differs from the appropriate region of the native protein by at least, or by less than, 2, 5, 10, 20, 40, 50 or 60 mutations (each of which may be a substitution, insertion or deletion). The variant sequences of the invention may have a percentage identity with a particular region of the full-length native protein that is the same as any of the specified percentage homology values ​​over any length of the sequence mentioned above (i.e., at least 40%, 55%, 80% or 90%, and more preferably at least 95%, 97% or 99% identity).

[0133] Mutants of proteins also include truncations. Any truncation may be used as long as the mutant is still functional. Truncations will typically be made to remove sequences that are non-essential for activity / function, particularly for the formation of isopeptide bonds, and / or do not affect the conformation of the folded protein, particularly the folding of any immunogenic sites. Truncations can also be selected to improve the ease of production of the construct. Suitable truncations can be routinely identified by systematic truncation of sequences of various lengths from the N- or C-terminus.

[0134] Variants of native proteins further include mutants having one or more, e.g., 2, 3, 4, 5-10, 10-20, 20-40 or more, amino acid insertions, substitutions or deletions with respect to a particular 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 the sequence from the native protein, e.g., for recombinant expression. Substitutions are also typically made in regions that are non-essential for 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, which replace amino acids with other amino acids of similar chemical structure, similar chemical properties, or similar side chain volume. The introduced amino acids may have similar polarity, hydrophilicity, hydrophobicity, basicity, acidity, neutrality, or charge to the amino acids they replace. Alternatively, conservative changes 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 or is created from a parent entity by substitution of some part of the parent entity. EXAMPLES

[0137] Example 1 Generation of an Exemplary Multimer-VLP Composition (HCMV Pentamer-HBsAg VLP) ExpiFectamine TM HCMV pentamers were transiently expressed in Expi293F cells using 293 transfection reagent (ThermoFisher Scientific) and five separate plasmids encoding the following sequences: The HCMV pentamers described below are approximately 162 kDa without glycosylation (including tags and linkers, but not including the signal peptide).

[0138] Nucleotide sequence The HCMV pentamer sequence expressed the native sequence (including introns) from the Merlin strain (GenBank: AY446894.2; a low-passage (i.e., attenuated) HCMV strain), except for 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 the GeneArt® synthesis, as the native sequence CACCTGC around this nucleotide was flagged as potentially problematic. The construct comprises: signal peptide (nt 1-69), ectodomain (nt 70-2151), transmembrane domain (truncated) (nt 2152-2157) (signal peptide, ectodomain and transmembrane domain (truncated) are both shown in SEQ ID NO: 13), linker (nt 2158-2175; SEQ ID NO: 14), SpyTag (nt 2176-2214; SEQ ID NO: 15), 6xHis tag (nt 2215-2232), 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) In this sequence: signal peptide (nt 1-90), ectodomain (nt 91-834), stop codon (nt 835-837).

[0141] UL130-C-tag nucleotide sequence (SEQ ID NO: 17) In this sequence: signal peptide (nt 1-75), ectodomain (nt 76-642), linker (nt 643-687), C-tag (nt 688-699), stop codon (nt 700-702).

[0142] UL128 nucleotide sequence (SEQ ID NO:20) (including two introns present in the native sequence) In this sequence: signal peptide (nt 1-81), introns: nt 165-287, nt 423-542, ectodomain exons (nt 82-164, nt 288-422, nt 543-756), stop codon (nt 757-759).

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

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

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

[0146] Amino acid sequence 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) The predicted molecular weight is 81.852 kDa (excluding signal peptide) and 84.364 kDa (including signal peptide).

[0147] In this sequence: signal peptide (aa 1-23), ectodomain (aa 24-717), transmembrane domain (truncated) (aa 787-719) (the signal peptide, ectodomain and transmembrane domain (truncated) are both set forth in SEQ ID NO:28), linker (aa 720-725; SEQ ID NO:29), SpyTag (aa 726-738; SEQ ID NO:30), 6xHis tag (aa 739-744). Amino acid residues 1-719 correspond to the native Merlin strain gH amino acid sequence including the truncated TM domain (SEQ ID NO:28).

[0148] gL amino acid sequence (SEQ ID NO:31) The predicted molecular weight is 27.522 kDa (excluding signal peptide) and 30.815 kDa (including signal peptide).

[0149] In this sequence: signal peptide (aa 1-30), ectodomain (aa 31-278). Amino acid residues 1-278 correspond to the native Merlin strain gL amino acid sequence. UL130-C-tag amino acid sequence (SEQ ID NO:32) The predicted molecular weight is 23.167 kDa (excluding signal peptide) and 26.081 kDa (including signal peptide).

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

[0151] UL128 amino acid sequence (SEQ ID NO:35) The predicted molecular weight is 16.659 kDa (excluding signal peptide) and 19.717 kDa (including signal peptide).

[0152] In this sequence: signal peptide (aa 1-27), ectodomain (aa 28-171). Amino acid residues 1-171 correspond to the native merlin strain UL128 amino acid sequence. UL131A amino acid sequence (SEQ ID NO:36) The predicted molecular weight is 12.985 kDa (excluding signal peptide) and 14.989 kDa (including signal peptide).

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

[0154] SpyCatcher-HBsAg amino acid sequence (SEQ ID NO:37) Predicted molecular weight including tag and linker: 36.824 kDa. In this sequence: SpyCatcher delta N1 (aa 1-92; SEQ ID NO: 38), flexible 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), C-tag (aa 332-335).

[0155] Purification of pentamers Pentamer-SpyTag was expressed in EXPI293F cells and secreted into the supernatant (due to deletion of (part of) the TM domain from the gH subunit). Initial attempts to purify HCMV pentamers using affinity purification relied on expression of the gH subunit containing a C-tag, which resulted in the isolation of the gH / gL heterodimer as well as the pentamer. In an alternative strategy, a C-tag was added to the UL130 subunit (SEQ ID NO:17 (nucleotide) and SEQ ID NO:32 (amino acid)) to allow purification of the pentamer from the supernatant using C-tag affinity purification (ThermoFisher) and size-exclusion chromatography. The pentamer appeared as expected under non-reducing and reducing conditions when analyzed by SDS-PAGE (Figure 1A), and reacted with an anti-HCMV pentamer antibody (Native Antigen Company (AbCMV2450)) (Figure 1B), with only a small amount of contaminant 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 major protein band corresponded to the expected size of the monomer (approximately 37 kDa), with larger bands indicating the presence of oligomeric species, indicating that excellent cross-linking of the particles had occurred (Figure 2A, lane "R"). Under non-reducing conditions (lane "NR"), the material remained mainly at the top of the gel with some smear, indicating that the 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), indicating that the presence of SpyCatcher does not negatively affect the reactive epitope. Both HCMV pentamer-SpyTag and SpyCatcher-HBsAg eluted as a single peak when assessed by HPLC size-exclusion analysis on a s200increase 3.2 / 300 column (Figure 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 pentamer structure is not globular and in such cases the retention time of the protein is known to be altered during size-exclusion chromatography. SpyCatcher-HBsAg eluted in the void volume of the column, indicating that the particles were properly formed and no monomer was detectable in solution (Figure 3B).

[0157] Antigen-VLP conjugation HCMV pentamer-SpyTag was conjugated to SpyCatcher-HBsAg overnight at 4° C., resulting in HCMV-pentamer-coated HBsAg VLPs. A buffer containing Tris-buffered saline (TBS; 20 mM Tris and 150 mM NaCl, pH 7.4) supplemented with 5 mM EDTA was used for conjugation. Conjugation was monitored using SDS-PAGE and Western blot analysis as well as HPLC. Comparing the conjugation reaction to either pentamer-SpyTag or SpyCatcher-HBsAg alone, a ∼130 kDa fragment was observed under reducing conditions. There was a novel band present (Fig. 4A, lane 2) that was reactive with both monoclonal anti-HBsAg (Fig. 4B) and polyclonal anti-HCMV pentamer (Fig. 4C) antibodies, indicating that the band contained at least conjugated HBsAg-gH. When analyzed by HPLC size-exclusion chromatography, 97% eluted in a major peak corresponding to the predicted size of conjugated HCMV pentamer-HBsAg monomer (Fig. 5).

[0158] Example 2 In vivo testing of HCMV-Spy-Tag--SpyCatcher-HBsAg VLPs (adjuvanted) Conjugated HCMV pentamer-HBsAg VLPs, as well as unconjugated HCMV pentamer-SpyTag, were used in an immunization schedule with BALB / c mice to (i) confirm the immunogenicity of the produced HCMV pentamer-SpyTag, and (ii) compare the immunogenicity of unconjugated HCMV pentamer-SpyTag versus conjugated HCMV pentamer-HBsAg VLPs.

[0159] A prime-boost-boost schedule at 3 week intervals was used as follows: Day 0: immunization (prime); day 20: tail bleed; day 21: immunization (boost 1); day 41: tail bleed; day 42: immunization (boost 2); day 63: cardiac bleed.

[0160] The immunization groups were as follows: For each group, n=10: 1) AddaVax TM 1 μg HCMV pentamer-SpyTag in (Invivogen) 2) AddaVax TM 1 μg HCMV pentamer-SpyTag--SpyCatcher-HBsAg VLP (equivalent of 1 μg pentamer) 3) AddaVax TM SpyCatcher-HBsAg VLPs in (normalized to the amount of SpyCatcher-HBsAg in group 2) 4) AddaVax TM 0.1 μg HCMV pentamer in 5) AddaVax TM 0.1 μg HCMV pentamer-SpyTag--SpyCatcher-HBsAg VLP (equivalent to 0.1 μg of pentamer) 6) TBS (20 mM Tris and 150 mM NaCl, pH 7.4) AddaVax TM is a squalene-based oil-in-water nanoemulsion that contains a formulation similar to MF59®, 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 assessed using ELISA. A standardized ELISA against HCMV pentamers was used to determine the titers of antisera raised in each group. Plates were coated overnight with 5 μg / ml pentamers (without SpyTag), 50 μL / well; washed; blocked with milk for 1 hour; washed; applied mouse serum (approximate dilutions in PBS) for 1 hour; washed; applied goat anti-mouse-alkaline phosphatase antibody (1:10,000) for 1 hour; washed; developed.

[0162] Different doses of both unconjugated (groups 1 and 4) and conjugated HCMV pentamer-HBsAg (groups 2 and 5) were included to allow for a comparison of immunogenicity between the conjugated HCMV pentamer-HBsAg VLP vaccine and the unconjugated HCMV pentamer-SpyTag, which allows for extrapolation to other HCMV pentamer vaccines (e.g., soluble pentamers). Groups 3 and 6 represent negative controls.

[0163] At each time point, the OD values ​​of the samples were read at the appropriate dilution and the ELISA units were determined using the standard curve generated on each plate. Data showing the post-prime results for groups 1, 2, 4 and 5 are shown in Figure 6. HCMV pentamer-HBsAg immunized mice show substantially stronger serum IgG antibody responses with both the 1 μg and 0.1 μg doses compared to mice immunized with 1 μg or 0.1 μg doses of unconjugated HCMV pentamer. As also 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 examined using a microneutralization assay based on Wang et al. (Vaccine 33(2015) 7254-7261; DOI: 10.1016 / j.vaccine.2015.10.110). Neutralization titers for groups 1, 2, 4 and 5 are shown in Figure 7. Sera from mice immunized with pentamer-HBsAg VLPs are substantially more neutralizing than those from mice immunized with pentamer-SpyTag protein alone.

[0165] Example 3 Stable construct sequences Two stable constructs (adopted from Hofmann et al., (2015) Biotech and Bioeng, 112(12):2505-2515) were optimized for CHO expression of the HCMV pentamer-SpyTag construct. The intron was 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 contain the gH-SpyTag-His (SEQ ID NO:42) and gL (SEQ ID NO:43) components upstream and downstream of the EV71 IRES, respectively. The coding sequences used in this construct are listed below.

[0167] Nucleotide sequence gH-(GSG)2-SpyTag-His (without intron) inserted upstream of the EV71 IRES (SEQ ID NO: 42) In this sequence: signal peptide (nt 1-69), ectodomain (nt 70-2151), truncated transmembrane domain (nt 2152-2157), (GSG)2 linker (nt 2158-2175), SpyTag (nt 2176-2214), His-tag (nt 2215-2232), stop codon (nt 2233-2235).

[0168] gL (without intron) inserted downstream of EV71 IRES (SEQ ID NO: 43) In this sequence: signal peptide (nt 1-90), ectodomain (nt 91-834), stop codon (nt 835-837). HCMV UL128 / UL130 / UL131A stable expression constructs 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 of 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 (without intron) (SEQ ID NO: 44) In this sequence: signal peptide (nt 1-81), ectodomain (nt 82-51 3), the stop codon (nt 514–516).

[0170] UL130-(G4S)3-C-tag (without intron) (SEQ ID NO: 45) In this sequence: signal peptide (nt 1-75), ectodomain (nt 76-642), (G4S)3 linker (nt 643-687), C-tag (nt 688-699), stop codon (nt 700-702).

[0171] UL131A (without intron) (SEQ ID NO: 46) In this sequence: signal peptide (nt 1-54), ectodomain (nt 55-387), stop codon (nt 388-390).

[0172] Example 4 In vivo testing of HCMV-SpyTag--SpyCatcher-HBsAg (non-adjuvanted) Conjugated HCMV pentamer-HBsAg VLPs, as well as unconjugated HCMV pentamer-SpyTag, were used in an immunization schedule with BALB / c mice to further investigate the immunogenicity of conjugated pentamer-HBsAg VLPs versus unconjugated pentamer-SpyTag protein.

[0173] A prime-boost-boost schedule at 3 week intervals was used as follows: Day 0: immunization (prime); day 20: tail bleed; day 21: immunization (boost 1); day 41: tail bleed; day 42: immunization (boost 2); day 63: cardiac bleed.

[0174] The immunization groups were as follows: For each group, n=10: 1) 1 μg non-adjuvanted HCMV pentamer-SpyTag 2) 1 μg unadjuvanted HCMV pentamer-SpyTag--SpyCatcher-HBsAg VLP (equivalent to 1 μg of pentamer) 3) 0.1 μg unadjuvanted HCMV pentamer-SpyTag--SpyCatcher-HBsAg VLP (equivalent to 0.1 μg of pentamer) Immunogenicity was assessed using ELISA. A standardized ELISA against HCMV pentamers was used to determine the titers of antisera raised in each group. Plates were coated overnight with 5 μg / ml pentamers (without SpyTag), 50 μL / well; washed; blocked with milk for 1 hour; washed; applied mouse serum (approximate dilutions in PBS) for 1 hour; washed; applied goat anti-mouse-alkaline phosphatase antibody (1:10,000) for 1 hour; washed; developed.

[0175] At each time point, the OD values ​​of the samples were read at the appropriate dilution and the ELISA units were determined using the standard curve generated on each plate. The post-prime and post-boost data are shown in Figure 8. HCMV pentamer-HBsAg immunized mice show substantially stronger serum IgG antibody responses using both the 1 μg and 0.1 μg doses compared to mice immunized with only 1 μg of HCMV pentamer as 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 prime and boost are shown in Figure 9. Sera from mice immunized with non-adjuvanted pentamer-HBsAg VLPs are substantially more neutralizing than those from mice immunized with non-adjuvanted pentamer-SpyTag protein alone.

[0177] Example 5 Expression and purification of RSV-F-SpyTag The sequence from antigen RSV-F Sc9-10 DS-Cav1 A149C Y458C was fused to SpyTag to generate RSV-F-SpyTag and then used in ExpiCHO. TM Expression System Kit and ExpiFectamine TM The nucleotide sequence of SEQ ID NO: 47 in the plasmid pcDNA3.4 was transfected using the ExpiCHO transfection reagent (ThermoFisher Scientific). TM Expression was achieved by transiently transfecting cells.

[0178] RSV-F Sc9-10 DS-Cav1 A149C Y458C (National Institutes of Health) was developed based on the results of Joyce et al. (2016) (Iterative structure-based improvement of a respiratory syncytial virus fusion glycoprotein vaccine. Nat Struct Mol Biol. 2016 Sep; 23(9): 811-820) is a mutant of the respiratory syncytial virus fusion protein (pre-fusion RSV-F). This mutant is a pre-fusion stabilized form of the fusion (F) glycoprotein that contains a genetically linked F subunit, the fusion peptide is deleted, it contains a T4 fibritin trimerization motif (foldon domain), and inter-promoter movement is stabilized by an additional inter-promoter disulfide bond (A149C Y458C).

[0179] Nucleotide sequence RSV-F-SpyTag-C tag nucleotide sequence (SEQ ID NO: 47) The original sequence of Sc9-10 DS-Cav1 A149C Y458C was modified at 3' by deletion of the thrombin site, 6xHis-tag and Strep-tag®II. These deleted domains were replaced with a linker-SpyTag-C-tag sequence to generate a 1587 nt cassette (SEQ ID NO: 47) containing Sc9-10 DS-Cav1 A149C Y458C (nt 1-1515, including signal peptide (nt 1-75) and T4 fibritin 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). The Sc9-10 DS-Cav1 A149C Y458C nucleotide sequence, excluding the linker, SpyTag, C-tag and stop codon, is included in SEQ ID NO: 48. The Sc9-10 DS-Cav1 A149C Y458C nucleotide acid sequence, excluding the signal peptide, linker, SpyTag or C-tag, is included in SEQ ID NO: 49.

[0180] Amino acid sequence The expression of the nucleotide sequence, SEQ ID NO: 47, is determined by the following domains: Sc9-10 DS-Cav1 A149C Y458C ((aa 1-505, including the signal peptide (aa 1-25) and the foldon domain (aa 479-505)), linker (aa The amino acid sequence of Sc9-10 DS-Cav1 A149C Y458C, excluding the linker, SpyTag or C-tag, is included in SEQ ID NO:51. The amino acid sequence of Sc9-10 DS-Cav1 A149C Y458C, excluding 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 the cells and purified from the supernatant using C-tag affinity purification and size exclusion chromatography. The pyTag appeared as expected under nonreducing and reducing conditions when analyzed by SDS-PAGE (Fig. 10A ) and reacted with anti-RSV-F[2F7] monoclonal antibody (ab43812; Abcam) (Fig. 10B ).

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

[0183] Conjugation of RSV-F-SpyTag to SpyCatcher-HBsAg RSV-F-SpyTag was conjugated to SpyCatcher-HBsAg overnight at 4° C. to generate HBsAg VLPs coated with RSV-F trimers (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 (FIG. 11). Comparing the conjugation reaction to either RSV-F-SpyTag or SpyCatcher-HBsAg alone, there was the presence of a novel band of ∼105 kDa (lane 2) under reducing conditions (Figure 11A), which was reactive with both an anti-HBsAg monoclonal antibody (MCA4658, Bio-Rad) (Figure 11B) and an anti-RSV-F[2F7] monoclonal antibody (ab43812; Abcam) (Figure 11C), indicating that the band contained conjugated RSV-F-SpyTag--SpyCatcher-HBsAg.

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

[0185] Mice immunized with post-prime RSV-F-SpyTag--SpyCatcher-HBsAg showed no significant difference in efficacy when the vaccine was unadjuvanted (Figure 6) or when the vaccine was combined with Addavax TM In comparison to mice immunized with the RSV-F-SpyTag protein alone, mice immunized with either the RSV-F-SpyTag or RSV-F-SpyTag protein alone (FIG. 6) showed substantially stronger serum IgG antibody responses.

[0186] Sequence Listing

[0187] [Table 4]

Claims

1. 1. A composition comprising a virus-like particle displaying an antigenic component: i) an antigenic component comprising a first peptide tag, and ii) a portion comprising a second peptide tag Including, the antigenic entity and the moiety are linked through an isopeptide bond between said first and second peptide tags, and the antigenic entity is greater than 50 kDa and the moiety is a hepatitis B virus surface antigen (HBsAg); The composition.

2. 2. The composition of 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. 3. The composition of claim 1 or claim 2, wherein the antigenic component is a monomer or a multimer, and the multimer may be a trimer, tetramer, pentamer, hexamer, heptamer, octamer, nonamer or decamer.

4. 4. The composition of any of claims 1 to 3, wherein the first and second peptide tags are selected from a SpyTag and SpyCatcher pair; a SnoopTag or SnoopTagJr and SnoopCatcher pair; a RrgATag, RrgATag2 or DogTag and RrgACatcher pair; an Isopep tag Pilin-C pair; an IsopepTag-N and Pilin-N pair; a PsCsTag and PsCsCatcher pair; and a SnoopTagJr and DogTag pair mediated by SnoopLigase; or any one of their variants, derivatives or modifications, optionally wherein the first and second peptide tags are a SpyTag / SpyCatcher pair.

5. 5. The composition of any one of claims 1 to 4, wherein the antigenic entity comprises any one of the immunogenic entities of the pentameric complex from human cytomegalovirus (HCMV), gB glycoprotein from human cytomegalovirus (HCMV), G glycoprotein from RSV, F glycoprotein from RSV, hemagglutinin (HA) antigen from influenza A virus, neuraminidase (NA) antigen from influenza A virus, Plasmodium falciparum Pfs230 protein, Plasmodium falciparum CSP proteins, 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.

6. The composition of any one of claims 1 to 5, wherein the antigenic entity comprises an immunogenic entity of an HCMV pentamer.

7. 7. The composition of claim 6, wherein the immunogenic components of the HCMV pentamer comprise one or more of the gH, gL, pUL128, pUL130 or pUL131 subunits.

8. 8. The composition of claim 7, wherein the immunogenic components of the HCMV pentamer include all of the gH, gL, pUL128, pUL130 and pUL131 subunits.

9. The composition of any one of claims 6 to 8, wherein the immunogenic component of the HCMV pentamer comprises a gH subunit containing a truncated transmembrane domain.

10. The composition of any one of claims 8 to 10, wherein the gH, gL, pUL128, pUL130 and pUL131 subunits have the amino acid sequences set forth in SEQ ID NOs: 28, 31, 35, 33 and 36, respectively, or functional equivalents thereof.

11. The composition of any of claims 7 to 10, wherein the first peptide tag is attached to the gH subunit, preferably to the C-terminus of the gH subunit.

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

13. The composition of claim 12, wherein the immunogenic component of the RSV-F protein is a pre-fusion F protein, and optionally a stabilized pre-fusion F protein.

14. The immunogenic component of the RSV pre-fusion F protein is F １ and F ２ The composition of claim 13 comprising a trimer of subunits.

15. The composition according to any one of claims 12 to 14, wherein the RSV-F protein has an amino acid sequence as set forth in any one of SEQ ID NOs: 50 to 52 or 56 to 58.

16. The composition of any one of claims 13 to 15, wherein the first peptide tag is attached to the C-terminus of the pre-fusion F protein.

17. The composition of any one of claims 1 to 16, wherein the first peptide tag is a SpyTag.

18. The composition of claim 17, wherein the SpyTag has the amino acid sequence set forth in SEQ ID NO:

30.

19. 19. The composition of claim 17 or 18, wherein the SpyTag is attached through a linker.

20. The composition of claim 19, wherein the linker has the amino acid sequence shown in SEQ ID NO:

29.

21. An antigenic component comprising a first peptide tag, (i) comprising the amino acid sequence set forth in any of SEQ ID NOs: 27, 50, or 56; or (ii) encoded by a nucleotide sequence set forth in any one of SEQ ID NOs: 12, 42, 47, or 53; The composition of claim 1.

22. A nucleic acid molecule encoding the amino acid sequence set forth in any of SEQ ID NOs: 27, 50 or 56.

23. 23. The nucleic acid molecule of claim 22, comprising a sequence as set forth in any of SEQ ID NOs: 12, 42, 47 or 53.

24. The composition of any one of claims 1 to 21, wherein the second peptide tag is SpyCatcher.

25. 25. The composition of claim 24, wherein SpyCatcher has the amino acid sequence set forth in SEQ ID NO:

38.

26. 26. The composition of claim 24 or 25, wherein the moiety is attached to the SpyCatcher through a linker, preferably a flexible linker.

27. The composition of claim 26, wherein the linker has the amino acid sequence shown in SEQ ID NO:

39.

28. The portion comprising the second peptide tag is (i) comprising the amino acid sequence set forth in SEQ ID NO: 37; or (ii) encoded by the nucleotide sequence set forth in SEQ ID NO:22; The composition of claim 1.

29. A nucleic acid molecule encoding the amino acid sequence shown in SEQ ID NO:

37.

30. 30. The nucleic acid molecule of claim 29 comprising the sequence set forth in SEQ ID NO:

22.

31. The composition of any one of claims 1 to 21 or claims 24 to 28, which is an immunogenic or vaccine composition.

32. A vaccine comprising a composition according to any one of claims 1 to 21 or claims 24 to 28 for use in the prevention and / or treatment of a disease.

33. A method for producing a composition according to any one of claims 1 to 21 or claims 24 to 28, or for producing a vaccine according to claim 32, comprising: - introducing into a first host cell a first nucleic acid encoding a first genetic fusion of a first protein to a first peptide tag; - incubating said first host cell under conditions for expressing said first gene fusion; - introducing into a second host cell a second nucleic acid encoding a second genetic fusion of a second protein to a second peptide tag; - incubating said second host cell under conditions for expressing said second gene fusion; - optionally purifying the expressed components; - incubating the expressed construct under conditions for the formation of an isopeptide bond between the first peptide tag and the second peptide tag; and optionally purifying the resulting composition. The method comprises the steps of: The method, wherein the first protein comprises the antigenic component and the second protein comprises the moiety.

34. A vector comprising a nucleic acid molecule according to any one of claims 22, 23, 29 or 30.

35. A host cell comprising a nucleic acid molecule according to any one of claims 22, 23, 29 or 30, or a vector according to claim 34.

36. 32. A kit comprising a composition comprising a first immunogenic composition and optionally one or more booster compositions comprising a second immunogenic composition, wherein said first and / or second immunogenic composition comprises a composition according to any of claims 1 to 21 or claims 24 to 28 or a vaccine according to claim 32.

37. A vaccine for use in the prevention and / or treatment of HCMV infection comprising a composition according to any one of claims 6 to 11.

38. A vaccine for use in the prevention and / or treatment of RSV infection comprising a composition according to any one of claims 12 to 16.

39. 39. A pharmaceutical composition comprising the vaccine of claim 32, 37 or 38, and a pharma- ceutically acceptable buffer, excipient, carrier, adjuvant, or combination thereof.

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