Vaccine composition

Abstract

The present invention relates to vaccine compositions, most particularly vaccine compositions in which the antigen component is large (e.g., more than 50 kDa), or multimeric (i.e., consisting of subunits). Such antigen components are particularly interesting in that they may represent antigen components from pathogens against which they cannot be vaccinated currently. The present invention relates to a composition comprising particles displaying an antigen component wherein the composition comprises an antigen component comprising a first peptide tag and a moiety comprising a second peptide tag wherein the antigen component and the moiety are linked by an isopeptide bond between the first and second peptide tags, and wherein the antigen component exceeds 50 kDa, or optionally a multimer.

Description

[0001] This application is a divisional application of an application filed on May 3, 2019, with application number 201980044956.2 and invention name “Vaccine Composition”. Background of the Invention

[0003] Vaccines are a safe and effective way to combat and eradicate infectious diseases. Vaccine development has been highly successful, but a range of disease challenges remain for which vaccines do not currently exist, including many important pathogens that represent daunting immune barriers. It is generally believed that an effective vaccine must be transported to the lymph nodes and persist long enough to generate an immune response.

[0004] Vaccine development has shifted from using attenuated or dead pathogens to using smaller antigenic components of these pathogens in order to generate the desired protective immune response while avoiding the inherent risks of using such attenuated strains. Efforts have focused on attempting to express the immunogenic portions of pathogen components (such as those required for pathogen infection of cells), which, for simplicity, are limited to short / small peptides and proteins due to technical issues in expressing large or multi-component antigens. However, a potential problem with using very short or small peptides is the risk of antigenically variable pathogens that escape the immune response induced by vaccination through changes in specific portions of the antigen.

[0005] The expression of large / multi-component antigens will bring immunological advantages, including the ability to allow the production of antibodies against multiple neutralizing epitopes of a pathogen. However, the expression of one or more large antigens that can form a complex (in a manner where the relevant antigenic epitopes are maintained and presented to produce an effective antibody response) remains a huge challenge. Therefore, there is still a demand for improved methods of expressing large antigens and / or multi-component antigens so that they can cause a clinically significant immune response.

[0006] Previous recombinant vaccines have been designed to elicit immune responses against multiple antigenic components, which rely on each component being expressed and packaged separately into different particles, such as in the case of the anti-HPV vaccines Cervarix and Gardasil, in which recombinant major capsid L1 proteins of specific HPV strains are expressed and assembled into virus-like particles (VLPs) separately, and then different types of VLPs are combined into vaccine formulations. Alternatively, multiple short epitopes are selected and combined into a single recombinant vaccine (e.g., the multimer-001 influenza vaccine), but these epitopes are essentially short linear peptides that were selected to avoid preparation complexities involving three-dimensional structure or refolding, and therefore do not attempt to represent the natural pathogen presented to the immune system in an active infection.

[0007] For example, beta-herpes human cytomegalovirus (HCMV, also known as human herpesvirus-5 (HHV-5)) is the leading viral cause of neonatal developmental disorders. This ubiquitous virus has infected more than 60% of the general population, and the initial infection is usually only mild or asymptomatic. After infection, the virus remains latent in the body, but can cause serious disease in immunocompromised people (i.e., HIV patients, transplant patients, and people undergoing chemotherapy) or the elderly. HCMV is the leading infectious cause of birth defects in developed countries. Up to 4 / 200 infants are born with HCMV due to congenital infection, and up to 10% of these infants will suffer long-term consequences. HCMV infection is also associated with adult hypertension and atherosclerosis (Cheng et al. (May 2009). Früh K, ed. "Cytomegalovirus infection causes an increase of arterial blood pressure". PLoS Pathog. 5 (5): e1000427). Therefore, HCMV is a public health priority. However, despite extensive efforts, no successful HCMV vaccine has been developed to date.

[0008] Respiratory syncytial virus (RSV) is another ubiquitous virus that causes minimal discomfort in healthy adults and older children who are infected. However, it is the second leading cause of death in infants under one year of age worldwide, second only to malaria. It is estimated that this virus causes 160,000 deaths worldwide each year. This virus causes serious respiratory infections and complications, including pneumonia and bronchiolitis. High-risk groups include infants under one year of age and immunocompromised patients, the elderly, and those with cardiopulmonary conditions. Again, despite years of active research and development, there is currently no licensed RSV vaccine.

[0009] For diseases such as those caused by RSV and HCMV, there are currently no vaccines available and often current vaccine production methods do not show the desired efficacy, indicating a large unmet need in providing alternative types of vaccines to combat diseases with such devastating consequences.

[0010] Several genetically encoded systems capable of spontaneous or assisted formation of amide bonds have recently been described. For example, SpyTag is a peptide that has been engineered so that when mixed with its protein partner, SpyCatcher, the two components spontaneously and irreversibly form an isopeptide bond. The positions of the SpyTag and SpyCatcher components in the protein chain can be designed at different locations and are reactive under a wide range of pH, buffer and temperature conditions. The SpyTag / SpyCatcher pair and its variants and derivatives have been used in vaccine development, but so far only for the presentation of simple antigens. Other genetically encoded systems capable of 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.

[0011] The present inventors have shown that the use of a genetically encoded system capable of forming amide bonds enables the use of large / multicomponent antigens in vaccine compositions, which can improve responses to large / multicomponent antigens. This is a surprising result. SUMMARY OF THE INVENTION

[0013] In a first aspect of the present invention, a composition is provided, comprising particles displaying a protein component, wherein the composition comprises:

[0014] i) a protein component comprising a first peptide tag, and

[0015] ii) a portion comprising a second peptide tag,

[0016] wherein the protein component and the portion are linked via an isopeptide bond between the first peptide tag and the second peptide tag, and wherein the protein component exceeds 50 kDa.

[0017] In another aspect of the present invention, a composition is provided, comprising particles displaying a protein component, wherein the composition comprises:

[0018] i) a protein component comprising a first peptide tag, and

[0019] ii) a portion comprising a second peptide tag,

[0020] wherein the protein component and the moiety are linked via an isopeptide bond between the first peptide tag and the second peptide tag, and wherein the protein component is a multimer.

[0021] The protein component may have any function, for example it may be an enzyme or have the properties of an enzyme. The protein component may be a full-length protein, or it may be a portion, segment, domain or truncation of a full-length protein. The protein component may be an antigen or an immunogen. The protein component may also be referred to as an antigenic component.

[0022] In another aspect of the present invention, a composition is provided, comprising particles displaying an antigen component, wherein the composition comprises:

[0023] i) an antigenic component comprising a first peptide tag, and

[0024] ii) a portion comprising a second peptide tag,

[0025] wherein the antigenic component and the portion are linked by an isopeptide bond between the first peptide tag and the second peptide tag, and wherein the antigenic component exceeds about 50 kDa.

[0026] In some embodiments of any aspect of the invention, the protein component or antigenic component may be 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 or greater, such as greater than 200 kDa, greater than 300 kDa, or greater than 400 kDa.

[0027] The polymer can comprise any number of subunits, which may be covalently linked or not covalently linked in the protein component or antigen component. The polymer can comprise 2-20 subunits, optionally 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more subunits. Alternatively, the polymer can be a dimer, trimer, tetramer, pentamer, hexamer, heptamer, octamer, nonamer or decamer. The polymer can be from any suitable pathogen, but is preferably a viral polymer.

[0028] Non-limiting examples of large protein components, i.e., larger than 50 kDa or as described above, include the pentameric complex (PC) and gB glycoprotein from human cytomegalovirus (HCMV), the G glycoprotein and F glycoprotein from RSV, the hemagglutinin (HA) antigen and neuraminidase (NA) antigen from influenza A virus, Plasmodium falciparum Pfs230, Plasmodium falciparum CSP, human HER2 receptor, PCSK9, VAR2CSA, Plasmodium falciparum RIPR, varicella zoster virus (VZV) glycoprotein E, rabies virus glycoprotein, and Epstein-Barr virus (EBV) gH / gL complex.

[0029] In some embodiments of any aspect of the invention, the protein component or antigenic component may be a monomer or a polymer, such as a dimer, trimer, tetramer or pentamer. In some embodiments of any aspect of the invention, the protein component or antigenic component may be a protein or peptide complex.

[0030] The limiting examples of polymer antigen components include pentamer complex (PC) and gB trimer from human cytomegalovirus (HCMV), G glycoprotein and F glycoprotein from RSV, hemagglutinin (HA) antigen and neuraminidase (NA) antigen from influenza A virus, some of which are described in this article. Other examples include components of pathogens such as viruses, bacteria, fungal pathogens, parasites or other disease vectors. Suitable polymer antigen components include, for example, derive from polymer antigen components of viruses such as influenza virus (such as influenza hemagglutinin (HA) (for example influenza trimer)), respiratory syncytial virus (RSV) etc.

[0031] The protein component can be attached to the first peptide tag by genetic fusion and recombinantly expressed in a suitable cell. For components that include post-translational modifications such as glycosylation, expression of the recombinant protein in eukaryotic or mammalian cell lines may be preferred.

[0032] In one embodiment, a "part" is a component on which a protein component or an antigen component can be displayed, for example, so that it can be used for the immune system. In one embodiment, the part is polymerized to form the particle. Suitably, such a part can be a protein component of a virus, a bacterium, a polymerized scaffold or a polymerized VLP (virus-like particle) for vaccination. Suitably, the part can be a component of a bacteriophage, a tobacco mosaic virus particle, an adeno-associated virus-like particle (AAVLP), an Escherichia coli (E. coli), etc. In one embodiment, the part itself is a component of a virus, a bacterium, etc., so that the polymerization (e.g., self-assembly) of the part forms a particle for displaying a protein component or an antigen component. In one embodiment, the part can be a viral structural protein, such as 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 phage AP205 virus coat protein (CP3), hemagglutinin-neuraminidase from a variety of viruses (including mumps), etc. Suitable viral structural proteins will be known to those skilled in the art. In other embodiments, the part can be a protein or peptide, such as a multimerization domain such as IMX313 forming a nanoparticle, or a particle such as MI3 in the computational source. In other embodiments, the part can be a synthetic nanoparticle or a synthetic VLP, such as gold, lipopeptides or poly (lactic acid-co-glycolic acid) (PLGA) nanoparticle. Other suitable parts can include liposomes or outer membrane vesicles. Suitably, the part comprising the second peptide tag is the part to which the second peptide tag is attached.

[0033] The use of the structural surface antigen from virus can be preferred.Therefore, the second peptide tag is attached to the structural surface antigen, allowing the formation of virus-like particles (VLP), the second peptide tag is attached to the virus-like particles, or the second peptide tag is displayed on the virus-like particles.VLP is a non-infectious self-assembling nanoparticle, and its repeated molecular definition structure is attractive to engineering multivalent particularly vaccination.VLP has been produced from the components of many virus families, and the virus family includes hepatitis B virus (including hepatitis B small surface antigen (HBsAg)), Parvoviridae (Parvoviridae) (for example adeno-associated virus), Retroviridae (Retroviridae) (for example HIV), Flaviviridae (Flaviviridae) (for example hepatitis C virus) and bacteriophage (for example Q β, AP205).Any of these can be suitable for use as the part in the present invention.

[0034] The second peptide tag can be attached to the part by genetic fusion. This genetic fusion can be at any suitable point in the sequence, not just limited to the end. Those skilled in the art will understand that the fusion protein can be recombinantly expressed in a suitable cell.

[0035] Alternatively, the second peptide tag may be displayed on or attached to the moiety by means of chemical conjugation. This will require, for example, the presence of a reactive amine group to allow conjugation to occur.

[0036] Accordingly, in one embodiment, the part is the surface antigen of the hepatitis B virus (HBsAg). Suitably, as described herein, the HBsAg has the amino acid sequence set out in SEQ ID NO: 41 (or a functional equivalent thereof).

[0037] In one embodiment of any aspect of the present invention, the protein component or antigenic component is an immunogenic component of HCMV pentamer. Suitably, the antigenic component or immunogenic component is a component that can produce an immune response such as an antibody response to the component when introduced into a subject such as a patient. Accordingly, for example, "the immunogenic component of HCMV pentamer" is a component that can produce an anti-HCMV antibody response in a subject. Suitably, the immunogenic component comprises one or more (at least one) HCMV pentamer subunit components selected from gH, gL, pUL128, pUL130 and pUL131 (also referred to as pUL131A). In some embodiments, the immunogenic component comprises one or more of those "pUL" components or "UL" components. In other embodiments, the immunogenic component comprises one or more of those gH components or gL components. In one embodiment, the immunogenic component comprises a combination of one or more "UL" components and one or more components selected from gH components or gL components. In another embodiment of the present invention, the immunogenic component of the HCMV pentamer is a HCMV pentamer comprising all gH / gL / pUL128 / pUL130 / pUL131 subunits. Suitably, the gH / gL / pUL128 / pUL130 / pUL131 subunits have amino acid sequences corresponding to those derived from any known HCMV strains (including laboratory strains and / or clinical isolates), including Towne (GI: 239909366), AD169 (GI: 219879600), Toledo (GI: 290564358) and Merlin (GI: 155573956) or their functional equivalents. Functional equivalents mean that there is some homology in common and only differs in some amino acids, but retains an amino acid sequence that is capable of forming an antigenic subunit or pentamer that provides protective antibodies. Suitable variants of the components gH / gL / pUL128 / pUL130 / pUL131A are described, for example, in WO2014 / 005959 (see pages 4 to 10), which is hereby incorporated by reference. Advantageously, the use of HCMV pentamer subunits in vaccine approaches can provide immunogenic protection against infection from a wide range of HCMV strains due to the high homology between strains at the pentamer amino acid sequence level.

[0038] In some embodiments, the antigenic component may correspond to a component or a portion thereof of a pathogen or a vector. For example, for ease of preparation, the antigenic component may lack a transmembrane domain. Suitably, in the HCMV pentamer, for example, the immunogenic component of the HCMV pentamer comprises a gH subunit with a truncated transmembrane domain (truncated by deleting one or more amino acids from the region), so that the subunit is secreted into the cell supernatant during protein production in the host cell for purification.

[0039] In one embodiment, the gH / gL / pUL128 / pUL130 / pUL131A subunits have the amino acid sequences listed in SEQ ID NOs: 28, 31, 35, 33, 36, respectively (or functional equivalents thereof) (with or without the indicated signal peptides). Functional equivalents mean amino acid sequences that share some homology and differ only in some amino acids, but retain functional properties, such as the ability to form antigenic subunits or pentamers that provide protective antibodies. In some embodiments, functional equivalents may share 70%, 80%, 90% or more homology with the relevant amino acid sequences. In another embodiment, the gH / gL / pUL128 / pUL130 / pUL131A subunits are encoded by nucleic acid sequences such as those listed in SEQ ID 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 the signal peptide, or a signal peptide derived from different HCMV strains. In some embodiments, any one of the gH / gL / pUL128 / pUL130 / pUL131A subunits can have a signal peptide derived from a heterologous protein. The selection of the signal peptide can be determined so that the expressed protein is targeted to a specific cell (or extracellular) position, or other functions are conferred. After the subunit is expressed, the signal peptide can be by the natural cell mechanism or in vitro enzymatic cleavage (for example, by a signal peptidase) in the expression system used. In some embodiments, any one of the gH / gL / pUL128 / pUL130 / pUL131A subunits can be expressed without a signal peptide. In some embodiments, native sequences can be used, including introns, wherein these sequences can result in higher expression levels. Suitably, the native nucleic acid sequence of UL128 comprises 2 introns. In another embodiment, the nucleic acid sequence of UL131A comprises an intron. In some embodiments, the intron can be removed. In some embodiments, the native sequence can be codon optimized for relevant expression systems.

[0040] In one embodiment of any aspect of the invention, the protein component or antigenic component is an immunogenic component of the RSV virus, such as an attachment glycoprotein (G protein) or a fusion glycoprotein (F protein), both of which control the initial stage of infection. G is a highly glycosylated 90 kDa type II integral membrane protein and can mediate viral attachment to host cell membranes by interacting with heparan sulfate on proteoglycans and is a good candidate for a protein component.

[0041] F protein is an integral membrane protein composed of three 0 It is composed of monomers and is processed into F during the assembly process. 1 and F 2 subunits, which are covalently linked by two disulfide bonds. The F protein is highly conserved among RSV isolates of subgroups A and B, and the amino acid sequence shows 90% or more identity. F is a 574 amino acid class I fusion protein consisting of a 50 kilodalton (kDa) carboxyl-terminal F1 fragment and a 20 kDa amino-terminal F2 fragment; making it a trimer of heterodimers. It is characterized by two furin protease cleavage sites, which release a 27 amino acid glycopeptide and expose a hydrophobic fusion peptide at the F1 amino terminus. 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 F ectodomain consists of 472 amino acids. Only 25 amino acids differ in the F ectodomain between subtypes A and B.

[0042] In order to develop antigen compositions from RSV-F protein, some studies have focused on variants of pre-fusion proteins prepared as trimers. Variant is produced by genetically fusing two subunits of mature pre-F into a single chain. DS-Cav1 variants with genetic fusion of F2 and F1 and fusion peptide and pep27 region deletion have been prepared. The difference of joints between F2 and F1 subunits shows as affecting immunogenicity, and therefore variants can use the selection of different joints. Natural RSV-F protein sequence can be found in accession number P03420.1.

[0043] Several forms of pre-fusion F protein have been studied and developed and subsequently published. These pre-fusion trimers can all be suitable for the present invention. The fusion glycoprotein stabilized by DS-Cav1 is derived from a natural protein. EP2222710, incorporated herein by reference, also discloses a recombinant RSV antigen comprising a soluble F protein polypeptide comprising an F2 domain and an F1 domain and a trimerization domain of an RSV-F protein polypeptide. In Nat. Commun. 2015; 6: 8143, Krarup et al., a highly stable pre-fusion RSV-F protein is described, again incorporated by reference.

[0044] WO2014 / 160463, incorporated herein by reference, describes isolated recombinant RSV-F proteins stabilized in a prefusion conformation and nucleic acid molecules encoding recombinant RSV-F proteins.

[0045] WO2017 / 172890, incorporated herein by reference, describes substitution-modified pre-fusion RSV-F proteins and encoding nucleic acids thereof. Further description is given in Nat Struct Mol Biol. 2016 Sep; 23(9): 811-820, Iterative structure-based improvement of a respiratory syncytial virus fusion glycoprotein vaccine, M. Gordon Joyce, Baoshan Zhang, Li Ou, Man Chen, Gwo-Yu Chuang, Aliaksandr Druz, Wing-Pui Kong, Yen-Ting Lai, Emily J. Rundlet, Yaroslav Tsybovsky, Yongping Yang, Ivelin S. Georgiev, Miklos Guttman, Christopher R. Lees, Marie Pancera, Mallika Sastry, Cinque Soto, Guillaume BEStewart-Jones, Paul V. Thomas, Joseph G. Van Galen, Ulrich Baxa, Kelly K. Lee, John R. Mascola, Barney S. Graham and Peter D. Kwong, which is also incorporated herein by reference.

[0046] Encoding a recombinant F linked to the T4 Fibritin trimerization domain 2 -F 1 Exemplary nucleic acid sequences of ectodomain protomers are available under the following accession numbers: LP884611.1, LP884610.1, LP884609.1 and LP884608.1.

[0047] In some embodiments, the protein component or antigenic component may correspond to a component or a portion thereof of a pathogen or vector. For example, for ease of preparation, the antigenic component may lack a transmembrane domain. Suitably, in the RSV-F protein or its pre-fusion conformation, for example, the immunogenic component of the F protein comprises an F having a truncated transmembrane domain (truncated by deleting one or more amino acids from the region). 2 -F 1The RSV-F protein lacks a functional TM domain. Alternatively, genetic fusion with the first peptide tag can indeed prevent the F protein from residing in the membrane despite the presence of a functional transmembrane domain.

[0048] In one embodiment, the pre-fusion stabilized subunits have the amino acid sequences listed in SEQ ID NO: 50-58 (or their functional equivalents). Functional equivalents mean that there is some homology in common and only differ in some amino acids, but retain functional properties such as amino acid sequences that can form antigenic subunits that provide protective antibodies. In some embodiments, functional equivalents may have 70%, 80%, 90% or more homology with the relevant amino acid sequences. In one embodiment, the pre-fusion stabilized RSV-F trimer may not include a heterologous trimerization domain.

[0049] The protein component comprises a first peptide tag. The first peptide tag can be attached to the protein component by expressing a recombinant fusion protein. Those skilled in the art will know the techniques for genetically expressing fusion peptide sequences so as to express recombinant proteins in suitable cell systems. For parts including post-translational modifications such as glycosylation, it is preferred to express the recombinant protein in a eukaryotic or mammalian cell line.

[0050] Advantageously, using the first peptide tag and the second peptide tag that form isopeptide bonds, such as the SpyTag-SpyCatcher system described herein, allows large antigens and / or multimeric antigens such as HCMV pentamers or their immunogenic components to be "decorated" to the part showing the large antigens, such as VLPs, with the correct formation and direction, so that the antigen is presented to the immune system in a manner that can produce anti-antigen (e.g., anti-HCMV) antibodies, which can provide protection / neutralization / immunogenic effects. Traditional vaccination methods using soluble antigens (even large antigens, such as polymers / pentamers) may be less effective in producing protection / neutralization / immunogenic effects. Advantageously, antigens (e.g., multimeric antigens) are displayed on particles such as VLPs or nanoparticles, resulting in the presentation of geometric repeat arrays of the same antigen, which can trigger immune responses robustly compared to soluble antigens. Compared to "free" antigens, larger-sized VLPs or other suitable particles can also have larger immunogenic effects. In addition, the display direction of multimeric antigens such as HCMV pentamers may be very important to immunogenicity. The use of paired tags, such as the SpyTag-SpyCatcher system described herein, to attach multimeric antigens to particles allows the antigens to be attached to the particles in a particularly favorable orientation. For example, in the case of HCMV, the gH / gL subunits are less likely to have neutralizing epitopes than the "UL" subunits. Thus, advantageously, the present invention allows the orientation of the display of the HCMV pentamer on the particle to be determined by appropriate positioning of the first peptide tag, such that, for example, the "UL" subunit is displayed toward the outside of the particle and is therefore more easily accessible to the individual's immune system. Alternatively / additionally, the positioning of the first peptide tag on the antigen can be determined so as to produce an antigen orientation similar to that on the natural virus, thereby presenting to the immune system particles that display the antigen in an orientation that is more likely to induce an immune response to an invading live virus.

[0051] In contrast, the traditional method of presenting proteins on VLPs can involve chemical ligation, the disadvantage of which is that such chemical reactions may be more random, making it impossible to obtain the correct (e.g., immunologically preferred) antigen orientation with certainty, and may represent only a small part of the obtained ligation reaction. In addition, the process involved in chemical conjugation may make it unlikely that the 3-D structure required for suitable antigen presentation is maintained. For example, 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 describe some shortcomings of traditional methods.

[0052] Similarly, genetic fusion of antigens to viral coat proteins has proven challenging and time consuming because of problems with misfolding and determining conditions for optimal expression of both components. In addition, genetic fusions are not suitable for expressing large or multicomponent antigens because efficient expression in the correct conformation is too difficult to achieve.

[0053] In order to present protein component or antigen component in an immunogenic manner, it is necessary to carefully design the position of the first peptide tag so that the native protein conformation is maintained, and if appropriate, any post-translational modification is optionally retained. For some antigens, the retention of glycosylation will not affect the presentation of the epitope, but for other antigens, retention or removal of glycosylation can improve efficacy. For protein components that are transmembrane proteins, the transmembrane portion of the protein component provides a good target for positioning the first peptide tag, because for example, the sequence does not involve the conformation of the protein component that is antigenic, and the effect provided is no longer needed in the vaccine. If the protein component does not comprise a transmembrane protein, the first peptide tag is fused to the C-terminal or N-terminal of this component or its subunit (in the case of a polymer) and can prove to be useful, but the first peptide tag may also be included in any part of the sequence. Alternatively, the first peptide tag may be positioned in a loop on the protein component or the antigen component.

[0054] In order to present immunogenic components, such as immunogenic components of HCMV pentamers, it is necessary to carefully design the position of the first peptide tag so that the native protein conformation is maintained. For HCMV, in one embodiment, attachment is by gH subunit, suitably by the C-terminus of gH subunit, or the transmembrane domain (or a part thereof) of gH subunit. In addition to maintaining the conformation of pentamers (or pentamer components), this rational design also makes the target region of pentamers presented toward the outside of particles as discussed above. As used herein, target region is a part of a protein known to cause antibodies with neutralizing effects, and may also be referred to as an immunogenic portion.

[0055] In order to present immunogenic components, such as the immunogenic components of RSV pre-fusion F protein, it is necessary to carefully design the position of the first peptide tag so that the native protein conformation is maintained. For RSV-F pre-fusion protein, in one embodiment, suitable attachment is by the C-terminal of F pre-fusion protein, by the 3 ' end of its nucleic acid encoding. Except keeping the conformation of pre-fusion F protein (or its components), this rational design also makes most of the neutralization epitopes of pre-fusion F protein as discussed above presented towards the outside of particle. The same consideration is applicable to any other variant of F protein. The inventor has shown that it is effective to include the first peptide tag at the C-terminal, leaving the immunogenic protein component to correctly fold.

[0056] In one embodiment, the first and second peptide tags are part of a peptide tag / binding partner pair that can form an isopeptide bond. The isopeptide bond can be spontaneous, i.e., without assistance, or requires assistance, i.e., assistance from a ligase or other auxiliaries. Suitably, the first and second peptide tags are SpyTag / SpyCatcher pairs. Suitably, the first and second peptide tags are selected from a 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 variants, derivatives and modifications of all these systems.

[0057] Suitably, the first peptide tag is a peptide tag from a peptide tag / binding partner pair, such as SpyTag, and the second peptide tag is a binding partner, such as SpyCatcher. In another embodiment, the first peptide tag is a binding partner, such as SpyCatcher, and the second peptide tag is a peptide tag component from a peptide tag / binding partner pair, such as SpyTag. Suitably, the first peptide tag is a peptide tag from a peptide tag / binding partner pair, such as SnoopTag, and the second peptide tag is a binding partner, such as SnoopCatcher. In another embodiment, the first peptide tag is a binding partner, such as SnoopCatcher, and the second peptide tag is a peptide tag component from a peptide tag / binding partner pair, such as SnoopTag. It can be seen from this that the first peptide tag can be a "tag" or a "catcher"; the second peptide tag is the partner of this pair, which is a "catcher" or a "tag", respectively. 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.

[0058] In one embodiment, the protein component or antigenic component is attached to any one of SpyTag, SnoopTag, RrgATag, RrgATag2, DogTag, IsopepTag, IsopepTag-N, PsCsTag and SnoopTagJr as a first peptide tag.

[0059] If desired, the first peptide tag can be attached via a linker, which can be rigid or flexible. One skilled in the art will recognize which linker is suitable.

[0060] In another embodiment, the moiety is attached to any one of SpyCatcher, SnoopCatcher, RrgACatcher, Pilin-C, Pilin-N, PsCsCatcher, and DogTag (mediated by SnoopLigase) as a second peptide tag.

[0061] As previously discussed, the moiety can be any suitable moiety, including a synthetic multimerization platform.

[0062] The second peptide tag can be attached to any suitable position of the part that does not affect the ability of the part to fold and form a suitable conformation. Genetic fusion may be preferred. It may be preferred to include a second peptide tag at the C-terminus or N-terminus of the part, but the second peptide tag may also be included in any part of the sequence. Alternatively, the second peptide tag may be positioned in a loop on the part. For example, SpyCatcher is genetically fused to the N-terminus of the viral coat protein (CP3) of RNA bacteriophage AP205, as described in Brune et al., Scientific Reports volume 6, Article number: 19234 (2016). Using self-assembling synthetic proteins as an alternative fusion of a polymerized platform is discussed in Bruun et al., ACS Nano, 2018, 12 (9), pp 8855-8866. Alternatively, the second peptide tag may be attached by chemical conjugation.

[0063] If desired, the second peptide tag can be attached via a linker, which can be rigid or flexible. One skilled in the art will recognize which linker is suitable.

[0064] In one embodiment, an antigenic component, such as a HCMV pentamer or an immunogenic component thereof, is attached to a SpyTag.A suitable SpyTag has the amino acid sequence set forth in SEQ ID NO:30.

[0065] The SpyTag can be attached via a linker. Suitable linkers include linkers having the amino acid sequence set forth in SEQ ID NO:29.

[0066] 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 set forth in SEQ ID NO:38. In one embodiment, the SpyCatcher is attached via a linker. The linker may be a rigid linker or a flexible linker, suitably wherein the linker has the amino acid sequence set forth in SEQ ID NO:39.

[0067] In another embodiment, the protein composition or antigenic composition according to any aspect or embodiment of the invention further comprises another (preferably different) protein comprising the first peptide tag.

[0068] In another embodiment, the composition according to any aspect or embodiment of the invention further comprises another (preferably different) antigen comprising a first peptide tag, such as another HCMV antigen. Suitably, the other HCMV antigen is glycoprotein B. Suitably, the glycoprotein B sequence is described, for example, in WO2014 / 005959, see SEQ ID NO: 21, 22, 23 or 36. In one embodiment, the composition comprises particles (e.g., VLPs) displaying both the HCMV pentamer and another HCMV antigen.

[0069] In one embodiment, the composition is an immunogenic composition or a vaccine composition. Preferably, the immunogenic composition or vaccine composition is a composition that can induce an immune response, such as an antibody response, after administration to an individual. Suitably, the immune response can be a protective immune response. Suitable immunogenic compositions may further comprise additional components, including adjuvants, immunostimulants and / or pharmaceutically acceptable excipients.

[0070] For example, suitable adjuvants may be based on aluminum, peptides, squalene, liposomes, oil-in-water emulsions, and saponins, and may include MF59, AS01, MatrixM, muramyl dipeptide and Quil A. Water-in-oil adjuvants are also suitable. Squalene oil-in-water emulsions, such as Addavax TM , is appropriate.

[0071] Accordingly, in another aspect or embodiment of the present invention, an immunogenic composition or vaccine composition comprising a composition according to the present invention is provided. Suitably, the vaccine composition comprises a vaccine dose, which is an amount of the composition according to the present invention, which provides immunogenicity from an infectious agent / vector, preferably an immunoprotective effect against an infectious agent / vector, such as neutralization against HCMV infection. Suitably, the vaccine composition comprises a vaccine dose, which is an amount of the composition according to the present invention, which provides neutralization against an infectious agent / vector, such as neutralization against RSV infection. Antibodies, preferably neutralizing antibodies produced against an immunogenic composition, can be detected and measured by methods familiar to those skilled in the art, including, for example, standardized ELISA assays or microneutralization assays described herein.

[0072] In another aspect, there is provided a VLP comprising:

[0073] i) The portion comprising the first peptide tag

[0074] ii) Protein containing a second peptide tag

[0075] wherein the first peptide tag and the second peptide tag form an isopeptide bond. In some embodiments, the moiety is HBsAg. However, as previously described, any suitable moiety may be used.

[0076] Suitably, the first peptide tag is a peptide tag from a peptide tag / binding partner pair, such as SpyTag, and the second peptide tag is a binding partner, such as SpyCatcher. In another embodiment, the first peptide tag is a binding partner, such as SpyCatcher, and the second peptide tag is a peptide tag from a peptide tag / binding partner pair, such as SpyTag. Other suitable peptide tag / binding partner pairs are described herein and will be known to those skilled in the art. 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 variants, derivatives and modifications of all these systems.

[0077] Suitably, the protein comprising the second peptide tag is a protein or peptide complex greater than 50 kDa. The protein comprising the second peptide tag can be a protein or peptide complex greater than 50 kDa, 60 kDa, 70 kDa, 80 kDa, 90 kDa, 100 kDa, 110 kDa, 120 kDa, 130 kDa, 140 kDa, 150 kDa or 160 kDa, 170 kDa, 180 kDa, 190 kDa or larger, such as more than 200 kDa, more than 300 kDa or more than 400 kDa.

[0078] 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 can be a HCMV pentamer as described herein. Suitably, the protein can be an RSV-F protein or a derivative thereof (such as a pre-fusion F protein). In one embodiment, the protein comprising the second peptide tag is an immunogenic component of the HCMV pentamer. As described herein and comprising a suitable linker and a label, the HCMV pentamer (gH / gL / pUL128 / pUL130 / pUL131) has a molecular weight of more than 160kDa. Other suitable large proteins or antigens or multimeric proteins or antigens include antigens from other infectious agents (including viruses such as influenza virus, RSV, etc.).

[0079] Advantageously, using HBsAg as a vector (VLP) in this manner may also result in a boost against HepB, or what is described as an anti-hepatitis B virus (HBV) response.

[0080] In another aspect, there is provided a VLP comprising:

[0081] i) Protein containing a first peptide tag

[0082] ii) a portion comprising a second peptide tag

[0083] wherein the first peptide tag and the second peptide tag form an isopeptide bond. In some embodiments, the moiety is HBsAg. However, as previously described, any suitable moiety may be used.

[0084] Suitably, the first peptide tag is a peptide tag from a peptide tag / binding partner pair, such as SpyTag, and the second peptide tag is a binding partner, such as SpyCatcher. In another embodiment, the first peptide tag is a binding partner, such as SpyCatcher, and the second peptide tag is a peptide tag from a peptide tag / binding partner pair, such as SpyTag. Other suitable peptide tag / binding partner pairs are described herein and will be known to those skilled in the art. 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 variants, derivatives and modifications of all these systems.

[0085] Suitably, the protein comprising the first peptide tag is a protein or peptide complex greater than 50kDa. The protein comprising the first peptide tag can be greater than 50kDa, 60kDa, 70kDa, 80kDa, 90kDa, 100kDa, 110kDa, 120kDa, 130kDa, 140kDa, 150kDa or 160kDa or larger, particularly 200kDa, 300kDa or even 400kDa or larger protein or peptide complex. 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 a HCMV pentamer as described herein. Suitably, the protein can be an RSV-F protein or a derivative thereof (such as a pre-fusion F protein). In one embodiment, the protein comprising the first peptide tag is an immunogenic component of a HCMV pentamer. HCMV pentamer (gH / gL / pUL128 / pUL130 / pUL131A) as described herein and comprising 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 agents, including viruses such as influenza, RSV, etc.

[0086] Advantageously, using HBsAg as a vector (VLP) in this manner may also result in a boost against HBV.

[0087] In another aspect of the invention, there is provided a HCMV pentamer linked to a SpyTag as described herein.

[0088] According to another aspect of the present invention, there is provided a method for producing a composition or VLP according to the present invention, the method comprising:

[0089] - introducing a first nucleic acid encoding a first genetic fusion of a first protein and a first peptide tag into a first host cell;

[0090] - incubating the first host cell under conditions whereby the first genetic fusion is expressed; optionally purifying the expressed components;

[0091] - introducing a second nucleic acid encoding a second genetic fusion of a second protein and a second peptide tag into a second host cell;

[0092] - incubating the second host cell under conditions whereby the second genetic fusion is expressed; optionally purifying the expressed components;

[0093] - incubating the expressed component under conditions where an isopeptide bond is formed between the first peptide tag and the second peptide tag; optionally purifying the resulting composition.

[0094] Suitably, the expressed components are incubated together to allow for the formation of the isopeptide bond. The formation of the isopeptide bond may require incubation with a ligase or the like.

[0095] Suitably, the method of producing a composition or VLP according to the invention may be used to produce a composition comprising an antigenic component displayed on a VLP.

[0096] In some embodiments, when the "immunogenic component of the HCMV pentamer" comprises the entire HCMV pentamer, the recombinant production of the HCMV pentamer component requires that each subunit be expressed in the correct stoichiometry to form the pentamer and folded correctly for assembly. In these embodiments, it is necessary to exclude from the final product only a portion of the desired pentamer complex (e.g., gH / gL dimers and tetramers, or tetramers lacking any of the five subunits). Advantageously, the present invention overcomes the problems that may be associated with expressing all vaccine components (i.e., HBsAg and the five subunits of the HCMV pentamer) in one system in other ways by providing a simple method for preparing the components separately and then conjugating them. Accordingly, in one embodiment, a purification tag is incorporated into UL130 (Hofmann et al., DOI 10.1002 / bit 25670). Similar principles will also apply to other immunogenic components.

[0097] In some embodiments, when the “immunogenic component of an RSV-F protein” comprises the entire F protein or a derivative thereof, recombinant production of the component of the F protein or its derivative requires its correct folding for assembly, wherein the derivative includes a prefusion F protein trimer.

[0098] Suitably, the method is used to produce a composition comprising HCMV pentamers displayed on HBsAg VLPs. Suitably, the method is used to produce a composition comprising RSV-F prefusion F protein trimers displayed on HBsAg VLPs.

[0099] In another aspect of the invention, a vaccine for preventing and / or treating a disease is provided. Suitably, the vaccine comprises a composition or VLP according to any aspect or embodiment of the invention. In one embodiment, the disease is HCMV infection. On the other hand, a preventive method for HCMV treatment is provided. Suitably, the vaccine is used in humans. Suitably, the vaccine is used in adults, such as women of childbearing age or pregnant women. On the other hand, the invention provides a method for inducing an immunogenic response (e.g., a protective immune response) against HCMV in an individual, wherein the method comprises administering a composition according to any aspect or embodiment of the invention.

[0100] In another aspect of the invention there is provided a composition according to any aspect of the invention for use as a medicament.

[0101] In another aspect of the invention, a composition according to any aspect of the invention is provided for use as a vaccine, preferably a vaccine for preventing and / or treating HCMV infection. The composition for use as a medicament or vaccine according to the invention can be administered to an adult, such as a woman of childbearing age or a pregnant woman.

[0102] In another aspect, the invention provides a nucleic acid molecule for use in a method according to the invention. In one embodiment, a nucleic acid molecule according to the invention comprises a nucleic acid sequence encoding an amino acid sequence listed in any one of SEQ ID NOs: 27 to 41. In one embodiment, a nucleic acid molecule according to the invention comprises a nucleic acid sequence listed in any one of SEQ ID NOs: 12 to 26 or 42 to 46.

[0103] In another aspect, the invention provides a variety of nucleic acid molecules, including those encoding the amino acid sequences set forth in SEQ ID NOs: 27 to 41. In one embodiment, nucleic acid molecules of the invention include those having a sequence set forth in any one of SEQ ID NOs: 12 to 26 or 42 to 46.

[0104] In another aspect, the invention provides a nucleic acid molecule for use in a method according to the invention. In one embodiment, a nucleic acid molecule according to the invention comprises a nucleic acid sequence encoding an amino acid sequence listed in any one of SEQ ID NOs: 50 to 58. In one embodiment, a nucleic acid molecule according to the invention comprises a nucleic acid sequence listed in any one of SEQ ID NOs: 47 to 55.

[0105] In another aspect, the present invention provides a variety of nucleic acid molecules, including those encoding the amino acid sequences set forth in SEQ ID NOs: 50 to 58. In one embodiment, nucleic acid molecules according to the present invention include those having a sequence set forth in any one of SEQ ID NOs: 47 to 55.

[0106] In another aspect, the invention provides a vector comprising a nucleic acid molecule or a plurality of nucleic acid molecules according to the invention.A suitable vector is an expression vector for expressing the amino acid sequence of any component of the composition according to the invention.

[0107] On the other hand, the invention provides host cells for expressing the components of the compositions according to the invention. Suitable host cells can be those host cells for transient or stable expression of these components. Methods and host cells for expressing CMV proteins are described in, for example, WO2014 / 005959 and WO2016 / 067239, both of which are incorporated by reference. In some embodiments, the components can be glycosylated.

[0108] In another aspect of the invention, a kit is provided, the kit comprising a composition according to the invention, for a primary immunization-boosting vaccination regimen. Suitably, the kit may comprise a primary immunization composition comprising a first immunogenic composition according to the invention and a booster composition comprising a second immunogenic composition according to the invention. Alternatively, a kit may be provided to provide a single-dose or multi-dose vaccination regimen, i.e. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 doses. Accordingly, in another aspect, the invention provides a dosage regimen comprising a dose administered at intervals of about 3 weeks.

[0109] The present invention also provides the following items:

[0110] 1. A composition comprising particles displaying an antigen component, wherein the composition comprises:

[0111] i) an antigenic component comprising a first peptide tag, and

[0112] ii) a portion comprising a second peptide tag,

[0113] wherein the antigenic component and the moiety are linked by an isopeptide bond between the first peptide tag and the second peptide tag, and wherein the antigenic component exceeds 50 kDa.

[0114] 2. A composition according to claim 1, wherein the antigenic component exceeds 60kDa, 70kDa, 80kDa, 90kDa, 100kDa, 110kDa, 120kDa, 130kDa, 140kDa, 150kDa, 160kDa, 170kDa, 180kDa, 190kDa, 200kDa, 300k Da or 400kDa.

[0115] 3. A composition according to item 1 or item 2, wherein the antigen component is a monomer or a multimer, optionally wherein the multimer is a trimer, a tetramer, a pentamer, a hexamer, a heptamer, an octamer, a nonamer or a decamer.

[0116] 4. A composition according to any of the preceding items, wherein the part is a virus, a bacterium, a polymerized scaffold, a protein component that polymerizes to form a virus-like particle (VLP), a viral structural protein, a polymerized domain that forms a nanoparticle, a synthetic nanoparticle or a synthetic VLP for vaccination.

[0117] 5. A composition according to any preceding item, wherein the moiety is HBsAg.

[0118] 6. A composition according to any of the preceding items, wherein the first peptide tag and the second peptide tag are selected from any one of the following: a SpyTag and SpyCatcher pair, a SnoopTag or SnoopTagJr and SnoopCatcher pair, a RrgATag, RrgATag2 or DogTag and RrgACatcher pair, an IsopepTag 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 variants, derivatives or modifications thereof, optionally wherein the first peptide tag and the second peptide tag are SpyTag / SpyCatcher pairs.

[0119] 7. The composition according to any preceding item, wherein the antigenic component comprises an immunogenic component of HCMV pentamer.

[0120] 8. The composition according to item 7, wherein the immunogenic component of the HCMV pentamer comprises one or more of the gH, gL, pUL128, pUL130 or pUL131 subunits.

[0121] 9. The composition according to item 8, wherein the immunogenic component of the HCMV pentamer comprises all of the gH, gL, pUL128, pUL130 and pUL131 subunits.

[0122] 10. A composition according to any one of items 7 to 9, wherein the immunogenic component of the HCMV pentamer comprises a gH subunit having a truncated transmembrane domain.

[0123] 11. A composition according to any one of items 8 to 10, wherein the gH, gL, pUL128, pUL130 and pUL131 subunits have the amino acid sequences listed in SEQ ID NOs: 28, 31, 35, 33 and 36, respectively, or functional equivalents thereof.

[0124] 12. The composition according to any one of items 8 to 11, wherein the first peptide tag is attached to the gH subunit, preferably to the C-terminus of the gH subunit.

[0125] 13. A composition according to any one of items 1 to 6, wherein the antigenic component comprises an immunogenic component of the RSV-F protein.

[0126] 14. A composition according to item 13, wherein the immunogenic component of the RSV-F protein is a prefusion F protein, optionally a stabilized prefusion F protein.

[0127] 15. The composition according to item 14, wherein the immunogenic component of the RSV prefusion F protein comprises F 1 Subunit and F 2 subunit trimer.

[0128] 16. A composition according to any one of items 13 to 15, wherein the RSV-F protein has a sequence listed in any one of SEQ ID NOs: 50 to 58.

[0129] 17. The composition according to items 13 to 16, wherein the first peptide tag is attached to the C-terminus of the pre-fusion F protein.

[0130] 18. The composition according to any preceding item, wherein the first peptide tag is a SpyTag.

[0131] 19. The composition according to item 18, wherein the SpyTag has the amino acid sequence listed in SEQ ID NO: 30.

[0132] 20. The composition of item 18 or item 19, wherein the SpyTag is attached via a linker.

[0133] 21. The composition according to item 20, wherein the linker has the amino acid sequence listed in SEQ ID NO: 29.

[0134] 22. A composition according to any preceding item, wherein the moiety is HBsAg, and optionally wherein the second peptide tag is SpyCatcher.

[0135] 23. The composition according to item 22, wherein the second peptide is SpyCatcher, and the SpyCatcher has the amino acid sequence listed in SEQ ID NO:38.

[0136] 24. A composition according to item 22 or item 23, wherein the moiety is attached to the SpyCatcher via a linker, preferably a flexible linker.

[0137] 25. The composition according to item 24, wherein the linker has the amino acid sequence listed in SEQ ID NO: 39.

[0138] 26. The composition according to any preceding item, wherein the composition is an immunogenic composition or a vaccine composition.

[0139] 27. A vaccine comprising the composition according to any one of items 1 to 26 for use in preventing and / or treating a disease.

[0140] 28. A method of producing a composition according to any one of items 1 to 26, the method comprising:

[0141] - introducing a first nucleic acid encoding a first genetic fusion of a first protein and a first peptide tag into a first host cell;

[0142] - incubating the first host cell under conditions where the first genetic fusion is expressed;

[0143] - introducing a second nucleic acid encoding a second genetic fusion of a second protein and a second peptide tag into a second host cell;

[0144] - incubating the second host cell under conditions where the second genetic fusion is expressed;

[0145] - optionally purifying the expressed components;

[0146] - incubating the expressed component under conditions where an isopeptide bond is formed between the first peptide tag and the second peptide tag; and optionally purifying the resulting composition.

[0147] 29. A method according to item 28, wherein the first protein comprises an antigenic component and the second protein comprises a portion.

[0148] 30. A nucleic acid molecule for use in the method according to item 28 or item 29, wherein the nucleic acid molecule encodes the amino acid sequence listed in any one of SEQ ID NOs: 27 to 41.

[0149] 31. The nucleic acid molecule according to item 30, which has a nucleotide sequence listed in any one of SEQ ID NOs: 12 to 26 or 42 to 46.

[0150] 32. A nucleic acid molecule for use in the method according to item 28 or item 29, wherein the nucleic acid molecule encodes the amino acid sequence listed in any one of SEQ ID NOs: 50 to 58.

[0151] 33. The nucleic acid molecule according to item 32, which has a nucleotide sequence listed in any one of SEQ ID NOs: 47 to 55.

[0152] 34. A vector comprising the nucleic acid molecule according to any one of items 30 to 33.

[0153] 35. A host cell comprising the nucleic acid molecule according to any one of items 30 to 32 or the vector according to item 34.

[0154] 36. A kit comprising a composition comprising a first immunogenic composition and optionally one or more boosting compositions comprising a second immunogenic composition, wherein the first immunogenic composition and / or the second immunogenic composition comprises a composition according to any one of items 1 to 26.

[0155] 37. A vaccine for preventing and / or treating HCMV infection, comprising the composition according to any one of items 7 to 12.

[0156] 38. A vaccine for preventing and / or treating RSV infection, comprising the composition according to any one of items 13 to 17. BRIEF DESCRIPTION OF THE DRAWINGS

[0158] Figure 1.SDS-PAGE and Western blot analysis of purified Pentamer-SpyTag under non-reducing and reducing conditions. Lane 1: ColorPlus pre-stained broad range protein ladder, size indicated in kDa; Lane 2: non-reducing sample; Lane 3: reduced sample. A) SDS-PAGE and Coomassie staining analysis indicating the location of HCMV pentamer components, the left side of the gel is non-reducing and the right side of the gel is reduced. B) Western blot analysis using anti-HCMV pentamer antibody.

[0159] Figure 2 . SDS-PAGE and Western blot analysis of purified SpyCatcher-HBsAg under non-reducing conditions (NR) and reducing conditions (R). A) SDS-PAGE and Coomassie staining analysis. B) Western blot analysis using anti-HBsAg monoclonal antibody.

[0160] Figure 3 : HPLC analysis using s200increase 3.2 / 300 column. A) 10 μl of purified HCMV pentamer-SpyTag was loaded and eluted as a single peak. B) 10 μl of purified SpyCatcher-HBsAg was loaded and eluted as a single major peak with the void volume of the column.

[0161] Figure 4 : SDS-PAGE and Western blot analysis of conjugated Pentamer-SpyTag and SpyCatcher-HBsAg under reducing conditions. 1: ColorPlus pre-stained broad range protein ladder, size indicated in kDa; 2: conjugate; 3: Pentamer-SpyTag; 4: SpyCatcher-HBsAg. A) SDS-PAGE and Coomassie staining analysis. B) Western blot using anti-HBsAg monoclonal antibody. C) Western blot using anti-pentamer polyclonal antibody.

[0162] Figure 5 : HPLC analysis using s200 increase 3.2 / 300 column. 30 μl of conjugated pentamer-SpyTag--SpyCatcher-HBsAg was loaded and eluted with the void volume of the column as the main peak.

[0163] Figure 6: Immunogenicity of HCMV pentamer-HBsAg vaccine vs. pentamer protein vaccine after single immunization with Addavax as adjuvant. BALB / c mice were immunized with 1 μg or 0.1 μg of HCMV pentamer-SpyTag as soluble protein or as pentamer-HBsAg VLPs. Titers were measured from mouse sera by standardized ELISA. Lines represent mean values ​​and error bars represent standard deviations (n=10). Mice immunized with HCMV pentamer-HBsAg VLPs showed significantly stronger serum IgG antibody responses compared to mice immunized with HCMV pentamer protein alone, even when the pentamer equivalent VLP dose was 10x lower.

[0164] Figure 7 : Neutralizing activity of sera from mice immunized with HCMV pentamer-HBsAg vaccine compared to pentamer vaccine. Addavax was used as an adjuvant for the vaccine, and responses after one immunization (prime) or two immunizations (boost) are shown. wt131 NT was measured in ARPE-19 cells infected with the virus strain (displaying functional pentamers) 50 In the same assay, the neutralization titers of Cytogam and a commercially available neutralizing anti-gH mAb (HCMV16 (51C1), from Bio-Rad Antibodies) are shown.

[0165] Figure 8 : Immunogenicity of HCMV pentamer-HBsAg vaccine versus pentamer protein vaccine after one or two immunizations without adjuvant. BALB / c mice were immunized with 1 μg or 0.1 μg of HCMV pentamer-SpyTag (pentamer-HBsAg) conjugated to SpyCatcher-HBsAg or with 1 μg of pentamer-SpyTag protein. Titers were measured from mouse sera by standardized ELISA. Lines represent mean values ​​and error bars represent standard deviations (n=10). Mice immunized with HCMV pentamer-HBsAg VLPs showed significantly stronger serum IgG antibody responses compared to mice immunized with HCMV pentamer protein alone, even when the pentamer equivalent VLP dose was 10x lower.

[0166] Fig. 9 : Neutralizing activity of sera from mice immunized with HCMV pentamer-HBsAg vaccine compared to pentamer protein vaccine. The vaccine was adjuvant-free and the responses were shown after one immunization (prime) or two immunizations (boost). wt131NT50 was measured for ARPE-19 cells infected with the strain (displaying functional pentamers). In the same assay, the neutralization titers of Cytogam and a commercially available neutralizing anti-gH mAb (HCMV16 (51C1), from Bio-Rad Antibodies) are shown.

[0167] Fig.10 . SDS-PAGE and Western blot analysis of purified RSV-F-SpyTag under non-reducing and reducing conditions. A) SDS-PAGE and Coomassie staining analysis, lane 1: ColorPlus pre-stained broad range protein ladder; lane 2: non-reducing sample; lane 3: reduced sample. B) Western blot analysis using anti-RSV-F monoclonal antibody, lane 1: ColorPlus pre-stained broad range protein ladder; lane 2: non-reducing sample; lane 3: reduced sample.

[0168] Fig.11 . SDS-PAGE and Western blot analysis of RSV-F-SpyTag conjugated with SpyCatcher-HBsAg under reducing conditions. 1: ColorPlus pre-stained broad range protein ladder, 2: RSV-F-SpyTag--SpyCatcher-HBsAg conjugate, 3: RSV-F-SpyTag, 4: SpyCatcher-HBsAg. A) SDS-PAGE and Coomassie staining analysis. B) Western blot using anti-HBsAg monoclonal antibody. C) Western blot using anti-RSV-F monoclonal antibody.

[0169] Fig.12 . Immunogenicity of conjugated RSV-F-SpyTag--SpyCatcher-HBsAg ('Sc9-10-HBsAg') versus non-conjugated RSV-F-SpyTag ('Sc9-10'). TM BALB / c mice (n=8) were immunized with 1 μg of RSV-F-SpyTag (RSV-F VLP) conjugated with SpyCatcher-HBsAg or 1 μg of RSV-F-SpyTag protein as adjuvant. RSV-F antigen was sc9-10 DS-Cav1 A149C Y458C-SpyTag. DETAILED DESCRIPTION OF THE INVENTION

[0171] Virus-like particles

[0172] Traditionally, vaccine approaches have used attenuated or killed whole pathogens, although this has been replaced by the use of recombinant subunit vaccines containing proteins from appropriate pathogens. More recently, approaches using virus-like particles (VLPs) have been developed. VLPs are particles that are similar in size (approximately 20-200 nm), shape, and their repeated protein arrangement to viruses, but lack any genetic material from the pathogen. Due to their size, VLPs are more likely to flow to lymph nodes, making them ideal for uptake and presentation by antigen presenting cells. In addition, their repetitive structure facilitates complement fixation and B cell receptor cross-linking (Kushnir et al. Vaccine 2012; Vol 31(1):58-83). However, their mechanism of action is not limited to theory.

[0173] HCMV

[0174] Human cytomegalovirus (HCMV, also known as human herpesvirus-5 (HHV-5)) is a virus to which most adults are exposed, and initial infection is usually only mild or asymptomatic. After infection, the virus remains latent in the body but can cause severe disease in immunocompromised or elderly people. HCMV is also the leading infectious cause of birth defects in developed countries. Up to 4 / 200 infants are born with HCMV due to congenital infection, and up to 10% of these infants will suffer long-term consequences. HCMV infection is also associated with hypertension and atherosclerosis in adults (Cheng et al. (May 2009). Früh K, ed. "Cytomegalovirus infection causes an increase of arterial blood pressure". PLoS Pathog. 5(5): e1000427).

[0175] Based on the observation that antibodies against the pentameric complex of HCMV containing viral proteins gH / gL / pUL128 / pUL130 / pUL131A can neutralize viral entry into epithelial cells and reduce the risk of perinatal transmission of HCMV, this complex has been identified as a potentially useful vaccine target for HCMV. However, despite extensive efforts, no successful HCMV vaccine has been developed to date.

[0176] HCMV pentamer

[0177] HCMV strains, including clinical isolates and laboratory strains, have different genome sequences. HCMV strains include Merlin (GI: 155573956), Towne (GI239909366) and AD169 (GI: 219879600), Toledo (GI290564358) and TB40 / E. HCMV contains a variety of membrane proteins and protein complexes. The pentamer protein gH / gL / pUL128 / pUL130 / pUL131A is important for HCMV infection of epithelial cells and endothelial cells, which is considered to be carried out through the endocytic pathway. Other combinations of components of the complex have been shown to be important for infection of, for example, fibroblasts. The "pUL" subunit / component is also referred to as "UL"; "pUL131" is also referred to as "pUL131A" and "pUL131a" or "UL131A".

[0178] Various HCMV strains have been deposited at the ATCC and can be found as: Merlin (VR-1590), Towne (VR-977), and AD169 (VR-538). The genome sequences can be referenced by the following accession numbers: Merlin (AY446894.2), Towne (GO121041.1), AD169 (FJ527563.1), Toledo (GU37742.2), and TB40 / E (KF297339.1).

[0179] RSV

[0180] Respiratory syncytial virus is the leading cause of severe respiratory illness in young children worldwide. An estimated 3.4 million children younger than 5 years are hospitalized each year due to severe RSV lower respiratory tract infections, with the highest incidence in children younger than 6 months of age. Most deaths occur in infants younger than 1 year of age and in developing countries. Currently, options for prevention and control are limited.

[0181] RSV prefusion trimer

[0182] The F glycoprotein is a type I viral fusion protein. The RSV F precursor (F0) is believed to be cleaved by a furin-like protease at two sites, which produces three fragments. The shorter N-terminal fragment (F 2 ) is covalently linked to a larger C-terminal fragment (F 1 ) is covalently attached. The intervening 27 amino acid fragment dissociates after cleavage and is not present in the mature protein.

[0183] As previously discussed, there are many stabilized pre-fusion F trimers available. In the examples submitted herein, exemplary sequences encoding these pre-fusion trimers can be found in SEQ ID NOs: 48, 49, 54 and 55. Sequences comprising fusions with SpyTag are included as SEQ ID NOs: 47 and 53. For the pre-fusion trimers, the amino acid sequences are shown in SEQ ID NOs: 51, 52, 57 and 58, and for those with SpyTag, the amino acid sequences are shown in SEQ ID NOs: 50 and 56. Other exemplary sequences are mentioned herein.

[0184] Peptide tag / binding partner pairs

[0185] Proteins capable of spontaneously forming isopeptide bonds (so-called "isopeptide proteins") have been advantageously used to develop peptide tag / polypeptide binding partner pairs (i.e., two-part linkers) that are covalently bound to each other and provide irreversible interactions (see, e.g., WO2011 / 098772 and WO 2016 / 193746, both of which are incorporated herein by reference, and WO2018 / 189517 and WO2018 / 197854, both of which are incorporated herein by reference). In this regard, proteins capable of spontaneously forming isopeptide bonds can be expressed as separate fragments to give a peptide tag and a polypeptide binding partner of the peptide tag, wherein the two fragments can be covalently recombined by the formation of an isopeptide bond, thereby connecting a molecule or component fused to the peptide tag and its polypeptide binding partner. The isopeptide bond formed by the peptide tag and its polypeptide binding partner is stable under conditions where non-covalent interactions would rapidly dissociate, such as over long periods of time (e.g., weeks), high temperatures (up to at least 95° C.), high forces, or harsh chemical treatments (e.g., pH 2-11, organic solvents, detergents, or denaturants).

[0186] Isopeptide bonds are amide bonds formed between carboxyl groups / carboxamido groups and amino groups, where at least one of the carboxyl groups or the amino group is outside the protein backbone (the backbone of the protein). Such bonds are chemically irreversible under typical biological conditions, and they are resistant to most proteases. Since isopeptide bonds are covalent in nature, they result in some of the strongest protein interactions measured.

[0187] In short, the two-part linker, i.e., a peptide tag and its polypeptide binding partner (so-called peptide tag / binding partner pair), can be derived from a protein (iso-peptide protein) capable of spontaneously forming an isopeptide bond, wherein the domains of the protein are expressed separately to produce a peptide "tag" and a peptide or polypeptide binding partner (or "catcher"), the peptide "tag" comprising one of the residues participating in the isopeptide bond (e.g., aspartic acid or asparagine, or lysine), and the peptide or polypeptide binding partner (or "catcher") comprising another residue participating in the isopeptide bond (e.g., lysine, or aspartic acid or asparagine) and at least one other residue required for the formation of an isopeptide bond (e.g., glutamic acid). Mixing the peptide tag and the binding partner results in the spontaneous formation of an isopeptide bond between the tag and the binding partner. Therefore, by incorporating the peptide tag and the binding partner into different molecules or components, such as proteins, respectively, it is possible to covalently link the molecules or components through the isopeptide bond formed between the peptide tag and the binding partner, i.e., forming a linker between the molecules or components into which the peptide tag and the binding partner are incorporated.

[0188] The spontaneous formation of the isopeptide bond may be isolated and does not require the addition of any other entity. For some peptide tag and tag partner pairs, the presence of an auxiliary entity such as a ligase may be required to generate the isopeptide bond.

[0189] The peptide tag / binding partner pair (two-part linker) called SpyTag / SpyCatcher is derived from the CnaB2 domain of the Streptococcus pyogenes FbaB protein (Zakeri et al., 2012, Proc Natl Acad Sci US A109, E690-697) and is used in a variety of applications including vaccine development (Brune et al., 2016, Scientific reports 6, 19234; Thrane et al., 2016, Journal of Nanobiotechnology 14, 30).

[0190] Suitably, the first and second peptide tags form a peptide tag / binding pair, referred to as SpyTag / SpyCatcher. Suitably, the SpyCatcher component is DeltaN1 (ΔN1) SpyCatcher (as described in Li, L., Fierer, JO, Rapoport, TA & Howarth, M. Structural analysis and optimization of the covalent association between SpyCatcher and a peptide Tag. J. Mol. Biol. 426, 309-317 (2014)), which has a 23 amino acid truncation at the N-terminus compared to "SpyCatcher" (SEQ ID No. 38).

[0191] In other embodiments, the first and second peptide tags form a peptide tag / binding pair that is a mutant form of SpyTag / SpyCather that exhibits an increased reaction rate of isopeptide bond formation, such as, for example, those described in co-pending application GB 1706430.4. In some embodiments, these mutant forms can be useful in situations where attachment of large proteins (e.g., >50 kDa or >100 kDa, such as the >160 kDa HCMV pentamer protein described herein) and / or slow reaction or steric hindrance may be a problem.

[0192] In other embodiments, the isopeptide protein may include a SnoopTag / SnoopCatcher, described in, for example, WO2016 / 193746.

[0193] In some embodiments, one or both isopeptide proteins may have an N-terminal truncation or a C-terminal truncation while still maintaining the reactivity of the isopeptide bond.

[0194] Exemplary first and second peptide tag pairs (peptide tag / binding partner pairs; reactive pairs) are described in the following table:

[0195]

[0196] Described in, for example, WO2011 / 098772, WO2016 / 193746, GB1706430.4, GB 1705750.6 or Li, L., et al., J. Mol. Biol. 426, 309-317 (2014).

[0197] Variants, derivatives and modifications of binding pairs can be made by any suitable means. Variants, derivatives and functional operational modifications may involve the addition, substitution, change or deletion of amino acids, which retain the same function in terms of the ability to form an isopeptide bond with the relevant binding partner.

[0198] For some binding pairs, mediation by a third entity such as an enzyme is required. For example, SnoopLigase can be used to mediate the bond formation between SnoopTagJr and DogTag. Therefore, pairing may require the help of an enzyme such as a ligase.

[0199] HBsAg

[0200] "HBsAg" means a surface antigen (HBsAg) or a portion thereof from hepatitis B virus. In one embodiment, HBsAg may refer to the N-terminus of HBsAg, such as the HBsAg sequence listed in SEQ ID NO: 41, comprising 226 amino acids of the S protein of hepatitis B virus (adw serotype). Suitably, HBsAg comprises a sequence of four amino acids Pro Val Thr Asn, representing the four carboxyl terminal residues of the preS2 protein of hepatitis B virus (adw serotype), as described in Valenzuela et al., (1979) 'Nucleotide sequence of the gene coding for the major protein of hepatitis B virus surface antigen' Nature 280: 815-819. VLPs formed from HBsAg have been approved for clinical use against hepatitis B (Kushnir et al. Vaccine 2012; Vol 31(1):58-83), including Recombivax HB (https: / / vaccines.procon.org / sourcefiles / recombivax_package_insert.pdf) and Energix B (https: / / au.gsk.com / media / 217195 / engerix-b_pi_006_approved.pdf). HBsAg is also used as the basis for the 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).

[0201] Connector Details

[0202] The distance between proteins (e.g., VLP and decorated antigen) can have an impact on the availability of antigenic epitopes in the protein, the stability of the protein, and can also have an impact on the conjugation efficiency due to the accessibility of any isopeptide bond partner (e.g., SpyTag / SpyCatcher). Therefore, a linker with suitable properties can be selected to optimize availability, stability, and / or accessibility. Linkers can be broadly subdivided into flexible and rigid subtypes.

[0203] Flexible joints

[0204] When the connected domain needs to move, a flexible linker can be used. They are usually composed of small non-polar (e.g., Gly) or polar (e.g., Ser, Thr) amino acids, wherein the small size provides flexibility (Chen et al., 2013 Adv Drug Deliv Rev. Oct 15; 65 (10): 1357-1369). The addition of Ser or Thr can help maintain stability in solution, and adjusting the length can affect the correct folding of protein (Chen et al., 2013). Any suitable flexible linker with properties and length suitable for related entities can be used. Suitably, a flexible linker can include a combination of amino acids of this type between 2 and 70.

[0205] Examples:

[0206]

[0207] Rigid joints

[0208] In some cases, rigid linkers can be preferred because they can help provide protein separation. Rigid linkers have secondary structures. One of the most common rigid linkers is (EAAAK) n (where n is the number of repeats), which adopts an α-helical structure (Arai et al., (2001) Protein Eng. Aug; 14(8):529-32). Other rigid linkers may include proline-rich sequences such as (XP) n , where X is any amino acid, but preferably Ala (A), Lys (K) or Glu (E), where proline provides conformational constraint (Chen et al., 2013).

[0209] Other suitable linkers are described, for example, by Klein et al. (2014) Protein Eng Des Sel. Oct; 27(10): 325-330. Any suitable rigid linker having properties and length suitable for the entities concerned may be used. Suitably, the rigid linker may comprise a combination of between 2 and 70 amino acids of this type.

[0210] Examples:

[0211] sequence name sequence SEQ ID No Rigid joint 1 EAAAK SEQ ID NO: 9 <![CDATA[Rigid joint 2-(EAAAK) 3 > EAAAKEAAAKEAAAK SEQ ID NO: 10 <![CDATA[Rigid joint 3-(AP) 7 > APAPAPAPAPAPAP SEQ ID NO: 11

[0212] Host cells and expression vectors

[0213] Those skilled in the art will be aware of suitable host cells for expressing nucleic acids to produce proteins and compositions according to the invention.

[0214] In one embodiment, host cells will be suitable for transient expression. In another embodiment, host cells will be those cells that can form stable cell lines. Suitably, coding sequences encoding antigenic components such as HCMV pentamers and RSV-F proteins (including those sequences of peptide tags that form isopeptide bonds) will be integrated into a host cell. In one embodiment, every kind of nucleic acid sequence encoding a subunit of a polymer such as pentamers will be contained in different plasmids / vectors, so that when cultivated under suitable conditions, host cells will be caused to produce pentamers with, for example, all 5 plasmids / vector transfection host cells. In other embodiments, plasmids / vectors can include a combination of one or more coding sequences so that at least 1, 2, 3, 4 or 5 plasmids can be introduced. Alternatively, a complete fusion peptide coding sequence can be provided in a vector so that complete protein components and the first peptide tag are encoded on the same vector.

[0215] In one embodiment, these vectors are used to stably integrate the coding sequence into the genome of the host cell. Suitable host cells for stable expression include mammalian cells, such as HEK cells (human embryonic kidney 293 cells) or rodent cells, including CHO (Chinese hamster ovary) cells. Suitable mammalian cells and vectors for expressing the protein components of the composition according to the present invention will be known to those skilled in the art, and are described in, for example, WO2016 / 067239 pages 15-16 and Hofmann et al., (2015) Biotech and Bioeng, 112 (12): 2505-2515. Exemplary stable construct sequences for expressing components according to the present invention can be found in Example 3 below.

[0216] Affinity purification

[0217] In some embodiments, those expression constructs for expressing the components of the compositions according to the present invention may include a "tag" sequence or a sequence that promotes purification such as affinity purification. Any suitable tag such as an affinity tag may be included to separate the protein component and the first peptide tag from the system that produces the protein component and the first peptide tag. The skilled person in the field of recombinant protein production knows that systems such as His-tags and Strep-tags that can be included for purification purposes. Such tags greatly contribute to protein purification, and rarely have an adverse effect on biological activity or biochemical activity, and are therefore ideal. 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.

[0218] Both the protein component and / or the portion may comprise an affinity purification tag. For ease of use these are usually genetically fused to the C-terminus or N-terminus of the protein.

[0219] Therefore, in some embodiments, for example, gH, gL, pUL128, pUL130, pUL131A (or its fragment) subunit of HCMV, RSV pre-fusion F protein or HBsAg peptide / protein can include additional amino acid residues at N-terminus or C-terminus, which is conducive to purification. Such additional amino acid residues can include tags, such as His-tags or C tags. In some embodiments, C tags can provide cleaner purification. Other suitable tag sequences include maltose binding protein (MBP), Strep-tags, glutathione-S-transferase (GST) and FLAG tags. In some embodiments, the tag can be connected to the amino acid sequence in such a way that the tag can be cleaved after purification by, for example, using, for example, a cleavable joint. In other embodiments, non-affinity purification methods can be used.

[0220] In other embodiments, the RSV prefusion F protein may comprise additional amino acid residues at the C-terminus or N-terminus, which facilitate purification. As exemplified herein, the RSV prefusion F protein has a C tag for affinity purification.

[0221] Conjugation of the first and second peptide tag pairs

[0222] The 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, because it is expected that the coupling speed will increase at room temperature. For a specific coupling reaction, the optimal first and second binding partner ratio depends on the size of each binding partner. For example, for smaller antigens (~20kDa), a molar ratio of VLP monomer to antigen of 1:1.5 may be sufficient, while for larger antigens (>100kDa), a mass ratio of 1:1 may be sufficient in combination with the same VLP monomer. However, both ratios will result in excess antigen (smaller binding partner). Any excess antigen can be removed by, for example, size exclusion chromatography (SEC) or by dialysis. Dialysis may be more suitable for smaller antigens because it is not as effective as SEC. Alternatively, the ratio of VLP / particle to antigen can be optimized so that all antigens are conjugated, and therefore no downstream purification is required. A suitable final protein concentration of about 1 mg / ml is optimal for the conjugation reaction because lower concentrations reduce the reaction rate. Many buffers 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 cases, as described by Brune et al. Sci Rep. (2016), a 10x stock solution (40 mM NaCl) with the addition of citrate buffer can be used. 2 HPO 4 , 200 mM sodium citrate, pH 6.2).

[0223] Pharmaceutical compositions and uses

[0224] The compositions of the invention may be incorporated into a vaccine or immunogenic composition. Suitably, the vaccine or immunogenic composition will comprise an immunogenic dose of the particles of the invention.

[0225] The pharmaceutical composition may comprise the particles or immunogenic compositions according to the invention provided together with a pharmaceutically acceptable carrier. Suitable carriers are well known to those of ordinary skill in the art. In one embodiment, the pharmaceutical composition comprises a buffer, an excipient or a carrier. Suitably, the pharmaceutical composition may comprise suitable excipients and formulations to maintain the stability of the composition. Suitably, the formulation may comprise an adjuvant. In one embodiment, the formulation may comprise a formulation similar to AddaVax TM or similar squalene-based oil-in-water nanoemulsions. Other suitable adjuvants include liposome-based adjuvants, such as Matrix M and AS01. Other suitable adjuvants include aluminum-based formulations, such as In one embodiment, the formulation may include EDTA, for example, at a concentration of 5 mM. The appropriate excipient or formulation may depend on the characteristics of the particle or immunogenic composition; for example, the choice of expression system may affect the stability, glycosylation or folding of the protein in the composition, which may in turn affect the optimal formulation of the composition. Methods for determining appropriate excipients, formulations or adjuvants will be known to those skilled in the art.

[0226] Various other aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure.

[0227] All documents mentioned in this specification are incorporated herein by reference in their entirety.

[0228] As used herein, "and / or" should be considered as a specific disclosure of each of the two specified features or components with or without the other. For example, "A and / or B" should be considered as a specific disclosure of each of (i) A, (ii) B, and (iii) A and B, as if each were listed separately herein.

[0229] Unless the context dictates otherwise, the descriptions and definitions of the features listed above are not limited to any particular aspect or embodiment of the invention, and apply equally to all aspects and embodiments described.

[0230] It will also be appreciated by those skilled in the art that while the invention has been described by way of example with reference to several embodiments, it is not limited to the disclosed embodiments and that alternative embodiments may be constructed without departing from the scope of the invention as defined in the appended claims.

[0231] As used herein, "recombinant" to describe a polynucleotide is intended to refer to a polynucleotide of genomic, cDNA, semisynthetic, or synthetic origin that, by virtue of its origin or manipulation: (1) is not associated with all or part of a polynucleotide with which it is associated in nature; and / or (2) is linked to a polynucleotide other than the one with which it is linked in nature. The term "recombinant" as used with respect to a protein or polypeptide is intended to refer to a polypeptide produced by expression of a recombinant polynucleotide.

[0232] Unless otherwise specified, the processes comprising steps may be performed in any suitable order. Therefore, the steps may be performed in any suitable order.

[0233] The sequence identity between polypeptide sequences is preferably determined by a pairwise alignment algorithm using the Needleman-Wunsch global alignment algorithm (Needleman and Wunsch 1970) with default parameters (e.g., using the EBLOSUM62 scoring matrix, gap open penalty = 10.0, and gap extension penalty = 0.5). The algorithm is conveniently implemented in the needle tool in the EMBOSS software package (Rice, Longden and Bleasby 2000). Sequence identity should be calculated over the entire length of the polypeptide sequence of the invention.

[0234] Any homologue of the components mentioned herein is generally a functional homologue, and is generally at least 40% homologous to the relevant region of the protein. Homology can be measured using known methods. For example, the UWGCG software package provides the BESTFIT program (e.g., using its default settings) that can be used to calculate homology (Devereux et al. (1984) Nucleic Acids Research 12, 387-395). The PILEUP algorithm and the BLAST algorithm can be used to calculate homology or line up sequences (usually based on their default settings), such as in Altschul SF (1993) J Mol Evol 36: 290-300; Altschul, S, F et al. (1990) J Mol Biol 215: described in 403-10. The software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ).

[0235] 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 total probability (P(N)), which provides an indication of the probability that a match between two nucleotide or amino acid sequences occurs by chance. For example, a sequence is considered similar to another sequence if the smallest total probability in a comparison of the first sequence to the second sequence is less than about 1, preferably less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.

[0236] Variant polypeptide comprises the sequence (or is made up of it) with at least 40% homology with native protein.In preferred embodiments, variant sequence can be at least 20, preferably at least 30, for example at least 40, 60, 100, 200, 300, 400 or more continuous amino acids, or even on the whole sequence of variant, with the specific region of native protein at least 55%, 65%, 70%, 75%, 80%, 85%, 90% and more preferably at least 95%, 97% or 99% homology.Alternatively, variant sequence can be at least 55%, 65%, 70%, 75%, 80%, 85%, 90% and more preferably at least 95%, 97% or 99% homologous with full-length native protein. Typically, the difference between the variant sequence and the relevant region of the native protein is at least less than 2, 5, 10, 20, 40, 50 or 60 mutations or less than 2, 5, 10, 20, 40, 50 or 60 mutations (each mutation can be a substitution, insertion or deletion). The variant sequence of the present invention can have a certain identity percentage with a specific region of the full-length native protein, and the identity percentage is the same as any of the specific homology value percentages of any length across the above-mentioned sequence (that is, it can have at least 40%, 55%, 80% or 90% and more preferably at least 95%, 97% or 99% identity).

[0237] Variants of proteins also include truncations. Any truncation can be used, provided that the variant is still functional. Truncations are usually performed to remove sequences that are not essential for activity / function, particularly 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 component production. Suitable truncations can usually be identified by systematically truncating sequences of different lengths from the N-terminus or the C-terminus.

[0238] The variant of native protein also comprises mutant, and mutant has one or more, for example, 2, 3, 4, 5 to 10, 10 to 20, 20 to 40 or more amino acid insertions, substitutions or deletions relative to the specific region of native protein. Deletion and insertion are preferably carried out outside the antigenic region. Insertion is usually carried out at the N-terminal or C-terminal of the sequence deriving from the native protein, for example, for the purpose of recombinant expression. Replacement is also usually carried out in the region that activity / function is not necessary and / or does not affect the conformation of folded protein. This replacement can be carried out to improve the solubility or other characteristics of protein. In order to increase the stability of protein, replacement can be carried out.

[0239] The substitution preferably introduces one or more conservative changes, which replace an amino acid with another amino acid 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 as the amino acids they replace. Alternatively, conservative changes may introduce another aromatic or aliphatic amino acid to replace a pre-existing aromatic or aliphatic amino acid. Conservative amino acid changes are well known in the art.

[0240] A derivative is an entity produced or prepared from a parent entity by replacing some of the parts of the parent entity. Example

[0241] Example 1

[0242] Production of an exemplary multimer-VLP composition (HCMV pentamer-HBsAg VLP)

[0243] Using 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 pentamer described below is approximately 162 kDa, aglycosylated (including tag and linker, but excluding signal peptide).

[0244] Nucleotide sequence

[0245] The expressed HCMV pentamer sequence represents the native sequence from the Merlin strain (GenBank: AY446894.2; a low-passage (ie, attenuated) HCMV strain) (including introns) except for two introduced mutations (one in gH and one in UL128), as described in the relevant paragraphs below.

[0246] gH-SpyTag-His nucleotide sequence (SEQ ID NO.12)

[0247] In this sequence (SEQ ID NO: 12), a silent mutation C>A was introduced at position 1146 for Synthesized because the native sequence CACCTGC around this nucleotide was marked as potentially problematic. The construct comprises: signal peptide (nt 1-69), extracellular domain (nt 70-2151), transmembrane domain (truncated) (nt 2152-2157), (the signal peptide, extracellular domain and transmembrane domain (truncated) together are represented by SEQ ID NO: 13), linker (nt 2158-2175; SEQ ID NO: 14), SpyTag (nt2176-2214; SEQ ID NO: 15), 6x His tag (nt 2215-2232), stop codon (nt2233-2235). Nucleotides 1 to 2157 (SEQ ID NO: 13) represent the gH coding sequence.

[0248] gL nucleotide sequence (SEQ ID NO.16)

[0249] In this sequence: signal peptide (nt 1-90), extracellular domain (nt 91-834), stop codon (nt835-837).

[0250] UL130-C tag nucleotide sequence (SEQ ID NO.17)

[0251] In this sequence: signal peptide (nt 1-75), extracellular domain (nt 76-642), linker (nt 643-687), C tag (nt 688-699), stop codon (nt 700-702).

[0252] UL128 nucleotide sequence (SEQ ID NO.20) (including 2 introns present in the native sequence)

[0253] In this sequence: signal peptide (nt 1-81), introns: nt 165-287, nt 423-542, extracellular domain exons (nt 82-164, nt 288-422, nt 543-756), stop codon (nt 757-759).

[0254] A T>C mutation was introduced at nucleotide 634. The T634 nucleotide was mentioned in the GenBank file to cause premature termination of UL128 in the Merlin strain, and therefore we used annotations from a different strain (GenBank: GQ396662.1, strain HAN38) to inform which base to replace in order to restore expression of the full-length protein.

[0255] UL131A nucleotide sequence (SEQ ID NO.21) (including introns present in the native sequence)

[0256] In this sequence: signal peptide (nt 1-54), intron (nt 237-344), extracellular domain exon (nt55-236, nt 345-495), stop codon (nt 496-498).

[0257] SpyCatcher-HBsAg nucleotide sequence (SEQ ID NO.22)

[0258] In this sequence: SpyCatcherDeltaN1 (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).

[0259] Amino acid sequence

[0260] Expression of the above nucleotide sequence is expected to produce the following amino acid sequence.

[0261] gH-SpyTag-His amino acid sequence (SEQ ID NO.27)

[0262] Expected mass 81.852 kDa (without signal peptide), 84.364 kDa (with signal peptide).

[0263] In this sequence: signal peptide (aa 1-23), extracellular domain (aa 24-717), transmembrane domain (truncated) (aa718-719), (the signal peptide, extracellular domain and transmembrane domain (truncated) are represented by SEQ ID NO: 28), linker (aa720-725; SEQ ID NO: 29), SpyTag (aa 726-738; SEQ ID NO: 30), 6x His tag (aa 739-744). Amino acid residues 1-719 represent the natural Merlin strain gH amino acid sequence with a truncated TM domain (SEQ ID NO: 28).

[0264] gL amino acid sequence (SEQ ID NO:31)

[0265] Expected mass 27.522 kDa (without signal peptide), 30.815 kDa (with signal peptide).

[0266] In this sequence: signal peptide (aa 1-30), extracellular domain (aa 31-278). Amino acid residues 1-278 represent the amino acid sequence of natural Merlin strain gL.

[0267] UL130-C tag amino acid sequence (SEQ ID NO: 32)

[0268] Expected mass 23.167 kDa (without signal peptide), 26.081 kDa (with signal peptide).

[0269] In this sequence: signal peptide (aa 1-25), extracellular domain (aa 26-214), (the signal peptide and extracellular domain together are represented by SEQ ID NO: 33), linker (aa 215-229; SEQ ID NO: 34), C tag (aa 230-233).

[0270] Amino acid residues 1-214 represent the natural Merlin strain UL130 amino acid sequence.

[0271] UL128 amino acid sequence (SEQ ID NO:35)

[0272] Expected mass 16.659 kDa (without signal peptide), 19.717 kDa (with signal peptide).

[0273] In this sequence: signal peptide (aa 1-27), extracellular domain (aa 28-171). Amino acid residues 1-171 represent the natural Merlin strain UL128 amino acid sequence.

[0274] UL131A amino acid sequence (SEQ ID NO:36)

[0275] Expected mass 12.985 kDa (without signal peptide), 14.989 kDa (with signal peptide).

[0276] In this sequence: signal peptide (aa 1-18), extracellular domain (aa 19-129). Amino acid residues 1-129 represent the natural Merlin strain UL131A amino acid sequence.

[0277] SpyCatcher-HBsAg amino acid sequence (SEQ ID NO: 37)

[0278] Expected mass 36.824 kDa including tag and linker.

[0279] In this sequence: SpyCatcherDeltaN1 (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).

[0280] Purification of pentamers

[0281] Pentamer-SpyTag was expressed in EXPI293F cells and secreted into the supernatant (due to the deletion of the TM domain (a portion thereof) of the gH subunit). Initial attempts to purify HCMV pentamers using affinity purification relied on the expression of the gH subunit with a C tag, but this resulted in the separation of gH / gL heterohomodimers as well as pentamers. In an alternative strategy, a C tag was added to the UL130 subunit (SEQ ID NO: 17 (nucleotides) and SEQ ID NO: 32 (amino acids)), which allowed the pentamer to be purified from the supernatant using C tag affinity purification (ThermoFisher) and size exclusion chromatography. When analyzed by SDS-PAGE, the pentamer appeared as expected under non-reducing and reducing conditions ( Figure 1 A), and reacted with anti-HCMV pentamer antibody (Native Antigen Company (AbCMV2450)) ( Figure 1 B), Only a small amount of contaminant was observed at ~14 kDa.

[0282] Purification of HBsAg VLP monomers

[0283] SpyCatcher-HBsAg was expressed in Pichia pastoris and purified from cell homogenates. On SDS-PAGE gels under reducing conditions, the major protein band corresponded to the expected size of the monomer (approximately 37 kDa), and larger bands indicated the presence of oligomeric material, indicating that the particles were well cross-linked ( Figure 2 A, lane 'R'). Under non-reducing conditions (lane 'NR'), material remained primarily at the top of the gel with some smearing, indicating that the VLP particles were well formed and therefore too large to migrate completely into the gel ( Figure 2 A). Both non-reduced and reduced SpyCatcher-HBsAg reacted strongly with mouse anti-HBsAg monoclonal antibody (obtained from Bio-Rad (MCA4658)) ( Figure 2 B), indicating that the presence of SpyCatcher did not negatively affect the reactive epitopes. Both HCMV pentamer-SpyTag and SpyCatcher-HBsAg eluted as a single peak as assessed by HPLC size exclusion analysis on an s200increase 3.2 / 300 column ( Figure 3 A-3B). HCMV pentamer-SpyTag eluted at approximately 400 kDa ( Figure 3A), which is larger than expected. However, this can be explained by the fact that the pentamer is not spherical, which is known to alter the retention time of proteins during size exclusion chromatography. SpyCatcher-HBsAg eluted in the void volume of the column, indicating that the particles were correctly formed with no detectable monomers in solution ( Figure 3 B).

[0284] Antigen-VLP conjugation

[0285] HCMV pentamer-SpyTag was conjugated with SpyCatcher-HBsAg at 4°C overnight to obtain HBsAg VLPs coated with HCMV pentamer. 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. When the conjugation reaction was compared with pentamer-SpyTag or SpyCatcher-HBsAg alone, a new band was present at ∼130 kDa under reducing conditions ( Figure 4 A, lane 2), which is compared with monoclonal anti-HBsAg ( Figure 4 B) and polyclonal anti-HCMV pentamer ( Figure 4 C) antibodies reacted, indicating that it contained at least conjugated HBsAg-gH. When analyzed by HPLC size exclusion chromatography, 97% of the elution in the main peak corresponded to the expected size of the conjugated HCMV pentamer-HBsAg monomer ( Figure 5 ).

[0286] Example 2

[0287] In vivo testing of HCMV-SpyTag--SpyCatcher-HBsAg VLP (with adjuvant)

[0288] Conjugated HCMV pentamer-HBsAg VLPs as well as non-conjugated HCMV pentamer-SpyTag were used in an immunization protocol using BALB / c mice to (i) confirm the immunogenicity of the generated HCMV pentamer-SpyTag and (ii) compare the immunogenicity of non-conjugated HCMV pentamer-SpyTag with that of conjugated HCMV pentamer-HBsAg VLPs.

[0289] Use a prime-boost-boost schedule with 3-week intervals as follows:

[0290] Day 0: immunization (primary immunization); Day 20: tail bleeding; Day 21: immunization (boost 1); Day 41: tail bleeding; Day 42: immunization (boost 2); Day 63: heart bleeding.

[0291] The immunized groups were as follows. For each group n=10:

[0292] 1) AddaVax TM 1 μg HCMV pentamer-SpyTag (Invivogen)

[0293] 2) AddaVax TM 1 μg HCMV pentamer-SpyTag--SpyCatcher-HBsAg VLP (1 μg pentamer equivalent)

[0294] 3) AddaVax TM SpyCatcher-HBsAg VLPs in Group 2 (normalized to the amount of SpyCatcher-HBsAg in Group 2)

[0295] 4) AddaVax TM 0.1 μg HCMV pentamer-SpyTag

[0296] 5) AddaVax TM 0.1 μg HCMV pentamer-SpyTag--SpyCatcher-HBsAg VLP (0.1 μg pentamer equivalent)

[0297] 6) TBS (20 mM Tris and 150 mM NaCl, pH 7.4)

[0298] AddaVax TM is a formula similar to Squalene-based oil-in-water nanoemulsions have been licensed in Europe as influenza vaccine adjuvants. Squalene oil-in-water emulsions are known to elicit cellular (Th1) and humoral (Th2) immune responses. Other suitable adjuvants will be known to those skilled in the art.

[0299] Immunogenicity was assessed using ELISA. A standardized ELISA for HCMV pentamer was used to determine the titer of the antisera generated in each group. Plates were coated overnight with 50 μL / well of 5 μg / ml pentamer (without SpyTag); washed; blocked with milk for 1 hour; washed; applied mouse serum (appropriately diluted in PBS) for 1 hour; washed; applied goat anti-mouse-alkaline phosphatase antibody (1:10,000) for 1 hour; washed; developed.

[0300] Different doses of non-conjugated (Groups 1 and 4) and conjugated HCMV pentamer-HBsAg (Groups 2 and 5) were included to allow comparison of immunogenicity between conjugated HCMV pentamer-HBsAg VLP vaccines and non-conjugated HCMV pentamer-SpyTag, which allows extrapolation to other HCMV pentamer vaccines (e.g., soluble pentamer). Groups 3 and 6 represent negative controls.

[0301] 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 run on each plate. Data showing the results for Groups 1, 2, 4, and 5 after priming are shown in Figure 6 Mice immunized with 1 μg and 0.1 μg doses of HCMV pentamer-HBsAg showed significantly stronger serum IgG antibody responses compared to mice immunized with 1 μg or 0.1 μg doses of non-conjugated HCMV pentamer. The ELISA units of groups 3 and 6 provided the baseline for this assay and were also Figure 6 Shown in.

[0302] The functional activity of the generated antibodies was investigated using a microneutralization assay based on Wang et al. (Vaccine 33 (2015) 7254-7261; DOI: 10.1016 / j.vaccine.2015.10.110). The neutralization titers of groups 1, 2, 4, and 5 were Figure 7 Sera from mice immunized with pentamer-HBsAg VLPs were significantly more neutralizing than sera from mice immunized with pentamer-SpyTag protein alone.

[0303] Example 3

[0304] Stable construct sequence

[0305] Two stable constructs (adapted from Hofmann et al., (2015) Biotech and Bioeng, 112(12):2505-2515) were optimized for CHO expression of components of HCMV pentamer-SpyTag. Introns were removed from the HCMV pentamer sequence, but the signal sequence was retained.

[0306] HCMV gH-SpyTag / gL stable expression construct

[0307] Stable vector construct HCMV-gH-(GSG) 2-SpyTag-His-IRES-gL was designed to contain a gH-SpyTag-His component (SEQ ID NO: 42) and a gL component (SEQ ID NO: 43) upstream and downstream of the EV71 IRES, respectively. The coding sequences used in this construct are described below.

[0308] Nucleotide sequence

[0309] gH-(GSG)2-SpyTag-His (no intron) (SEQ ID NO: 42) inserted upstream of EV71 IRES

[0310] In this sequence: signal peptide (nt 1-69), extracellular domain (nt 70-2151), truncated transmembrane domain (nt2152-2157), (GSG) 2 Linker (nt 2158-2175), SpyTag (nt 2176-2214), His-tag (nt 2215-2232), stop codon (nt 2233-2235).

[0311] gL (no intron) (SEQ ID NO: 43) inserted downstream of EV71 IRES

[0312] In this sequence: signal peptide (nt 1-90), extracellular domain (nt 91-834), stop codon (nt835-837).

[0313] HCMV UL128 / UL130 / UL131A stable expression construct

[0314] The stable construct HCMV-UL128-IRES-UL130-(G4S)3-C tag-IRES-UL131A was designed to include a UL128 component (SEQ ID NO: 44), a UL130 component (SEQ ID NO: 45), and a UL131A component (SEQ ID NO: 46). The UL130 component 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 described below.

[0315] Nucleotide sequence

[0316] Ul128 (no intron) (SEQ ID NO: 44)

[0317] In this sequence: signal peptide (nt 1-81), extracellular domain (nt 82-513), stop codon (nt514-516).

[0318] UL130-(G4S)3-C tag (no intron) (SEQ ID NO: 45)

[0319] In this sequence: signal peptide (nt 1-75), extracellular domain (nt 76-642), (G4S) 3 Adapter (nt643-687), C tag (nt 688-699), stop codon (nt 700-702).

[0320] UL131A (no intron) (SEQ ID NO: 46)

[0321] In this sequence: signal peptide (nt 1-54), extracellular domain (nt 55-387), stop codon (nt388-390).

[0322] Example 4

[0323] In vivo detection of HCMV-SpyTag--SpyCatcher-HBsAg VLP (without adjuvant)

[0324] Conjugated HCMV pentamer-HBsAg VLPs, as well as non-conjugated HCMV pentamer-SpyTag, were used in an immunization protocol using BALB / c mice to further investigate the immunogenicity of conjugated pentamer-HBsAg VLPs versus non-conjugated pentamer-SpyTag protein.

[0325] Use a prime-boost-boost schedule with 3-week intervals as follows:

[0326] Day 0: immunization (primary immunization); Day 20: tail bleeding; Day 21: immunization (boost 1); Day 41: tail bleeding; Day 42: immunization (boost 2); Day 63: heart bleeding.

[0327] The immunized groups were as follows. For each group n=10:

[0328] 1) 1 μg HCMV pentamer-SpyTag, no adjuvant

[0329] 2) 1 μg HCMV pentamer-SpyTag--SpyCatcher-HBsAg VLP (1 μg pentamer equivalent), no adjuvant

[0330] 3) 0.1 μg HCMV pentamer-SpyTag--SpyCatcher-HBsAg VLP (0.1 μg pentamer equivalent), no adjuvant

[0331] Immunogenicity was assessed using ELISA. A standardized ELISA for HCMV pentamer was used to determine the titer of the antisera generated in each group. Plates were coated overnight with 50 μL / well of 5 μg / ml pentamer (without SpyTag); washed; blocked with milk for 1 hour; washed; applied mouse serum (appropriately diluted in PBS) for 1 hour; washed; applied goat anti-mouse-alkaline phosphatase antibody (1:10,000) for 1 hour; washed; developed.

[0332] 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 run on each plate. The data after the prime and boost are shown in Figure 8 Mice immunized with both 1 μg and 0.1 μg doses of HCMV pentamer-HBsAg showed significantly stronger serum IgG antibody responses compared to mice immunized with 1 μg HCMV pentamer alone as a soluble protein.

[0333] The functional activity of the generated antibodies was investigated using a microneutralization assay based on Wang et al. (2015). Neutralization titers after priming and boosting were Fig. 9 Sera from mice immunized with unadjuvanted pentamer-HBsAg VLPs were significantly more neutralizing than sera from mice immunized with unadjuvanted pentamer-SpyTag protein alone.

[0334] Example 5

[0335] Expression and purification of RSV-F-SpyTag

[0336] The sequence from the antigen RSV-F Sc9-10 DS-Cav1 A149C Y458C was fused to SpyTag to generate RSV-F-SpyTag and expressed using ExpiCHO TM Expression System Kit and ExpiFectamine TM Transfection reagent (ThermoFisher Scientific), by transiently transfecting ExpiCHO with plasmid pcDNA3.4 containing the nucleotide sequence SEQ ID NO:47 TM cells to express.

[0337] RSV-F Sc9-10 DS-Cav1 A149C Y458C (National Institutes of Health) is a variant of the respiratory syncytial virus fusion protein (pre-fusion RSV-F) described by 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). The variant is a fusion (F) glycoprotein in a pre-fusion stabilized form, with genetically linked F subunits, deleted fusion peptides, T4 fibritin trimerization motifs (folding domains) and protomer inter-stabilized by disulfide bonds (A149C Y458C) between other protomers.

[0338] Nucleotide sequence

[0339] RSV-F-SpyTag-C tag nucleotide sequence (SEQ ID NO: 47)

[0340] By deleting the thrombin site, 6x His-tag and II The original sequence of Sc9-10DS-Cav1A149C Y458C was modified. These deleted domains were replaced by the linker-SpyTag-C tag sequence to generate a 1587nt cassette (SEQ ID NO:47) containing Sc9-10 DS-Cav1 A149C Y458C (nt 1-1515, including the signal peptide (nt 1-75) and the T4 fibritin foldon domain (nt 1435-1515)), (GSG) 2 Linker (nt1516-1533; 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 not including the linker, SpyTag, C tag and stop codon is contained in SEQ ID NO: 48. The Sc9-10 DS-Cav1 A149C Y458C nucleotide sequence not including the signal peptide, linker, SpyTag or C tag is contained in SEQ ID NO: 49.

[0341] Amino acid sequence

[0342] Expression of the nucleotide sequence SEQ ID NO:47 is expected to produce an RSV-F-SpyTag-C tag amino acid sequence (SEQ ID NO:50) having the following domains: Sc9-10 DS-Cav1 A149CY458C ((aa 1-505, including a signal peptide (aa1-25) and a foldon domain (aa 479-505)), a linker (aa 506-511; SEQ ID NO:29), a SpyTag (aa 512-524; SEQ ID NO:30), and a C tag (aa 525-528). The expected mass of the protein with a signal peptide is 57.9 kDa, and the expected mass of the protein without a signal peptide is 55.3 kDa. The Sc9-10 DS-Cav1 A149CY458C amino acid sequence that does not include a linker, SpyTag, or C tag is included in SEQ ID The amino acid sequence of Sc9-10DS-Cav1 A149C Y458C without a signal peptide, linker, SpyTag or C tag is contained in SEQ ID NO:52.

[0343] Purification of RSV-F-SpyTag

[0344] RSF-F-SpyTag antigen was secreted from cells and purified from the supernatant using C-tag affinity purification and size exclusion chromatography. When analyzed by SDS-PAGE ( Fig.10 A) and reacted with anti-RSV-F[2F7] monoclonal antibody (ab43812; Abcam) ( Fig.10 B), RSV-F-SpyTag appears as expected under non-reducing and reducing conditions.

[0345] Purification of HBsAg VLP monomers

[0346] SpyCatcher-HBsAg (VLP monomer) was prepared and purified as described in Example 1 above, see also Figure 2 .

[0347] Conjugation of RSV-F-SpyTag to SpyCatcher-HBsAg

[0348] RSV-F-SpyTag was conjugated with SpyCatcher-HBsAg at 4°C overnight to obtain 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.11When the conjugation reaction was compared to RSV-F-SpyTag or SpyCatcher-HBsAg alone, a new band was present at -105 kDa under reducing conditions ( Fig.11 A), which is combined with anti-HBsAg monoclonal antibody (MCA4658, Bio-Rad) ( Fig.11 B) and anti-RSV-F[2F7] monoclonal antibody (ab43812; Abcam) ( Fig.11 C) All reacted, indicating that they contained conjugated RSV-F-SpyTag--SpyCatcher-HBsAg.

[0349] Example 6

[0350] Immunogenicity of conjugated RSV-F-SpyTag--SpyCatcher-HBsAg

[0351] An immunization program using BALB / c mice was designed to confirm the immunogenicity of the RSV-F antigen produced and to compare the immunogenicity of the conjugated RSV-F-SpyTag--SpyCatcher-HBsAg VLP with the non-conjugated RSV-F-SpyTag protein. Each group was dosed based on the amount of RSV-F-SpyTag in the sample, and a prime-boost schedule with a 3-week interval was selected, with the final time point being 2 weeks after the booster immunization.

[0352] Whether the vaccine is unadjuvanted ( Figure 6 ) or Addavax TM Prepared ( Figure 6 ), mice immunized with RSV-F-SpyTag--SpyCather-HBsAg showed a significantly stronger serum IgG antibody response than mice immunized with RSV-F-SpyTag protein alone.

[0353] Sequence table

[0354]

Claims

1. A composition, the composition comprising particles presenting an antigenic component, wherein the composition comprises: i) an antigenic component comprising a first peptide tag, and ii) a moiety comprising a second peptide tag, wherein the antigenic component and the moiety are linked by an isopeptide bond between the first peptide tag and the second peptide tag, and wherein the antigenic component is over 50 kDa, optionally wherein the composition is an immunogenic composition.

2. The composition according to claim 1, wherein the antigenic component is: (i) over 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; and / or (ii) monomeric or polymeric, optionally wherein the polymer is a trimer, tetramer, pentamer, hexamer, heptamer, octamer, nonamer or decamer.

3. The composition according to claim 1 or claim 2, wherein the moiety is a virus for vaccination, a bacterium, a polymerized scaffold, a protein component that polymerizes to form virus-like particles (VLPs), a viral structural protein, a polymerized domain that forms nanoparticles, a synthetic nanoparticle or a synthetic VLP, optionally wherein the moiety is the surface antigen of hepatitis B virus (HBsAg).

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

5. The composition according to any one of claims 1 to 4, wherein the antigen component comprises an immunogenic component of any of the following: a 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 protein, human HER2 receptor, PCSK9, VAR2CSA, Plasmodium falciparum RIPR protein, varicella-zoster virus (VZV) glycoprotein E, rabies virus glycoprotein or Epstein-Barr virus (EBV) gH / gL complex.

6. The composition according to claim 1, wherein the antigen component comprises a first peptide tag, and / or wherein the moiety comprises a second peptide tag. The first peptide tag: (i) comprises the amino acid sequence listed in any one of SEQ ID NO: 27, 50 or 56; or (ii) is encoded by the nucleotide sequence listed in any one of SEQ ID NO: 12, 42, 47 or 53. The second peptide tag: (i) comprises the amino acid sequence listed in SEQ ID NO: 37; or (ii) is encoded by the nucleotide sequence listed in SEQ ID NO:

22.

7. A vaccine comprising the composition according to any one of claims 1 to 6 for preventing and / or treating a disease.

8. A method for producing the composition according to any one of claims 1 to 6 or the vaccine according to claim 7, the method comprising: - introducing a first nucleic acid encoding a first genetic fusion of a first protein and a first peptide tag into a first host cell; - incubating the first host cell under conditions for expressing the first genetic fusion; - introducing a second nucleic acid encoding a second genetic fusion of a second protein and a second peptide tag into a second host cell; - incubating the second host cell under conditions for expressing the second genetic fusion; - optionally purifying the expressed components; - incubating the expressed components under conditions for forming an isopeptide bond between the first peptide tag and the second peptide tag; and optionally purifying the resulting composition, wherein the first protein comprises the antigen component and the second protein comprises the moiety.

9. A nucleic acid molecule encoding the amino acid sequence listed in any one of SEQ ID NO: 27, 37, 50 or 56, optionally wherein the nucleic acid molecule comprises the sequence listed in any one of SEQ ID NO: 12, 22, 42, 47 or 53.

10. A vector comprising the nucleic acid molecule according to claim 9.

11. A host cell comprising the nucleic acid molecule according to claim 9 or the vector according to claim 10.

12. A kit, the kit comprising a composition containing a first immunogenic composition and optionally one or more booster compositions containing a second immunogenic composition, wherein the first immunogenic composition and / or the second immunogenic composition comprises the composition according to any one of claims 1 to 6 or the vaccine according to claim 7.

13. A pharmaceutical composition, the pharmaceutical composition comprising the vaccine according to claim 7 and a pharmaceutically acceptable buffer, excipient, carrier, adjuvant or combination thereof.

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