Chimeric VLP forming polypeptides comprising beta-retroviral gag

Abstract

The present invention relates to a platform concept for presenting antigenic polypeptides as part of a virus like particle (VLP) construct, which comprises a Gag (group-specific antigen) protein of a beta-retrovirus, for instance of a human endogenous retrovirus K (HERV-K) or of IAPE. Surprisingly it was found that antigenic polypeptide expression in a VLP comprising a Gag protein of HERV-K or of murine endogenous retrovirus IAPE (Intracisternal A-type Particles elements with an Envelope) promotes antigenic polypeptide display and immunogenicity.

Description

[0001] Hervolution Therapeutics

[0002] ZSP ref.: 1195-9 PCT

[0003] Chimeric VLP forming polypeptides comprising beta-retroviral Gag

[0004] To boost and / or activate the body’s own defence mechanisms against harmful disease triggers that enter from outside of or even originate in the body itself, vaccination and or immune therapy can be used to stimulate the immune system’s response to certain target antigens which are otherwise overlooked by the bodies surveillance mechanisms or which the body has not previously been confronted with, in a safe and non-infectious manner.

[0005] One elegant vaccination strategy is the presentation of antigens (e.g. viral proteins such as viral envelope proteins) to the immune system on virus-like particles (VLPs), which are encoded in a nucleotide comprised by a vaccine. These particles do not contain viral nucleic acids and are therefore non-infectious. Nevertheless, VLPs are highly immunogenic and displayed proteins are presented in a natural context. For example, a viral envelope (Env) protein integrated in VLPs is presented on a virus-like surface, which promotes correct folding and conformation. In addition to the advantage of a strong immunogenicity, the vaccination strategy with VLPs includes also practical benefits. Thus, VLPs are relatively easy to produce as they are built from few proteins and production can be performed in cell cultures. Bayer et al. (2010) showed that the combination of encoded and capsid presented antigens was able to increase the level of functional antibodies on capsid based viral vaccines. This observation was assigned to the fact that while the presentation on the adenoviral capsid helped to cross-link B cell receptors, encoded antigens were required for an essential CD4+ T cell responses promoting affinity maturation of B cells. With this vaccination strategy Bayer et al. were able to reduce viral load of F-MLV after challenge. However, no indication of increased CD8+ T cell responses against the target antigen could be observed (Bayer W, Tenbusch M, Lietz R, Johrden L, Schimmer S, Uberla K, Dittmer U, Wildner O, J. Virol. 2010;84:1967-1976). Vaccination with an adenoviral vector that encodes and displays a retroviral antigen induces improved neutralizing antibody and CD4+ T-cell responses and confers enhanced protection (J Virol. 2010 Feb;84(4):1967-76). Shoji et al. primarily focused on the optimization of an adenovirus-based HIV vaccine and investigated the in-situ formation of group specific antigen (Gag) protein based VLPs. In their study such a setting showed the highest immune responses compared to other display strategies that did not promote in situ formation of VLPs (Shoji M, Yoshizaki S, Mizuguchi H, Okuda K, Shimada M. Immunogenic comparison of chimeric adenovirus 5 / 35 vector carrying optimized human immunodeficiency virus clade C genes and various promoters. PLoS One. 2012;7(l):e30302).One particularly interesting field of application for vaccinations sensitizing the body to fight otherwise undetected or previously unknown diseases is to vaccinate against the antigens of corresponding disease causing agents (e.g. such as viruses).

[0006] Suitable antigenic polypeptide targets for vaccinations exist. However, additional vaccination platforms are needed to allow swift exchange of a present antigen for a new antigen, allowing for example the vaccination against a new mutated disease strain. Also vaccination platforms are needed that have the capacity of effective antigen expression to allow an effective and even enhanced immune response. The present inventors had previously found that antigenic peptides of endogenous retroviruses (ERVs) and especially of human endogenous retroviruses (HERVs) could be effectively displayed using a chimeric VLP platform, wherein the VLP components may be encoded by a nucleic acid molecule that can be used as a vaccinating agent. These nucleic acids can be in the form of DNA, mRNA in LNPs, viral vectors and perceivably any kind of delivery method leading to the translation of the VLP forming nucleotide message. ERVs are the evidence of ancient infections with retroviruses in distant ancestors. Upon infection, viral RNA was reverse transcribed into parvoviral DNA, which was integrated into the host genome. Eventually, the provirus was integrated into cells of the germ line and became inheritable, giving rise to endogenous retroviruses. Over millions of years the viral DNA was passed down generations and became fixed in the populations. Today, every human genome consists of about 8% endogenous retroviral DNA, but these are just relics of the former retrovirus. Due to mutations, deletions and insertions most of the retroviral genes became inactivated or got completely lost from the genome. Today, no functional, full-length endogenous retrovirus is present in humans anymore. However, ERVs underwent duplication processes leading to the integration of several copies into the host genome with distinct functional proteins. Thus, in some cases the multitude of homologous ERVs has still the potential to produce viral particles.

[0007] Among the different families of HERVs present in the human genome, the human ERV type K (HERV-K, HML2) is one of the most recently acquired ERVs in the human genome and members of this family remained full-length open reading-frames for almost all viral proteins. HERV-K is a comparatively intact ERV family. Unique factors of the HERV-K family include the presence of functional Gag (Group-specific antigen) proteins, which are responsible for virus-like particle (VLP) formation, that were observed to robustly induce exosome formation.To protect against infectious diseases, a practical, cheap and efficient strategy is the induction of immune responses by vaccination. A simple approach is the vaccination with virus-encoded antigens. Vaccination directly administered to a patient can be tricky, since in order to vaccinate against viruses or virus-related disease ideally the whole antigenic protein of a virus should be displayed to the immune system to ensure an immune response against a full protein target.

[0008] Although immunotherapy approaches are constantly improving, broadly acting, universally applicable and highly efficient vaccines and vaccination strategies are still missing, particularly regarding the vaccination against one or more different antigenic polypeptides via a universally applicable strategy. In particular, the field is in need of alternative vaccine solutions that allow an increased immune response against the antigen are being sought. In particular, there is a need to sufficiently display and therefore effectively express antigenic polypeptides to be presented to the immune system. In view of the particularly suitable option of using virus like particles (VLPs) for antigenic display on VLP surfaces, there is a need for effective VLP based antigen display systems, allowing optimal VLP formation and sufficient antigenic polypeptide display on VLP surfaces. There is also, a need for a platform for efficient delivery of a wide range of antigens related to a variety of diseases, wherein preferably such platform should be universally applicable. In summary, it is desirable to optimize antigen expression and / or antigen display so that the maximum immune response may be elicited. Efficient vaccination technologies and particular VLP-based vaccine platform technologies are needed.

[0009] SUMMARY OF THE INVENTION

[0010] The above mentioned challenges have been successfully addressed by the present invention, which aims at producing an effective and universally applicable vaccine technology suitable for the prophylaxis and / or treatment of disease (preferably cancer and infectious diseases) caused by a variety of triggers, which is based on the unique properties of beta-retroviral VLP polypeptide components.

[0011] The present inventors unexpectedly found that beta-retroviral Gag and envelope (Env) polypeptides (for instance HERV-K Gag and HERV-K Env or Gag and Env of IAPE) surprisingly provide particularly suitable polypeptides and / or polypeptide domains, which are able to enhance the display of co-expressed antigenic polypeptides on VLP or cell surfaces. Expressing antigenic polypeptides along with said useful beta-retroviral polypeptides and / or comprising said useful beta-retroviral polypeptide domains further enhances immunogenicity of said antigenic polypeptide. The technology described herein is applicable of beta-retroviral Gag polypeptides from different types toa variety of antigenic polypeptides, non-retroviral and non-viral, and therefore the technology is suitable for a wide range of vaccination approaches. In particular, the present invention is advantageous when used in connection with a variety of different antigens, preferably that are surface displayed, and particularly such antigens that are otherwise poorly displayed, i.e. when displayed without the VLPs of the invention. To broaden applicability of a variety of surface-presentable antigenic polypeptides in disease treatment and / or prophylaxis, HERV-K Gag-based virus like particles (VLPs) disclosed herein as well as VLPs based on other beta-retroviral Gag proteins were investigated. The tested VLP constructs comprising HERV-K or IAPE Gag were found to effectively promote display of antigens, such as Influenza A HA protein and hMPV L-protein which are unrelated to retroviruses, let alone endogenous retroviruses. Betaretroviral Gag-based VLPs present a versatile beta-retrovirus-based, nucleic acid encodable vaccine technology, providing improved vaccination efficiency.

[0012] Furthermore, it was surprisingly found that surface display and immunogenicity of antigenic polypeptides, and particularly of viral envelope proteins, could be increased by fusing the cytoplasmic tail sequence of HERV-K or IAPE Env or a part thereof to antigenic polypeptides or by replacing the cytoplasmic tail sequence of a polypeptide comprising said antigenic polypeptide with the cytoplasmic tail sequence of HERV-K or IAPE Env or a part thereof. In some experiments, the observed effects were further enhanced when not only the cytoplasmic tail but also the transmembrane domain of HERV-K Env was expressed as part of a polypeptide comprising the antigenic polypeptide of interest, such as a viral envelope or other surface displayed protein.

[0013] The VLP modifications disclosed herein can be combined, e.g. a VLP construct was encoded comprising for instance both a Gag protein from the betaretrovirus IAPE and an antigenic polypeptide fused to the transmembrane domain and the cytoplasmic tail of said IAPE, whereby the immunogenicity enhancing effect was further enhanced.

[0014] Antigenic polypeptides can be modified to encompass a HERV-K Env cytoplasmic tail allowing integration into HERV-K Gag based VLPs, which rapidly induce antibodies to the antigenic target sequence. Without wishing to be bound by theory the introduction of said cytoplasmic tail sequence of HERV-K Env may allow improved incorporation of the polypeptide components defined herein into chimeric HERV-K Gag VLPs.

[0015] Although the present examples exemplarily used HERV-K or IAPE Gag and cytoplasmic tail and transmembrane domain sequences of the Env protein of HERV-K or IAPE, experiments regarding beta-retrovirus VLPs in general herein indicate that the described technology is broadly applicable toVLP-based antigen presentation generally employing beta-retroviral components. Furthermore, as the VLP constructs work with unrelated viral envelopes, the invention is suitable to present any antigenic polypeptide, that can be transmembrane-anchored (e.g. at the C-terminus), to the immune system. Thus, in a first aspect, the present invention relates to at least one nucleic acid molecule encoding (1) a first polypeptide A, wherein the polypeptide A is a group-specific antigen (Gag) protein of a beta-retrovirus; and

[0016] (2) a second polypeptide, wherein the second polypeptide comprises the polypeptides B, C and D in the order B-C-D or D-C-B; wherein

[0017] polypeptide B is an antigenic polypeptide, wherein the antigenic polypeptide is not an endogenous retrovirus (ERV) envelope protein,

[0018] polypeptide C is a transmembrane domain (TMD), and

[0019] polypeptide D is a cytoplasmic tail (CT) of a viral envelope protein or fragment thereof, wherein said first polypeptide and second polypeptide is encoded by the same nucleic acid molecule or by two different nucleic acid molecules.

[0020] In a further aspect, the present invention relates to at least one nucleic acid molecule encoding (1) a first polypeptide A, wherein the polypeptide A is a group-specific antigen (Gag) protein of a beta-retrovirus; and

[0021] (2) a second polypeptide, wherein the second polypeptide comprises the polypeptides B, C and D in the order B-C-D or D-C-B; wherein

[0022] polypeptide B is an antigenic polypeptide of a virus causing an infectious disease,

[0023] polypeptide C is a transmembrane domain (TMD), and

[0024] polypeptide D is a cytoplasmic tail (CT) of a viral envelope protein or fragment thereof, wherein said first polypeptide and second polypeptide is encoded by the same nucleic acid molecule or by two different nucleic acid molecules.

[0025] In a further aspect, the present invention relates to at least one nucleic acid molecule encoding (1) a first polypeptide A, wherein the polypeptide A is a group-specific antigen (Gag) protein of a beta-retrovirus; and

[0026] (2) a second polypeptide, wherein the second polypeptide comprises the polypeptides B, C and D in the order B-C-D or D-C-B; wherein

[0027] polypeptide B is an antigenic polypeptide of a respiratory virus, preferably a respiratory virus selected from the group consisting of influenza virus, respiratory syncytial virus, parainfluenza virus, metapneumovirus, rhinovirus, coronavirus, adenovirus and bocavirus,polypeptide C is a transmembrane domain (TMD), and

[0028] polypeptide D is a cytoplasmic tail (CT) of a viral envelope protein or fragment thereof, wherein said first polypeptide and second polypeptide is encoded by the same nucleic acid molecule or by two different nucleic acid molecules.

[0029] In a further aspect, the present invention relates to at least one nucleic acid molecule encoding (1) a first polypeptide A, wherein the polypeptide A is a group-specific antigen (Gag) protein of a beta-retrovirus; and

[0030] (2) a second polypeptide, wherein the second polypeptide comprises the polypeptides B, C and D in the order B-C-D or D-C-B; wherein

[0031] polypeptide B is an antigenic polypeptide of a negative sense RNA virus, preferably an antigen of Orthornavirae, more preferably an antigen of Negarnaviricota, even more preferably an antigen of Orthomyxoviridae, Paramyxoviridae or Pneumoviridae, and preferably wherein the Orthomyxoviridae are selected from the group consisting of Alphainfluenzavirus, Betainfluenzavirus, Gammainfluenzavirus and Deltainfluenzavirus, and preferably wherein the Paramyxoviridae are selected from the group consisting of Parainfluenza Virus 1, Parainfluenza Virus 2, Parainfluenza Virus 3, Parainfluenza Virus 4, and preferably wherein the Pneumoviridae are selected from the group consisting of human metapneumovirus (HMPV), human respiratory syncytial virus A2 (HRSV-A2) and human respiratory syncytial virus B1 (HRSV-B1),

[0032] polypeptide C is a transmembrane domain (TMD), and

[0033] polypeptide D is a cytoplasmic tail (CT) of a viral envelope protein or fragment thereof, wherein said first polypeptide and second polypeptide is encoded by the same nucleic acid molecule or by two different nucleic acid molecules.

[0034] In a further aspect, the present invention relates to a composition comprising at least one nucleic acid molecule or a pharmaceutically acceptable salt thereof, encoding

[0035] (1) a first polypeptide A, wherein the polypeptide A is a group-specific antigen (Gag) protein of a beta-retrovirus; and

[0036] (2) a second polypeptide, wherein the second polypeptide comprises the polypeptides B, C and D in the order B-C-D or D-C-B; wherein

[0037] polypeptide B is an antigenic polypeptide,

[0038] polypeptide C is a transmembrane domain (TMD), and

[0039] polypeptide D is a cytoplasmic tail (CT) of a viral envelope protein or fragment thereof,wherein said first polypeptide and second polypeptide is encoded by the same nucleic acid molecule or by two different nucleic acid molecules,

[0040] wherein the composition comprises a buffering agent, buffering the composition at a pH in the range of 7 to 8.

[0041] In a further aspect, the present invention relates to at least one nucleic acid molecule encoding at least three polypeptides

[0042] (1) a first polypeptide A, wherein the polypeptide A is a group-specific antigen (Gag) protein of a beta-retrovirus;

[0043] (2) a second polypeptide, wherein the second polypeptide comprises the polypeptides Bl, Cl and DI in the order B1-C1-D1 or D1-C1-B1; wherein

[0044] polypeptide Bl is an antigenic polypeptide,

[0045] polypeptide Cl is a transmembrane domain (TMD), and

[0046] polypeptide DI is a cytoplasmic tail (CT) of a viral envelope protein or fragment thereof; and (3) a third polypeptide, wherein the third polypeptide comprises the polypeptides B2, C2 and D2 in the order B2-C2-D2 or D2-C2-B2; wherein

[0047] polypeptide B2 is an antigenic polypeptide,

[0048] polypeptide C2 is a transmembrane domain (TMD), and

[0049] polypeptide D2 is a cytoplasmic tail (CT) of a viral envelope protein or fragment thereof, wherein said first polypeptide, said second polypeptide and said third polypeptide are encoded by the same nucleic acid molecule,

[0050] wherein the polypeptides Bl and B2 are different antigenic polypeptides, preferably wherein the different antigenic polypeptides are from different pathogens.

[0051] In a further aspect, the present invention relates to a VLP comprising the polypeptides A, B, C and D of the invention.

[0052] In a further aspect, the present invention relates to a viral vector comprising the at least one nucleic acid molecule of the invention, preferably wherein the vector is an adenoviral vector, more preferably a human adenoviral vector, even more preferably human adenoviral vector is selected from subtype C and subtype D human adenoviral vectors, even more preferably wherein the subtype C human adenoviral vector is Ad5F35 and wherein the subtype D human adenoviral vector is Adi 9a, most preferably Adl9a / 64.In a further aspect, the present invention relates to a method of producing the VLP according to the invention comprising the step of transfecting a nucleic acid molecule according to the invention into a cell and preferably a cell of a A549 cell line.

[0053] In a further aspect, the present invention relates to a pharmaceutical composition comprising the at least one nucleic acid molecule, the VLP, or the viral vector according the to the invention, wherein the pharmaceutical composition comprises a pharmaceutically acceptable excipient.

[0054] In a further aspect, the present invention relates to at least one nucleic acid molecule, the VLP or the viral vector, or the pharmaceutical composition of the invention for use as a medicament.

[0055] In a further aspect, the present invention relates to at least one nucleic acid molecule, the VLP, the viral vector, or the pharmaceutical composition according to the invention for the manufacture of a medicament.

[0056] In a further aspect, the present invention relates to at least one nucleic acid molecule, the VLP, or the viral vector, or the pharmaceutical composition according to the invention for use in the prophylaxis and / or treatment of a disease.

[0057] In a further aspect, the present invention relates to the pharmaceutical composition according to the invention for use in the manufacture of a medicament for the prophylaxis and / or therapeutic treatment of a disease.

[0058] In a further aspect, the present invention relates to a method of treatment or prophylaxis of a disease comprising administering the at least one nucleic acid molecule according, the VLP, or the viral vector, or the pharmaceutical composition according to the invention to a subject.

[0059] DETAILED DESCRIPTION

[0060] The objective problem underlying the present invention is solved by the claimed subjectmatter. For the sake of clarity and readability the following definitions are provided. Any technical feature mentioned for these definitions may be read on each and every embodiment of the present disclosure. Additional definitions and explanations may be specifically provided in the context of the claimed embodiments.

[0061] Administering'. As used herein, "administering" refers to a method of delivering a composition to a subject or patient. A method of administration may be selected to target delivery (e.g., to specifically deliver) to a specific region or system of a body. For example, an administration may be parenteral (e.g., subcutaneous, intracutaneous, intravenous, intraperitoneal, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional, or intracranialinjection, as well as any suitable infusion technique), oral, trans- or intra-dermal, inter dermal, rectal, intravaginal, topical (e.g., by powders, ointments, creams, gels, lotions, and / or drops), mucosal, nasal, buccal, enteral, vitreal, intratumoral, sublingual, intranasal; by intratracheal instillation, bronchial instillation, and / or inhalation; as an oral spray and / or powder, nasal spray, and / or aerosol, and / or through a portal vein catheter. Preferred means of administration are intramuscular, intravenous, intradermal or subcutaneous.

[0062] Amino acids: “Amino acids” as used herein include those L-amino acids commonly found in naturally occurring proteins. Amino acid residues are indicated in the present disclosure according to the standard three-letter or one-letter amino acid code. Any amino acid sequence that contains post-translationally modified amino acids may be described as the amino acid sequence that is initially translated and modifications e.g., hydroxylations or glycosylations, shall not be shown explicitly in the amino acid sequence. Any peptide or protein that can be expressed as a sequence modified linkages, cross links and end caps, non-peptidyl bonds, etc., is embraced herein, all as known in the art.

[0063] And / or. The term "and / or" wherever used herein includes the meaning of "and", "or" and "all or any other combination of the elements connected by said term".

[0064] Antigenic polypeptide: As used herein, the term "antigenic polypeptide" refers to a polypeptide or a portion of a polypeptide that is suitable to provoke an immune response. The antigenic polypeptide may be specifically recognized by B-cells or T-cells and the immune response may involve antibody production or activation of specific immunologically-competent cells or both. Recognition of the antigenic polypeptide by said cells is facilitated by recognition of a specific epitope of the antigenic polypeptide, the term “epitope” being defined elsewhere herein. B-cells respond to recognized foreign antigenic determinants via antigen-specific antibody production, whereas T-lymphocytes are mediate cellular immunity upon antigen recognition via T-cell receptors. A person skilled in the art is aware that any macromolecule, including virtually all proteins or polypeptides, can serve as an antigenic polypeptide. Antigenic polypeptides can be encoded by recombinant or genomic nucleic acid molecules, such as RNA and DNA. A skilled person will understand that any nucleic acid molecule, which comprises a nucleotide sequences or a partial nucleotide sequence encoding a protein that is suitable to elicit an immune response encodes an "antigenic polypeptide" as used herein. Furthermore, one skilled in the art is aware that an antigenic polypeptide need not be encoded solely by a full length nucleotide sequence of a gene. It is readily apparent that the present invention includes, but is not limited to, the use of partial nucleotide sequences of more than one gene or encodingnucleotide sequence and that these nucleotide sequences may be arranged in various combinations to elicit a desired immune response (e.g. when encoded). It is readily apparent that an antigenic polypeptide can be generated, synthesized or can be derived from a biological sample. Such a biological sample can include, but is not limited to a tissue sample, a tumor sample, a cell or a biological fluid. In the context of the present disclosure and the experiments presented herein, tumor antigens or tumor related antigens may be of particular interest as antigenic polypeptides, and particularly cancer and cancer related antigens. An antigenic polypeptide preferably has a length of at least 5 amino acids.

[0065] A tumor antigen may for instance be a tumor-specific antigen (TSA) or a tumor-associated antigen (TAA). A TSA is unique to tumor cells and does not occur on other cells in the body. A TAA (associated antigen) is not unique to a tumor cell and instead is also expressed on a normal cell under conditions that fail to induce a state of immunologic tolerance to the antigen. The expression of the antigen on the tumor may occur under conditions that enable the immune system to respond to the antigen. TAAs may be antigens that are expressed on normal cells during fetal development when the immune system is immature and unable to respond or they may be antigens that are normally present at extremely low levels on normal cells but which are expressed at much higher levels on tumor cells. Non-limiting examples of TSA or TAA antigens include the following: Differentiation antigens such as MART-l / MelanA (MART-1), gplOO (PMEL17), tyrosinase, TRP-1, TRP-2 and tumor-specific multilineage antigens such as MAGE-1, MAGE-3, BAGE, GAGE- 1, GAGE-2, pi 5; overexpressed (carcinogenic) embryonic antigens such as CEA; overexpressed oncogenes and mutated tumorsuppressor genes such as p53, p21, Mucl, Ras, HER-2 / neu; unique tumor antigens resulting from chromosomal translocations; such as BCR-ABL, E2A-PRL, H4-RET, 1GH-IGK, MYL-RAR; and viral antigens, such as the Epstein Barr virus antigens EBVA and the human papillomavirus (HPV) antigens E6 and E7. Other large, protein-based antigens include TSP- 180, MAGE-4, MAGE-5, MAGE- 6, RAGE, NY-ESO, pl 85erbB2, p 1 80erbB-3, c-met, nm-23H 1, PSA, TAG- 72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, beta-Catemn, CDK4, Mum-1, pl5, pl6, 43-9F, 5T4 (791Tgp72), alpha-fetoprotein (AFP), beta-HCG, BCA225, BTAA, CA125, CA15-3\CA 27.29\BCAA, CA195, CA242, CA-50, CAM43, CD68\I, CO-029, FGF-5, G250, Ga733VEpCAM, HTgp-175, M344, MA-50, MG7-Ag, M0V18, NB / 70K, NY-CO-1, RCAS1, SDCCAG16, TA-90\Mac-2 binding protein / cyclophilin C-associated protein, TAAL6, TAG72, TLP, and TPS.

[0066] As used herein, also part of an antigenic polypeptide or an antigenic part of a polypeptide may be used when referring to an antigenic polypeptide. An “antigenic part” as used herein is any part of,for instance, a polypeptide, which comprises an epitope that can be recognized by the immune mechanisms outlined above. This also includes antigenic parts comprising discontinuous epitopes, i.e. epitopes, which are composed of several smaller fragments that are scattered within a polypeptide sequence, but are close when the protein is structured, thus forming a recognizable site. The minimal amino acid length of a suitable epitope as used herein is typically at least 5 amino acid residues. This means that a suitable epitope as used herein comprises at least 5 amino acid residues. Thus, also an “antigenic part” as used herein comprises at least 5 amino acid residues.

[0067] Approximately, about: As used herein, the terms "approximately" or "about," in the context of a numerical value means that said numerical value ranges from - 10% of that value to + 10 % of that value, unless otherwise stated herein or unless the numerical value is a percentage and the + 10 % of that percentage would exceed 100 % (in that case the upper range would end with 100 %). For example, a particle dose having “about” 1 x 1011particles includes from 9 x 1010to 11 x 1010particles.

[0068] Comprise'. Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integer or step. When used herein the term "comprising" can be substituted with the term "containing" or "including" or sometimes when used herein with the term "having".

[0069] Conjugated: As used herein, the term "conjugated," when used with respect to two or more moieties, means that the moieties are physically associated or connected with one another, either directly or via one or more additional moieties that serves as a linking agent, to form a structure that is sufficiently stable so that the moieties remain physically associated under the conditions in which the structure is used, e.g., physiological conditions. In some embodiments, two or more moieties may be conjugated by direct covalent chemical bonding. In other embodiments, two or more moieties may be conjugated by ionic bonding or hydrogen bonding.

[0070] Conservative amino acid changes / substitutions: When applying amino acid changes to the polypeptide sequences described herein, a skilled person may preferably take into account “conservative amino acid changes / substitutions”, which can generally be described as amino acid substitutions in which an amino acid residue is replaced with another amino acid residue of similar chemical structure and which has little or essentially no influence on the function, activity or other biological properties of the polypeptide. Such conservative amino acid substitutions are well known in the art, and the skilled person knows how to select (preferred) types and / or combinations of suchsubstitutions on the basis of teachings provided therein. It is preferred that conservative amino acid changes / substitutions are substitutions in which one amino acid is substituted by another amino acid residue within the same group as defined herein by (a) - (e) (amino acids named according to three letter nomenclature): (a) small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr, Pro and Gly; (b) polar, negatively charged residues and their (uncharged) amides: Asp, Asn, Glu and Gin; (c) polar, positively charged residues: His, Arg and Lys; (d) large aliphatic, nonpolar residues: Met, Leu, He, Vai and Cys; and (e) aromatic residues: Phe, Tyr and Trp. Exemplary conservative amino acid changes / substitutions include: Ala into Gly or into Ser; Arg into Lys; Asn into Gin or into His; Asp into Glu; Cys into Ser; Gin into Asn; Glu into Asp; Gly into Ala or into Pro; His into Asn or into Gin; He into Leu or into Vai; Leu into He or into Vai; Lys into Arg, into Gin or into Glu; Met into Leu, into Tyr or into He; Phe into Met, into Leu or into Tyr; Ser into Thr; Thr into Ser; Trp into Tyr; Tyr into Trp; and / or Phe into Vai, into He or into Leu.

[0071] Contacting: As used herein, the term "contacting" means establishing a physical connection between two or more entities. For example, contacting a cell with a nucleic acid molecule, a VLP or a pharmaceutical composition means that the cell and a nucleic acid molecule, a VLP or a pharmaceutical composition are made to share a physical connection. Methods of contacting cells with external entities both in vivo, in vitro, and ex vivo are well known in the biological arts. In exemplary embodiments of the disclosure, the step of contacting a mammalian cell with a composition (e.g., a transfection agent or a nanoparticle or pharmaceutical composition of the disclosure) is performed in vivo. For example, contacting an organism (e.g., a mammal) a lipid nanoparticle composition and a cell (for example, a mammalian cell) which may be disposed within may be performed by any suitable administration route (e.g., parenteral administration to the organism, including intravenous, intramuscular, intradermal, and subcutaneous administration). For a cell present in vitro, a composition (e.g., a transfection agent, e.g. a lipid nanoparticle) and a cell may be contacted, for example, by adding the composition to the culture medium of the cell and may involve or result in transfection. Moreover, more than one cell may be contacted by a transfection agent or a nanoparticle composition.

[0072] Cytoplasmic tail (CT): The "cytoplasmic tail" or also ”CT” as used herein refers to a domain or portion of a transmembrane protein or of a protein comprising a portion which is anchored in a cell membrane or other membrane, which extends into the cytoplasm of a cell. The cytoplasmic tail often comprises specific amino acid sequences or motifs that may be recognized and / or bound by other cytoplasmic proteins, and particularly by proteins located at or close to the cell membrane. Associationand / or protein-protein interaction with the cytoplasmic tail may for instance allow for the transmission of signals from the extracellular environment to the cell interior and / or may facilitate the positioning of membrane associated polypeptides. In one embodiment a cytoplasmic tail as defined herein may be a cytoplasmic tail of a viral envelope protein, such as of a beta-retroviral envelope protein, such as of an Envelope protein of a human endogenous retrovirus, such as HERV-K. Although the cytoplasmic tail is originally defined as a portion / domain of a polypeptide extending into the cytoplasm of a cell, a cytoplasmic tail as defined herein may in some embodiments also face towards the outside of a cell or a virus like particle (VLP), i.e. extend into the extracellular space surrounding a cell or VLP. Thus, the term “cytoplasmic” is merely a nomenclature of the original wildtype protein portion orientation in its natural cellular environment, but does not limit the actual orientation of a polypeptide as defined herein, for example in a VLP.

[0073] Delivering: As used herein, the term "delivering" means providing an entity to a destination. For example, delivering a therapeutic and / or prophylactic to a subject may involve administering a composition including the therapeutic and / or prophylactic to the subject (e.g., by an intravenous, intramuscular, intradermal, subcutaneous route or intratumoral injection). Administration of a composition to a mammal or mammalian cell may involve contacting one or more cells with the composition.

[0074] Encapsulate: As used herein, the term "encapsulate" means to enclose, surround, or encase. In some embodiments, a compound, polynucleotide (e.g., an mRNA), or other composition may be fully encapsulated, partially encapsulated, or substantially encapsulated. For example, in some embodiments, an mRNA of the disclosure may be encapsulated in a lipid nanoparticle, e.g., a liposome.

[0075] Effective amount: The "effective amount" of an agent is that amount sufficient to effect beneficial or desired results, for example, clinical results, and, as such, an "effective amount" depends upon the context in which it is being applied. In the present context an effective amount may particularly be considered any amount that is sufficient to induce or at least contribute to an overall immunisation and / or immune stimulating effect. Non-limiting examples of beneficial or desired results in the subject include increasing the level of expression and / or display of a polypeptide encoded by a nucleic acid by / on a cell in vivo or in vitro and / or increasing a prophylactic or therapeutic effect in vivo of a nucleic acid, or its encoded polypeptide(s), as compared to a nucleic acid molecule or polypeptide(s) not comprising the specific features of the invention. In some embodiments, a therapeutically effective amount of nucleic acid molecule, VLP, vector or pharmaceutical compositionis administered to a subject suffering from or susceptible to an infection, disease, disorder, and / or condition, when the amount is sufficient to treat, improve symptoms of, diagnose, prevent, and / or delay the onset of the infection, disease, disorder, and / or condition. In another embodiment, an effective amount is sufficient to result in expression of a desired protein in at least about 5%, 10%, 15%, 20%, 25% or more of target cells. For example, an effective amount of can be an amount that results in transfection of at least 5%, 10%, 15%, 20%, 25%, 30%, or 3 5% of target cells after a single administration. A VLP vaccine could for example be administered to a subject in an amount of between 5 pg to 45 pg per dose.

[0076] Epitope: As used herein an “epitope” is a specific structural unit that resides in an antigenic polypeptide and comprises a set of specific amino acids of the antigenic polypeptide, wherein the epitope is the polypeptide’s unit specifically recognised and bound by an antibody directed against said unit. Epitope as used herein also includes discontinuous epitopes, i.e. epitopes, which are composed of several smaller fragments that are scattered within a polypeptide sequence, but are close when the protein is structured, thus forming a site recognizable by the immune system. The minimal amino acid length of a suitable epitope as used herein is typically at least from 5 to 8 amino acid residues. This means that a suitable epitope as used herein comprises at least 5 amino acid residues Expression / expressed: As used herein, "expression" of a nucleic acid sequence or polypeptide / protein being “expressed” refers to one or more of the following events: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of an RNA transcript (e.g., by splicing, editing, 5' cap formation, and / or 3' end processing); (3) translation of an RNA into a polypeptide or protein; and (4) post-translational modification of a polypeptide or protein.

[0077] Fragment: A "fragment," as used herein, refers to a portion. For example, fragments of proteins may include polypeptides obtained by digesting full-length protein isolated from cultured cells or obtained through recombinant DNA techniques. A fragment of a protein can be, for example, a portion of a protein that includes one or more functional domains such that the fragment of the protein retains the functional activity of the protein.

[0078] Isolated: As used herein, the term "isolated" refers to a substance or entity that has been separated from at least some of the components with which it was associated (whether in nature or in an experimental setting). Isolated substances may have varying levels of purity in reference to the substances from which they have been associated. Isolated substances and / or entities may be separated from at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or more of the other components with which they were initially associated. In someembodiments, isolated agents are more than about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% pure. As used herein, a substance is "pure" if it is substantially free of other components.

[0079] Metastasis'. As used herein, the term "metastasis" means the process by which cancer spreads from the place at which it first arose as a primary tumor to distant locations in the body. A secondary tumor that arose as a result of this process may be referred to as "a metastasis."

[0080] Modified: As used herein "modified" or "modification" refers to a changed state or a change in composition or structure of a molecule of the disclosure (e.g., polynucleotide, e.g., mRNA). Molecules (e.g., polynucleotides) may be modified in various ways including chemically, structurally, and / or functionally. For example, polynucleotides may be structurally modified by the incorporation of one or more nucleic acid, such as RNA, elements, which comprise a sequence and / or a nucleic acid secondary structure(s) that provides one or more functions (e.g., translational regulatory activity). Accordingly, polynucleotides of the disclosure may be comprised of one or more modifications (e.g., may include one or more chemical, structural, or functional modifications, including any combination thereof). In one embodiment, nucleic acid molecules of the present disclosure are modified by the introduction of non-natural nucleosides and / or nucleotides, e.g., as it relates to the natural ribonucleotides A, U, G, and C. Noncanonical nucleotides such as the cap structures are not considered "modified" although they differ from the chemical structure of the A, C, G, U ribonucleotides.

[0081] mRNA: As used herein, an "mRNA" refers to a messenger ribonucleic acid. An mRNA may be naturally or non-naturally occurring. For example, an mRNA may include modified and / or non-naturally occurring components such as one or more nucleobases, nucleosides, nucleotides, or linkers. An mRNA may include a cap structure, a chain terminating nucleoside, a stem loop, a poly-A sequence, and / or a polyadenylation signal. An mRNA may have a nucleotide sequence encoding a polypeptide. An mRNA may also have a nucleotide sequence encoding multiple (for example at least two or at least three) different polypeptides. Translation of an mRNA, for example, in vivo translation of an mRNA inside a mammalian cell, may produce a polypeptide. Traditionally, the basic components of an mRNA molecule include at least a coding region, a 5'-untranslated region (5'UTR), a 3'UTR, a 5' cap and a poly-A sequence.

[0082] Nanoparticle: As used herein, "nanoparticle" refers to a particle having any one structural feature on a scale of less than about 1000 nm that exhibits novel properties as compared to a bulk sample of the same material. Routinely, nanoparticles have any one structural feature on a scale ofless than about 500 nm, less than about 200 nm, or about 100 nm. Also, routinely, nanoparticles have any one structural feature on a scale of from about 50 nm to about 500 nm, from about 50 nm to about 200 nm or from about 70 to about 120 nm. In exemplary embodiments, a nanoparticle is a particle having one or more dimensions of the order of about 1-1000 nm. In other exemplary embodiments, a nanoparticle is a particle having one or more dimensions of the order of about 10-500 nm. In other exemplary embodiments, a nanoparticle is a particle having one or more dimensions of the order of about 50-200 nm. A spherical nanoparticle would have a diameter, for example, of between about 50-100 or 70-120 nanometres. A nanoparticle most often behaves as a unit in terms of its transport and properties. It is noted that novel properties that differentiate nanoparticles from the corresponding bulk material typically develop at a size scale of under 1000 nm, or at a size of about 100 nm, but nanoparticles can be of a larger size, for example, for particles that are oblong, tubular, and the like. Although the size of most molecules would fit into the above outline, individual molecules are usually not referred to as nanoparticles.

[0083] Nucleic acid (molecule): As used herein, the term "nucleic acid (molecule)" is used in its broadest sense and encompasses any compound and / or substance that includes a polymer of nucleotides, unless specified otherwise. These polymers are often referred to as polynucleotides. A nucleic acid (molecule) as used herein may also comprise non-standard nucleotides, such as modified nucleobases. Comprised is also a chemical derivatization of a nucleic acid on a nucleotide base, on the sugar or on the phosphate, and nucleic acids containing non-natural nucleotides and nucleotide analogues. Exemplary nucleic acids or polynucleotides of the disclosure include, but are not limited to, ribonucleic acids (RNAs), mRNA, deoxyribonucleic acids (DNAs), DNA-RNA hybrids, RNAi-inducing agents, RNAi agents, siRNAs, shRNAs, miRNAs, antisense RNAs, ribozymes, catalytic DNA, RNAs that induce triple helix formation, threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs, including LNA having a B-D-ribo configuration, a-LNA having an a-L-ribo configuration (a diastereomer of LNA), 2'-amino-LNA having a 2'-amino functionalization, and 2'-amino-a-LNA having a 2'-amino functionalization) or hybrids thereof.

[0084] RNA element: As used herein, the term " RNA element" refers to a portion, fragment, or segment of an RNA molecule that provides a biological function and / or has biological activity (e.g., translational regulatory activity). Modification of a polynucleotide by the incorporation of one or more RNA elements, such as those described herein, provides one or more desirable functional properties to the modified polynucleotide. RNA elements, as described herein, can be naturally-occurring, non-naturally occurring, synthetic, engineered, or any combination thereof. For example, naturally-occurring RNA elements that provide a regulatory activity include elements found throughout the transcriptomes of viruses, prokaryotic and eukaryotic organisms (e.g., humans). RNA elements in particular eukaryotic mRNAs and translated viral RNAs have been shown to be involved in mediating many functions in cells. Exemplary natural RNA elements include, but are not limited to, translation initiation elements (e.g., internal ribosome entry site (IRES), see Kieft et al., (2001) RNA 7(2): 194-206), translation enhancer elements (e.g., the APP mRNA translation enhancer element, see Rogers et al., (1999) J Biol Chem 274(10):6421-6431), mRNA stability elements (e.g., AU-rich elements (AREs), see Garneau et al., (2007) Nat Rev Mol Cell Biol 8(2): 113-126), translational repression element (see e.g., Blumer et al., (2002) Meeh Dev 110(1 -2): 97-112), protein-binding RNA elements (e.g., iron-responsive element, see Selezneva et al., (2013) J Mol Biol 425(18):3301-3310), cytoplasmic polyadenylation elements (Villalba et al., (2011) Curr Opin Genet Dev 21(4):452-457), and catalytic RNA elements (e.g., ribozymes, see Scott et al., (2009) Biochim Biophys Acta 1789(9-10):634-641).

[0085] Nucleobase: As used herein, the term "nucleobase" (alternatively "nucleotide base" or "nitrogenous base") refers to a purine or pyrimidine heterocyclic compound found in nucleic acids, including any derivatives or analogues of the naturally occurring purines and pyrimidines that confer improved properties (e.g., binding affinity, nuclease resistance, chemical stability) to a nucleic acid or a portion or segment thereof. Adenine, cytosine, guanine, thymine, and uracil are the nucleobases predominately found in natural nucleic acids. Other natural, non-natural, and / or synthetic nucleobases, as known in the art and / or described herein, can be incorporated into nucleic acids.

[0086] Nucleoside / Nucleotide: As used herein, the term "nucleoside" refers to a compound containing a sugar molecule (e.g., a ribose in RNA or a deoxyribose in DNA), or derivative or analogy thereof, covalently linked to a nucleobase (e.g., a purine or pyrimidine), or a derivative or analogue thereof (also referred to herein as "nucleobase"), but lacking an internucleoside linking group (e.g., a phosphate group). As used herein, the term "nucleotide" refers to a nucleoside covalently bonded to an internucleoside linking group (e.g., a phosphate group), or any derivative, analogue, or modification thereof that confers improved chemical and / or functional properties (e.g., binding affinity, nuclease resistance, chemical stability) to a nucleic acid or a portion or segment thereof.

[0087] Open Reading Frame: As used herein, the term "open reading frame", abbreviated as " ORF", refers to a segment or region of a nucleic acid molecule that encodes one or more polypeptides. TheORF comprises a continuous stretch of non-overlapping, in-frame codons, beginning with the initiation codon and ending with a stop codon, and is translated by the ribosome.

[0088] Patient: As used herein, "patient" refers to a subject who may seek or need treatment, requires treatment, is receiving treatment, will receive treatment, or a subject who is under care by a trained professional for a particular disease or condition. In particular embodiments, a patient is a human patient. In some embodiments, a patient is a patient suffering from an autoimmune disease, e.g., as described herein.

[0089] Pharmaceutically acceptable: The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0090] Pharmaceutically acceptable excipient: The phrase "pharmaceutically acceptable excipient," as used herein, refers any ingredient other than the compounds described herein (for example, a vehicle capable of suspending or dissolving the active compound) and having the properties of being substantially nontoxic and non-inflammatory in a patient. Excipients may include, for example: antiadherents, antioxidants, binders, coatings, compression aids, disintegrants, dyes (colors), emollients, emulsifiers, fillers (diluents), film formers or coatings, flavors, fragrances, glidants (flow enhancers), lubricants, preservatives, printing inks, sorbents, suspensing or dispersing agents, sweeteners, and waters of hydration. Exemplary excipients include, but are not limited to: butylated hydroxytoluene (BHT), calcium carbonate, calcium phosphate (dibasic), calcium stearate, croscarmellose, crosslinked polyvinyl pyrrolidone, citric acid, crospovidone, cysteine, ethyl cellulose, gelatin, hydroxypropyl cellulose, hydroxypropyl methylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methyl paraben, microcrystalline cellulose, polyethylene glycol, polyvinyl pyrrolidone, povidone, pregelatinized starch, propyl paraben, retinyl palmitate, shellac, silicon dioxide, sodium carboxymethyl cellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E, vitamin C, and xylitol.

[0091] Pharmaceutically acceptable salts'. As used herein, "pharmaceutically acceptable salts" refers to derivatives of the disclosed compounds wherein the parent compound is modified by converting an existing acid or base moiety to its salt form (e.g., by reacting the free base group with a suitable organicacid). Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. Representative acid addition salts include acetate, acetic acid, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzene sulfonic acid, benzoate, bisulfate, borate, butyrate, camphorate, camphor sulfonate, citrate, cyclopentane propionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3 -phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate salts, and the like. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like, as well as nontoxic ammonium, quaternary ammonium, and amine cations, including, but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like. The pharmaceutically acceptable salts of the present disclosure include the conventional non-toxic salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. The pharmaceutically acceptable salts of the present disclosure can be synthesized from the parent compound which contains a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, nonaqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. Lists of suitable salts are found in Remington 's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418, Pharmaceutical Salts: Properties, Selection, and Use, P. H. Stahl and C. G. Wermuth (eds.), Wiley-VCH, 2008, and Berge et al., Journal of Pharmaceutical Science, 66, 1-19 (1977), each of which is incorporated herein by reference in its entirety.

[0092] Polyadenyl / poly-A tail / sequence: " Polyadenyl sequence", "poly-A sequence " or "poly-Atail" refers to an RNA molecule’s sequence of adenyl residues typically located at the 3' end. Such a sequence may be attached during RNA transcription. A poly-A sequence is usually attached to the free 3' end of the RNA by a template independent RNA polymerase after transcription in the nucleus. Artificially, a poly-A may be attached by transcription from a DNA template containing complementary repeated thymidyl residues. Alternatively, the at least one nucleic acid molecule asdescribed herein may comprise a polyadenylation signal, which is defined herein as a signal, which conveys polyadenylation to a (transcribed) RNA by specific protein factors. The poly-A sequence is important for the nuclear export, translation, and stability of mRNA and is shortened over time, and eventually leading to enzymatic mRNA degradation.

[0093] Polypeptide: As used herein, the term "polypeptide" or "polypeptide of interest" refers to a polymer of amino acid residues typically joined by peptide bonds that can be produced naturally (e.g., isolated or purified) or synthetically.

[0094] Prevent / preventing: As used herein, “prevent” or "preventing" refers to partially or completely inhibiting the onset of one or more symptoms or features of a particular infection, disease, disorder, and / or condition.

[0095] Prophylaxis: As used herein, the term "prophylaxis" refers to partially or completely inhibiting the onset of one or more symptoms or features of a particular infection, disease, disorder, and / or condition. “Prophylactic” is also used in that sense.

[0096] RNA: As used herein, an " RNA" refers to a ribonucleic acid that may be naturally or non-naturally occurring. For example, an RNA may include modified and / or non-naturally occurring components such as one or more nucleobases, nucleosides, nucleotides, or linkers. An RNA may include a cap structure, a chain terminating nucleoside, a stem loop, a poly-A sequence, and / or a polyadenylation signal. An RNA may have a nucleotide sequence encoding a polypeptide of interest. For example, an RNA may be a messenger RNA (mRNA). The RNA may also be circular RNA or self-replicating RNA, both of which the skilled person is aware of from the prior art. Translation of an mRNA encoding a particular polypeptide, for example, in vivo translation of an mRNA inside a mammalian cell, may produce the encoded polypeptide.

[0097] Sequence'. The term “sequence” as used herein, should generally be understood to include both the relevant amino acid sequence as well as nucleic acids or nucleotide sequences encoding the same, unless the context requires a more limited interpretation. Amino acid sequences are interpreted to mean a single amino acid or an unbranched sequence of two or more amino acids, depending of the context. Nucleotide sequences are interpreted to mean an unbranched sequence of 3 or more nucleotides.

[0098] Substantially: As used herein, the term "substantially" refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. One of ordinary skill in the biological arts will understand that biological and chemical phenomena rarely, if ever, go to completion and / or proceed to completeness or achieve or avoid an absolute result. The term"substantially" is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

[0099] Suffering from: An individual who is "suffering from" a disease, disorder, and / or condition has been diagnosed with or displays one or more symptoms of a disease, disorder, and / or condition.

[0100] Transfection / transfect(ing): As used herein, the term " transfection" / ’’transfect(ing)” refers to methods to introduce a species (e.g., a polynucleotide, such as a mRNA) into a cell.

[0101] Subject: As used herein, the term "subject" refers to any organism to which a composition in accordance with the disclosure may be administered, e.g., for experimental, diagnostic, prophylactic, and / or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans) and / or plants. In some embodiments, a subject may be a patient.

[0102] Transmembrane domain (TMD): The term "transmembrane domain" as used herein refers to a region or segment of a polypeptide that spans across a biological membrane, such as a cell membrane or virus like particle (VLP) membrane. It is typically a structural component of membrane proteins, which are proteins that are embedded within or traverse the lipid bilayer of cell membranes. A transmembrane domain typically prevalently comprises or essentially consists of hydrophobic amino acids facilitating its anchoring within the hydrophobic lipid bilayer of the biological membrane. Membrane-spanning transmembrane domains may often adopt an alpha helix topological conformation. Thus, a transmembrane domain used herein may comprise at least one alpha helix structure. A skilled person is aware of methods to determine the extent and position of transmembrane domains, for instance by hydrophobicity analysis for identifying and predicting transmembrane domains within protein sequences. However, a transmembrane domain may also contain polar or charged amino acids, which may interact with specific lipid or protein components of a biological membrane. Among integral transmembrane proteins, which comprise a membrane integral transmembrane domain, it can be differentiated between, among other things, bitopic type I and bitopic type II integral membrane proteins, wherein type I proteins and their corresponding transmembrane domains comprise a C-terminus located on the cytosolic membrane side and an N-terminus located on the extracellular membrane side and wherein type II proteins and their corresponding transmembrane domains are reversely oriented within the biological membrane. Although both orientations may be possible in the context in the present disclosure, it is preferred that the transmembrane domain as defined herein is a transmembrane domain of a bitopic type I transmembrane protein.Treating: As used herein, the term "treating" refers to partially or completely alleviating, ameliorating, improving, relieving, delaying onset of, inhibiting progression of, reducing severity of, and / or reducing incidence of one or more symptoms or features of a particular infection, disease, disorder, and / or condition. For example, "treating" cancer may refer to inhibiting survival, growth, and / or spread of a tumor. Treatment may be administered to a subject who does not exhibit signs of a disease, disorder, and / or condition and / or to a subject who exhibits only early signs of a disease, disorder, and / or condition for the purpose of decreasing the risk of developing pathology associated with the disease, disorder, and / or condition.

[0103] Unmodified: As used herein, "unmodified" refers to any substance, compound or molecule prior to being changed in any way. Unmodified may, but does not always, refer to the wild type or native form of a biomolecule. Molecules may undergo a series of modifications whereby each modified molecule may serve as the "unmodified" starting molecule for a subsequent modification. Variant: As used herein, the term "variant" refers to a molecule having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% consensus or homology with a wild type molecule, e.g., as measured by an art-recognized assay.

[0104] Viral / virus: The term “viral” / ”virus” as used herein refers to any virus, exogenous or endogenous. Viruses are microscopic infectious agents that rely on living organisms, such as plants, animals, and microorganism cells to replicate and carry out their life cycle. The process of viral infection typically involves attachment and entry of the virus into the host cell, release of the viral genetic material, replication of viral components, assembly of new virus particles, and finally, the release of these particles to infect other cells and propagate the infection. Typically, viruses comprise genetic material, which can be DNA or RNA, enclosed in a capsid protein coat, and optionally an outer envelope.

[0105] The term “viral” / ”virus” encompasses among other virus families also the family of retroviruses, including for instance beta- and gamma- retroviruses within the Orthoretrovirinae subfamily. Retroviruses typically comprise a diploid RNA genome and possess a reverse transcriptase enzyme, which may convert (i.e. reverse-transcribe) the RNA genome into DNA. This viral DNA may be integrated into the host cell's DNA with the help of an integrase enzyme. Once integrated, the viral DNA can be transcribed and translated by the host cell, allowing the virus to replicate and produce new viral particles. The skilled person is familiar with the term “beta-retrovirus” and “gammaretrovirus” as used herein. Beta-retroviruses and gamma-retroviruses are distinct groups within the Retroviridae family, reflecting their separate evolutionary lineages. Both Retroviridae subgroups

[0106] 1comprise a single-stranded RNA genome characteristically encoding the three major genes Gag, pol, and env for viral reproduction, wherein the gag encodes for structural proteins (group-specific antigen proteins), pol encodes for enzyme functions - including reverse transcriptase, RNAse and integrase -involved in viral replication, and env encodes for envelope glycoproteins that may be involved in viral attachment and host cell entry. Beta-retroviruses are known to primarily infect mammals and are known to be associated, among other things, with mammary tumors. Gamma-retroviruses can infect a broader range of species, including mammals, birds, and reptiles and are known to be associated, among other things, leukaemia, immunodeficiencies, and neurological disorders. Human endogenous retroviruses (HERVs) of the present disclosure are classified on the basis of animal retroviral homologies. For instance, HERVs belonging to class I show, among other things, particular homologies to mammalian gamma-retroviruses (type C). In the context of the present disclosure, they are therefore also described as (human) gamma-endogenous retroviruses (gamma-ERVs / gamma-HERVs). For instance, HERVs belonging to class II show, among other things, particular homologies to mammalian beta-retroviruses (Type B). In the context of the present disclosure, they are therefore also described as (human) beta-endogenous retroviruses (beta-ERVs / beta-HERVs).

[0107] “Virus” antigens or “viral” antigens as used herein may be any antigenic polypeptide of a virus. This includes viruses capable of causing infectious diseases, as well as viruses capable of causing cancer, with or without a preceding infectious disease. Preferably, a viral antigen in the context of the present invention relates to an antigen from a virus that is not an endogenous retrovirus.

[0108] Percentage identity or % identity: this term refers to a percentage of nucleotides or amino acids, which are identical in an optimal alignment between two nucleotide or amino acid sequences to be compared. Comparisons of two sequences usually requires a step of optimal alignment, which may be carried out manually or with the aid of the local homology algorithm by Smith and Waterman, 1981, Ads App. Math. 2, 482, with the aid of the local homology algorithm by Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443, and with the aid of the similarity search algorithm by Pearson and Lipman, 1988, Proc. Natl Acad. Sei. USA 85, 2444 or with the aid of computer programs using said algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N and TFASTA in Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.). Then one or more local regions of the two sequences corresponding to each other are identified. Percentage identity throughout the sequences is then calculated by determining the number of identical positions shared by the two sequences, which is divided by length of the reference sequence. This result is then multiplied by 100. In other words, the percent sequence identity defines the total number of aminoacids (when amino acid sequences are compared) or total number of nucleotides (when nucleotide sequences are compared) that are identical in the query sequence over the entire length of the reference sequence. The program " BLAST 2 sequences" is an exemplary tool to perform this calculation, available on the website http: / / www.ncbi.nlm.nih.gov / blast / bl2seq / wblast2.cgi. If herein a particular SEQ ID NO sequence is mentioned in the context with any one of proteins A, B, C and / or D of the invention, then in preferred embodiments said protein A, B, C and / or D has at least 95% sequence identity with said respective sequence defined by said SEQ ID NO as indicated in the context of the respective protein A, B, C and / or D.

[0109] 5 73 ’ untranslated regions / UTRs'. As used herein, "3'” or “3’ end” of a nucleic acid is that end which has a free hydroxy group and "5'“ or “5’ end” end of a nucleic acid" that end which has a free phosphate group. In a diagrammatic representation of double-stranded nucleic acids, the 3 ' end is right-hand and the 5 ' end is on the left-hand side:

[0110] 5 ' end 5'-P-NNNNNNN-OH-3' 3' end

[0111] 3 HO-NNNNNNN-P— 5 '

[0112] “Untranslated regions ” or “UTRs ”, as used herein, refers to either of two sections, one on each side of a coding sequence of mRNA, which are not part of the protein coding region. On the 5' end the UTR is called 5' UTR (or leader sequence), on the 3' side the UTR is called 3' UTR (or trailer sequence). The UTRs may contain RNA elements regulating translation and / or transcription, as described elsewhere herein. For instance, the 5’ UTR facilitates translation initiation by allowing the ribosome to bind to the sequence and the 3 ’ UTR is for instance known to be involved in translation termination as well as post-transcriptional modification.

[0113] Nucleic acid molecule(s) encoding VLP forming polypeptides

[0114] The invention provides a nucleic acid molecule encoding an antigenic polypeptide fused to the cytoplasmic tail of a betaretro viral, such as HERV-K or IAPE, envelope protein. The invention also provides virus like particles (VLPs) comprising one or more antigenic proteins fused to the cytoplasmic tail of a betaretroviral, such as HERV-K or IAPE, envelope protein.

[0115] In a first aspect, the present invention relates to at least one nucleic acid molecule encoding (1) a first polypeptide A, wherein the polypeptide A is a group-specific antigen (Gag) protein of a beta-retrovirus; and (2) a second polypeptide, wherein the second polypeptide comprises the polypeptides B, C and D in the order B-C-D or D-C-B; wherein polypeptide B is an antigenic polypeptide, wherein the antigenic polypeptide is not an endogenous retrovirus (ERV) envelopeprotein, polypeptide C is a transmembrane domain (TMD), and polypeptide D is a cytoplasmic tail (CT) of a viral envelope protein or fragment thereof, wherein said first polypeptide and second polypeptide is encoded by the same nucleic acid molecule or by two different nucleic acid molecules. In one aspect, polypeptide B alternatively is an antigenic polypeptide of a virus causing an infectious disease.

[0116] In one aspect, polypeptide B alternatively is an antigenic polypeptide of a respiratory virus.

[0117] In one aspect, polypeptide B alternatively is an antigenic polypeptide of a negative sense RNA virus.

[0118] In the context of the present invention, the “order” indicates the placement of individual polypeptide components in the direction amino-terminus to C-terminus. The order B-C-D of polypeptides B, C and D of the first polypeptide is preferred, wherein antigenic polypeptide B is followed by a transmembrane domain (TMD) polypeptide C, which is followed by a cytoplasmic tail (CT) polypeptide D of a viral envelope protein or a fragment thereof.

[0119] By “the second polypeptide comprises the polypeptides A, B and C” is meant that between each of the polypeptides A, B and C there can be one or more amino acids or no amino acids. In a first option the polypeptides are linked to each other via a linker peptide or linker amino acid and in the second option the polypeptides A, B and C are joined to each other on the polypeptide directly without any linking amino acids or linking peptides, i.e. linked directly. As defined herein, the term “comprise” signifies that the (second) polypeptide may comprise further components including other polypeptides.

[0120] The at least one nucleic acid of the invention, which encodes the polypeptides described herein, is suitable to encode the polypeptides B, C and D as well as A (on the same or a different nucleic acid molecule) to be expressed as part of a virus like particle (VLP), which may form upon expression of the polypeptide B, C, D and A components in the same cell. VLPs closely resemble viruses made up of one or more different molecules with the ability to self-assemble and mimic formation, release, shape / appearance and size of a virus particle but lacking the genetic material component for infecting a host cell, which is usually comprised by a virus. In the context of the present invention, they can for instance form by encoding a viral Gag polypeptide along with any antigen suitable to be presented on a VLP surface, such as a viral surface antigen or a viral envelope (Env) polypeptide, a polypeptide comprising the minimal structural or sequential features, which are required for polypeptide association in the process of VLP assembly. The encoded Gag polypeptide genetic sequence is translated into a polyprotein, which mediates the formation of the VLP in the absence of other viral proteins, incorporating the antigenic polypeptide on the VLP’s surface.In another aspect the present invention relates to a virus like particle (VLP) comprising the polypeptides A, B, C and D as outlined above.

[0121] In one embodiment the invention also relates to the at least one nucleic acid molecule or the VLP of the invention or a pharmaceutically acceptable salt thereof.

[0122] In one embodiment polypeptide D is a cytoplasmic tail (CT) of a viral envelope protein or fragment thereof capable of binding to the first polypeptide A so that when the first and second polypeptides are expressed in the same cell, virus like particles (VLPs) are formed that comprise said first and second polypeptide. One exemplary experimental method to test binding of a Gag protein to the cytoplasmic tail of an Env protein is a GST (Glutathione S-transferase) pull-down assay, for instance as described by Cosson et al. (1996) (Cosson P. Direct interaction between the envelope and matrix proteins of HIV-1. EMBO J. 1996 Nov 1;15(21):5783-8. PMID: 8918455; PMCID: PMC452325). The assay utilizes the affinity between the glutathione S-transferase (GST) tag and its binding partner, allowing for the detection of protein-protein interactions. In principle a GST-tagged gag protein and the cytoplasmic tail section are expressed in isolation, purified and the GST-tagged gag is coupled to glutathione agarose bead. The GST-tagged gag protein coupled beads are then incubated with the cytoplasmic tail protein (which does not comprise a tag) in a suitable binding buffer as known in the art to enable binding of Gag and tail. Associated proteins are then eluted from the beads, collected, and analysed using techniques known in the art such as SDS-PAGE and western blotting, which indicate the association / binding between the two proteins by distinct changes in molecular mass as indicated by a band shift.

[0123] When incorporated in a biological membrane, the first polypeptide comprising polypeptides B, C and D may have either of the following orientations: (a) polypeptide D (cytoplasmic tail) may face towards the inner membrane side, e.g. the inside of a VLP or the intracellular or cytosolic compartment of a cell, polypeptide C (transmembrane domain) may span through the membrane section of the VLP or cell membrane and polypeptide B (antigenic polypeptide) may face towards the extracellular compartment or the exterior of the VLP, i.e. being displayed at the cell or VLP membrane surface; or (b) polypeptide B (antigenic polypeptide) may face towards the inner membrane side, e.g. the inside of a VLP or the intracellular or cytosolic compartment of a cell, polypeptide C (transmembrane domain) may span through the membrane section of the VLP or cell membrane and polypeptide D (cytoplasmic tail) may face towards the exterior of the membrane of the VLP or cell.

[0124] Further applicable and potentially beneficial features of VLPs to be encoded by the at least one nucleic acid molecule of the invention will be clear to a skilled person of the relevant technicalfield. Some exemplary beneficial encoded features are outlined herein further below. The present disclosure also provides further exemplary and preferred embodiments of polypeptides A, B, C and D herein further below.

[0125] Cytoplasmic tail (CT, polypeptide D)

[0126] There are at least two ways of antigenic display or antigen provision after cellular contact and uptake of a composition of the invention to initiate an immune response: as MHC class I antigens on the contacted cell’s surface, presented to CD8+ T cells, or as MHC class II antigens on professional antigen-presenting cells. Both mechanisms emphasize the importance of surface display for antigen detection in the immunization process.

[0127] In this context, surprisingly, it has been found in the present experiments that incorporation of the cytoplasmic tail of an envelope protein of a beta-retrovirus, such as of a human endogenous retrovirus (HERV) and for instance HERV-K, in a polypeptide which is capable to be displayed at a cell or other membrane surface, improves the surface display of said polypeptide and particularly of antigenic sections exposed at the biological membrane surface. Said polypeptide comprising a cytoplasmic tail of an envelope protein of a beta-retrovirus may comprise further polypeptide components, such as a transmembrane domain and / or a surface (displayed) subunit or domain, such as a displayed antigenic polypeptide, which may be of different origin than said envelope protein of a beta-retrovirus. Therefore, the polypeptide, described herein as second polypeptide comprising polypeptides B, C and D, may also be described as a chimeric polypeptide.

[0128] The surface display of a surface domain or surface unit, such as of the antigenic polypeptide described herein, of such a chimeric polypeptide investigated herein was particularly improved for a chimeric polypeptide comprising the cytoplasmic tail of an envelope protein of a beta-retrovirus (or fragment thereof) when said chimeric protein was co-expressed with a Gag protein (first polypeptide herein) from the same beta-retrovirus.

[0129] Without wishing to be bound by theory, this is thought to be the case due to particular compatibility of the cytoplasmic tail (or fragment thereof) and Gag components during the formation of a VLP. It is believed that said compatibility promotes the ease of interaction of these two components, promotes the assembly of these components at the cell membrane or VLP membrane and promotes the formation of VLPs. Interestingly analogously to the described improved surface display, also the immunogenicity raised in response to a displayed antigenic polypeptide B was improved, as quantified by an increase of antigen-specific antibody formation.The increased surface display and immunogenicity effects were, to the surprise of the inventors, even observed when the antigenic polypeptide B of the second polypeptide was not from the same beta-retrovirus as polypeptides D and A. Surface display and immunogenicity of antigenic polypeptide B of different origin than polypeptides A and D (of the same beta-retrovirus), was improved compared to the same antigenic polypeptide B being expressed with polypeptides C, D and A originating from the same virus as polypeptide B. The effect was exemplarily shown for antigenic polypeptide B being of a different virus than a beta-retrovirus, like a gamma-endogenous retrovirus, for instance HERV-W. This finding is surprising since it would be expected that, instead, an antigenic surface displayable polypeptide component from one virus would be displayed and recognized by the immune system best, if said polypeptide was displayed with its “own” compatible components allowing the surface display. However, in the case of HERV-W no VLP formation at all was observed for expression of a construct encoding both Gag and Env polypeptides from HERV-W.

[0130] Thus, in another embodiment, (a) polypeptides C, D and A are from the same beta-retrovirus and preferably of HERV-K or IAPE; and (b) polypeptide B is an antigenic part of a protein of a virus, preferably wherein the protein is Influenza A Hemagglutinin or human metapneumovirus glycoprotein F.

[0131] In another embodiment, (c) polypeptides B, C, D are from the same virus and preferably of Influenza A; and (d) polypeptide A is of a beta-retrovirus, preferably of HERV-K.

[0132] In a preferred embodiment (i) of the nucleic acid or the VLP, (a) polypeptides C, D and A are of HERV-K or of IAPE; and (b) polypeptide B is any antigen but not an antigen that is an endogenous retrovirus (ERV) envelope protein. In another preferred embodiment (a) polypeptides C, D and A are of HERV-K or of IAPE; and (b) polypeptide B is not an antigen of HERV-K. In another preferred embodiment (i) of the nucleic acid or the VLP, (a) polypeptides C, D and A are of HERV-K; and (b) polypeptide B is antigenic polypeptide of a virus causing an infectious disease. In another preferred embodiment (a) polypeptides C, D and A are of HERV-K or of IAPE; and (b) polypeptide B is an antigenic polypeptide of a negative sense RNA virus. In another preferred embodiment (a) polypeptides C, D and A are of HERV-K or of IAPE; and (b) polypeptide B is an antigenic polypeptide form a respiratory virus.

[0133] In another preferred embodiment (i) of the nucleic acid or the VLP, (a) polypeptides C, D and A are from the same beta-retrovirus and preferably of HERV-K or IAPE; and (b) polypeptide B is any antigen but not an endogenous retrovirus (ERV) envelope protein and preferably polypeptide B is an antigenic part of a virus protein, preferably wherein polypeptide B is a virus surface antigen. In anotherembodiment (a) polypeptides D and A are from the same beta- retrovirus and preferably of HERV-K or IAPE; and (b) polypeptide B is any antigen and preferably polypeptide B is a polypeptide or an antigenic part thereof of a respiratory virus, preferably being Influenza A HA or hMPV F-glycoprotein, or a part thereof. In an alternative embodiment (ii) of the nucleic acid or the VLP, (c) polypeptides B, C, D are from the same virus and preferably of a respiratory virus (more preferably of Influenza); and (d) polypeptide A is of a beta-retrovirus, preferably of HERV-K.

[0134] The VLP vaccination platform of the invention is useful for presenting a variety of different antigens to a subject’s immune system for immunization. While practically any antigen can be presented using the VLP platform of the invention, which is suitable to be presented on the surface of a cell membrane or on a VLP, certain antigens can preferably be used in the context of the present invention. Thus, the invention in particular further relates to the following aspects:

[0135] The platform of the present invention is suitable to present antigens of viruses causing an infectious disease. Thus, in one aspect, the present invention provides at least one nucleic acid molecule encoding (1) a first polypeptide A, wherein the polypeptide A is a group-specific antigen (Gag) protein of a beta-retrovirus; and (2) a second polypeptide, wherein the second polypeptide comprises the polypeptides B, C and D in the order B-C-D or D-C-B; wherein polypeptide B is an antigenic polypeptide of a virus causing an infectious disease, polypeptide C is a transmembrane domain (TMD), and polypeptide D is a cytoplasmic tail (CT) of a viral envelope protein or fragment thereof, wherein said first polypeptide and second polypeptide is encoded by the same nucleic acid molecule or by two different nucleic acid molecules.

[0136] The platform of the present invention is suitable to present antigens of respiratory viruses. Thus, in one aspect, the present invention provides at least one nucleic acid molecule encoding (1) a first polypeptide A, wherein the polypeptide A is a group-specific antigen (Gag) protein of a betaretrovirus; and (2) a second polypeptide, wherein the second polypeptide comprises the polypeptides B, C and D in the order B-C-D or D-C-B; wherein polypeptide B is an antigenic polypeptide of a respiratory virus, polypeptide C is a transmembrane domain (TMD), and polypeptide D is a cytoplasmic tail (CT) of a viral envelope protein or fragment thereof, wherein said first polypeptide and second polypeptide is encoded by the same nucleic acid molecule or by two different nucleic acid molecules. In a preferred embodiment, the respiratory virus is selected from the group consisting of influenza virus, respiratory syncytial virus, parainfluenza virus, metapneumovirus, rhinovirus, coronavirus, adenovirus and bocavirus.The platform of the present invention is suitable to present antigens of negative sense RNA viruses. Thus, in one aspect, the present invention provides at least one nucleic acid molecule encoding (1) a first polypeptide A, wherein the polypeptide A is a group-specific antigen (Gag) protein of a beta-retrovirus; and (2) a second polypeptide, wherein the second polypeptide comprises the polypeptides B, C and D in the order B-C-D or D-C-B; wherein polypeptide B is an antigenic polypeptide of a negative sense RNA virus, polypeptide C is a transmembrane domain (TMD), and polypeptide D is a cytoplasmic tail (CT) of a viral envelope protein or fragment thereof, wherein said first polypeptide and second polypeptide is encoded by the same nucleic acid molecule or by two different nucleic acid molecules. In a preferred embodiment the antigen is an antigen of Orthornavirae. In a more preferred embodiment the antigen is an antigen of Negarnaviricota. In an even more preferred embodiment, the antigen is an antigen of Orthomyxoviridae, Paramyxoviridae or Pneumoviridae. In a preferred embodiment, the Orthomyxoviridae are selected from the group consisting of Alphainfluenzavirus, Betainfluenzavirus, Gammainfluenzavirus and Deltainfluenzavirus. In a preferred embodiment, the Paramyxoviridae are selected from the group consisting of Parainfluenza Virus 1, Parainfluenza Virus 2, Parainfluenza Virus 3, Parainfluenza Virus 4. In a preferred embodiment, the Pneumoviridae are selected from the group consisting of human metapneumovirus (HMPV), human respiratory syncytial virus A2 (HRSV-A2) and human respiratory syncytial virus B1 (HRSV-B1). In the context of selection viral antigens in the context of the VLP vaccination platform of the present invention, antigens of the viruses RSV, hMPV and Influenza (A) are particularly preferred. Exemplary antigens of these viruses include RSV glycoprotein F, hMPV glycoprotein F and Influenza (A) hemagglutinin (HA).

[0137] In a preferred embodiment, polypeptide B is an antigenic polypeptide of a virus of Paramyxoviridae. In a more preferred embodiment, polypeptide B is an antigenic polypeptide of a Metapneumo virus. In an even more preferred embodiment, polypeptide B is an F-glycoprotein of a Metapneumovirus. In a more preferred embodiment, polypeptide B is an F-glycoprotein of a Metapneumovirus, wherein the F-gly coprotein has a sequence identity of at least 95% with SEQ ID NO: 67, most preferably a sequence identity of 100% with SEQ ID NO: 67.

[0138] In a preferred embodiment the polypeptide D (CT) is of a cytoplasmic tail of an envelope (Env) protein of said beta-retrovirus or a fragment thereof. In a more preferred embodiment of the embodiment (i), the polypeptide D (CT) is of a cytoplasmic tail of the envelope (Env) protein of HERV-K or a fragment thereof.The at least one nucleic acid molecule, the composition or the VLP according to claim 9, wherein polypeptide D (CT) is of a cytoplasmic tail of an envelope (Env) protein of said beta-retrovirus or a fragment thereof; and / or wherein the c-terminus of said first polypeptide comprises a GSG linker and wherein said nucleic acid molecule encodes downstream of said GSG linker a p2A peptide capable of inducing ribosomal skipping during translation, wherein said GSG-linker and p2A peptide preferably have the amino sequences of SEQ ID NO: 55 and SEQ ID NO: 56, respectively.

[0139] Particularly the cytoplasmic tail (CT) of a viral envelope protein (polypeptide D) or fragment thereof was found to be of relevance for a successful and / or improved surface display. A skilled person in the technical field is aware of methods to acquire further precision in defining the relevant boundaries of cytoplasmic tail sections to be included in antigenic polypeptides, such as Env proteins, e.g. by screening for cell surface expression and folding / display functionality of constructs with variable engrafted cytoplasmic tail lengths and sections, when co-expressed with beta-retroviral Gag, and preferably when co-expressed with homologous / corresponding beta-retroviral Gag (homologous / corresponding meaning in this context that the beta-retrovirus of origin for e.g. the Gag polypeptide and the cytoplasmic tail section is the same).

[0140] Regarding the parts / sections of a cytoplasmic tail of beta-retroviral envelope proteins, which are most useful for improving the VLP or cellular surface display of antigenic polypeptides, it was found that pseudo-typing of heterologous Env and Gag proteins (i.e. mixing Env and Gag expression association between different viral species of origin) is obstructed by changing or truncating the most distal (C-terminal) part of the Env’s cytoplasmic tail, e.g. about 20 amino acids (Hanke K, Kramer P, Seeher S, Beimforde N, Kurth R, Bannert N. Reconstitution of the ancestral glycoprotein of human endogenous retrovirus k and modulation of its functional activity by truncation of the cytoplasmic domain. J Virol. 2009 Dec;83(24): 12790-800. doi: 10.1128 / JVI.01368-09. Epub 2009 Oct 7. PMID: 19812154; PMCID: PMC2786854). It was found that progressive truncations of the Env protein's C-terminal tail had effects on particle incorporation and viral functions. Without wishing to be bound by theory, it is suspected that this C-terminal section of the Env cytoplasmic tail may be the section mostly interacting with HERV-K Gag during VLP formation. Particularly a short truncation at the C-terminal cytoplasmic tail end resulted in a maturation defect with no particle incorporation (see Hanke et al. 2009). It is envisioned that the C-terminal tail plays a role in the maturation, incorporation, andfusing ability of beta-retroviral envelope proteins such as, HERV-K Env. In view of VLP formation it is further believed that the last 6 such as the last 20 amino acid positions (terminal of C-terminus of the cytoplasmic tail) are likely relevant for the interaction with a Gag protein, e.g. during VLP formation involving beta- retroviral envelope proteins such as, HERV-K Env. For instance, in view of HERV-K Envelope protein, it is suspected that - again without wishing to be bound by theory - the C-terminal last 6 amino acid residues, IVTVSV, of the cytoplasmic HERV-K Env tail can interact with and possibly even be buried within the associating Gag protein. This is expected to work particularly well with the corresponding HERV-K Gag protein.

[0141] Thus, in another embodiment the polypeptide D comprises at least 6 continuous amino acid residues of the C-terminus of a beta-retroviral envelope protein and wherein the C-terminal amino acid of said continuous amino acid residues is the C-terminal amino acid of said envelope protein.

[0142] As mentioned, it was surprisingly found that especially the combination of Gag and cytoplasmic tail or a cytoplasmic tail section of an Env protein of HERV-K are particularly useful to improve surface display of antigenic polypeptides, on VLPs and / or cells, wherein these antigenic polypeptides and said HERV-K Env cytoplasmic tail or cytoplasmic tail section are part of the same polypeptide. Therefore, the polypeptide D, i.e. the cytoplasmic tail, may advantageously comprise at least certain suitable sequence portions of the cytoplasmic tail of HERV-K Env. Preferred amino acid sequences of the cytoplasmic tail or section thereof are for instance as defined herein by SEQ ID NO:s 9, 10, 11, 12 and 27.

[0143] The same principles as described above in the context of HERV-K VLPs and the interaction of HERV-K Gag and HERV-K Env via the cytoplasmic tail section apply for instance also in the context of IAPE VLPs, i.e. VLPs derived from the murine endogenous retrovirus “Intracisternal A-type Particles elements with an Envelope”, which is another - however not human - betaretrovirus. The present inventors found that IAPE VLPs, the IAPE Env protein and the IAPE Gag protein interact according to the same principles to form VLPs as described for HERV-K VLPs above. Accordingly, it was surprisingly found that also the combination of Gag of IAPE and the cytoplasmic tail or a cytoplasmic tail section of the Env protein of IAPE are particularly useful to improve surface display of antigenic polypeptides on VLPs and / or cells, wherein these antigenic polypeptides and said IAPE Env cytoplasmic tail or cytoplasmic tail section are part of the same polypeptide.

[0144] The above observations and principles were made for two betaretroviruses and their VLPs, wherein these betaretroviruses are rather remote from one another, i.e. one being human and one being murine. Thus, the above observed mechanism applies to betaretroviral VLP components in general. Itfollows that a betaretroviral Gag protein combined with a corresponding cytoplasmic tail (and optionally transmembrane domain) of an envelope (Env) protein of a betaretrovirus, such as a of the same betaretrovirus, can be used to effectively display a variety of different antigens, following the above observed principles and leading to the surprisingly improved display and immunization effects described in the examples herein.

[0145] Thus, in one embodiment the polypeptide D comprises at least 6 continuous amino acid residues of the C-terminus of a HERV-K envelope protein and wherein the C-terminal amino acid of said continuous amino acid residues is the C-terminal amino acid of said HERV-K envelope protein. In a preferred embodiment polypeptide D comprises the amino acid residues IVTVSV (SEQ ID NO: 9), or comprises the amino acid residues the amino acid residues QIVTVSV (SEQ ID NO: 10), or comprises the amino acid residues KRKGGNVGKSKRDQIVTVSV (SEQ ID NO: 11), or comprises the ammo acid residues AVLSKRKGGNVGKSKRDQIVTVSV (SEQ ID NO: 12), or comprises the ammo acid residues RAMMTMAVLSKRKGGNVGKSKRDQIVTVSV (SEQ ID NO: 27). It is understood that also sequence variations of the amino acid sequences defined by SEQ ID NO:s 9, 10, 11, 12 and 27 may be used in the polypeptide of the invention, preferably wherein the variations consist of conservative amino acid changes as defined herein. The skilled person is aware of which amino acid changes may be performed while maintaining relevant conformations and functions of the cytoplasmic tail. Thus, in an alternative embodiment, polypeptide D comprises a polypeptide according to any of SEQ ID NOs: 9, 10, 11, 12 or 27 in which the polypeptide comprises at least one conservative amino acid substitution compared with the respective reference sequence SEQ ID NO: 9, 10, 11, 12 or 27.

[0146] In an alternative embodiment, polypeptide D comprises a polypeptide according to SEQ ID NO: 47 in which the polypeptide comprises at least one conservative amino acid substitution compared with the respective reference sequence SEQ ID NO: 47. In a preferred embodiment, polypeptide D comprises a polypeptide according to SEQ ID NO: 47.

[0147] In a preferred embodiment the polypeptide D comprises at least 6 continuous amino acid residues of the C-terminus of a HERV-K envelope protein wherein the C-terminal amino acid of said continuous amino acid residues is the C-terminal amino acid of said HERV-K envelope protein. In a more preferred embodiment polypeptide D comprises the amino acid residues IVTVSV (SEQ ID NO: 9).In a preferred embodiment the polypeptide D comprises at least 6 continuous amino acid residues of the C-terminus of an IAPE envelope protein wherein the C-terminal amino acid of said continuous amino acid residues is the C-terminal amino acid of said IAPE envelope protein.

[0148] It was further surprisingly found that a truncated form of HERV Env proteins comprising the TMD-CT (transmembrane domain and cytoplasmic tail) domain of HERV-K Env, wherein the cytoplasmic tail or a C-terminal section of the cytoplasmic tail of the TMD-CT domain of HERV-K Env is deleted, is useful to improve surface expression and display of antigenic polypeptides, such as Env proteins, on VLPs and / or cells. Therefore, the polypeptide D, i.e. the cytoplasmic tail, may advantageously have the amino acid sequence of the cytoplasmic tail of the HERV-K Env and may be C-terminally truncated at least to some extent.

[0149] Thus, in another embodiment

[0150] (a) in polypeptides D at least the 6 continuous terminal amino acid residues of the C-terminus of polypeptide D are deleted, preferably wherein at least the 20 continuous terminal amino acid residues of the C-terminus of polypeptide D are deleted, more preferably wherein at least the 30 continuous terminal amino acid residues of the C-terminus of polypeptide D are deleted, or wherein polypeptide D is deleted; or

[0151] (b) polypeptides C and D together comprise the polypeptide sequence TIGSTTIINLILILVCLFCLLLVCRCTQQLRRDSDHRERAMMTMAVLSKRKGGNVGKSKRDQ

[0152] (SEQ ID NO: 29), or comprises the polypeptide sequence TIGSTTIINLILILVCLFCLLLVCRCTQQLRRDSDHRERAMMTMAVLSKRKGGNVGKSKRDQ

[0153] (SEQ ID NO: 29) in which the polypeptide comprises at least one conservative amino acid substitution compared with the reference sequence SEQ ID NO: 29; or

[0154] (c) polypeptides C and D together comprise the polypeptide sequence TIGSTTIINLILILVCLFCLLLVCRCTQQLRRDSDHRERAMMTMAVLS (SEQ ID NO: 30), or comprises the polypeptide sequence TIGSTTIINLILILVCLFCLLLVCRCTQQLRRDSDHRERAMMTMAVLS (SEQ ID NO: 30) in which the polypeptide comprises at least one conservative amino acid substitution compared with the reference sequence SEQ ID NO: 30; or

[0155] (d) polypeptides C and D together comprise the polypeptide sequence TIGSTTIINLILILVCLFCLLLVCRCTQQLRRDSDHRE (SEQ ID NO: 31), or comprises the polypeptide sequence TIGSTTIINLILILVCLFCLLLVCRCTQQLRRDSDHRE (SEQ ID NO: 31) inwhich the polypeptide comprises at least one conservative amino acid substitution compared with the reference sequence SEQ ID NO: 31; or

[0156] (e) polypeptide A is HERV-K Gag and polypeptide C and D are as defined in (a), (b), (c) or (d), or (f) polypeptide A is HERV-K Gag, polypeptide B is an antigenic part of a protein of a virus, and polypeptide C and D are as defined in (a), (b), (c) or (d). In another preferred embodiment polypeptide B is an antigenic part of a protein of a respiratory virus. In another preferred embodiment polypeptide B is an antigenic part of a protein of a negative sense RNA virus. In a particularly preferred embodiment polypeptide B is an antigenic part of a protein of a virus, wherein the protein is Influenza A Hemagglutinin (HA) or human metapneumovirus (hMPV) glycoprotein Fit was found that Env protein surface expression may particularly benefit from a truncation of the cytoplasmic tail of HERV-K-Env, replacing the cytoplasmic tail of the Env protein to be expressed, by 20 amino acid residues. Thus, in a preferred embodiment polypeptides C and D together comprise the polypeptide sequence TIGSTTIINLILILVCLFCLLLVCRCTQQLRRDSDHRERAMMTMAVLS (SEQ ID NO: 30), or comprises the polypeptide sequence TIGSTTIINLILILVCLFCLLLVCRCTQQLRRDSDHRERAMMTMAVLS (SEQ ID NO: 30) in which the polypeptide comprises at least one conservative amino acid substitution compared with the reference sequence SEQ ID NO: 30.

[0157] It was further found that it may also be beneficial for Env surface expression, if the full TMD-CT domain of HERV-K Env is included in the Env protein to be expressed and displayed. Thus, in another preferred embodiment polypeptides C and D together comprise the polypeptide TIGSTTIINLILILVCLFCLLLVCRCTQQLRRDSDHRERAMMTMAVLSKRKGGNVGKSKRDQ IVTVSV (SEQ ID NO: 8), or comprise the polypeptide sequence TIGSTTIINLILILVCLFCLLLVCRCTQQLRRDSDHRERAMMTMAVLSKRKGGNVGKSKRDQ IVTVSV (SEQ ID NO: 8) in which the polypeptide comprises at least one conservative amino acid substitution compared with the reference sequence SEQ ID NO: 8.

[0158] It was found to be particularly advantageous for an effective surface expression to exchange the last 6 C-terminal amino acid residues in the cytoplasmic tail of an Env protein to be expressed and displayed by the last 6 C-terminal amino acid residues in the cytoplasmic tail of an HERV-K Env. However, it may be also be advantageous to simply attach these last 6 C-terminal amino acid residues in the cytoplasmic tail of an HERV-K Env to the C-terminus of an Env protein to be expressed and displayed.Thus, in yet another embodiment, the at least one nucleic acid molecule or the VLP according to any of the preceding claims, wherein at the C-terminus of polypeptide D, 6 continuous amino acid residues of the C-terminus of a beta-retroviral envelope protein are attached, and preferably wherein the 6 continuous amino acid residues of the C-terminus of a beta-retroviral envelope protein comprise the amino acid sequence IVTVSV (SEQ ID NO: 9) or an amino acid sequence according to SEQ ID NO: 9 in which the amino acid sequence comprises at least one conservative amino acid substitution compared with the respective reference sequence SEQ ID NO: 9.

[0159] In a preferred embodiment polypeptides C and D encoded by a nucleic acid molecule of the invention or comprised by a VLP of the invention together comprise the polypeptide sequence TIGSTTIINLILILVCLFCLLLVCRCTQQLRRDSDHRERAMMTMAVLS (SEQ ID NO: 30), or comprises the polypeptide sequence TIGSTTIINLILILVCLFCLLLVCRCTQQLRRDSDHRERAMMTMAVLS (SEQ ID NO: 30) in which the polypeptide comprises at least one conservative amino acid substitution compared with the reference sequence SEQ ID NO: 30, wherein at the C-terminus of polypeptide D, 6 continuous amino acid residues of the C-terminus of a beta-retroviral envelope protein are attached, wherein the 6 continuous amino acid residues of the C-terminus of a beta-retroviral envelope protein comprise the amino acid sequence IVTVSV (SEQ ID NO: 9) or an amino acid sequence according to SEQ ID NO: 9 in which the amino acid sequence comprises at least one conservative amino acid substitution compared with the respective reference sequence SEQ ID NO: 9.

[0160] Transmembrane domain (TMD, polypeptide C)

[0161] In one embodiment, the polypeptide C, i.e. the transmembrane domain, comprises at least on alpha helical polypeptide structure. In a preferred embodiment, polypeptide C comprises from about 15 to 55 amino acid residues. In an even more preferred embodiment polypeptide C is a transmembrane domain (TMD) of a bitopic type I integral transmembrane protein. Bitopic type I integral transmembrane proteins are known to be single-pass transmembrane proteins, which comprise a cytoplasmic C-terminus and an extracellular / luminal N-terminus.

[0162] In the experiments performed herein it was found that it is not only particularly advantageous to include in a polypeptide comprising a surface-displayable antigenic polypeptide, such as a viral Env protein, a cytoplasmic tail or fragment thereof of a beta-retrovirus and preferably from the same beta-retrovirus, from which also a co-expressed Gag protein is derived, to achieve an improved surface expression on cells / VLPs and an increased immunogenicity of said antigenic polypeptide. It was alsofound to be particularly advantageous to include in the same polypeptide, comprising said surface-displayable antigenic polypeptide, the transmembrane domain (polypeptide C) from said virus and preferably from said same beta-retrovirus from which also the cytoplasmic tail (polypeptide D) and possibly even said Gag protein (first polypeptide A) is derived of.

[0163] Thus, in one embodiment, the polypeptide C is a transmembrane domain (TMD) of a virus. In a preferred embodiment, the polypeptide C is a transmembrane domain (TMD) of a beta-retrovirus. In an even more preferred embodiment the polypeptide C is a transmembrane domain (TMD) of the same beta-retrovirus as the first polypeptide A, i.e. the group-specific antigen (Gag) protein of the same beta-retrovirus. In a most preferred embodiment the polypeptide C is of an envelope (Env) protein of said same beta-retrovirus.

[0164] As found in the experiments herein, a polypeptide C transmembrane domain of a HERV-K envelope protein (Env) may advantageously be used. It was further found to be especially suitable to include both the polypeptide C transmembrane domain and the polypeptide D cytoplasmic domain of HERV-K in a (second) polypeptide to display an antigenic polypeptide (B) also comprised by said (second) polypeptide as defined herein. It may therefore be useful for the second polypeptide as defined herein, to comprise the (continuous) amino acid sequence of the transmembrane domain and the cytoplasmic tail of HERV-K Env domain as polypeptides C and D as defined by SEQ ID NO: 8 or to comprise said sequence as a portion of the polypeptides C and D. Thus, in another alternative embodiment polypeptide C and D together comprise the polypeptide TIGSTTIINLILILVCLFCLLLVCRCTQQLRRDSDHRERAMMTMAVLSKRKGGNVGKSKRDQ IVTVSV (SEQ ID NO: 8). It is understood that also sequence variations of the amino acid sequence defined by SEQ ID NO: 8 may be used in the polypeptide of the invention, preferably wherein the variations consist of conservative amino acid changes as defined herein. The skilled person is aware of which amino acid changes may be performed while maintaining relevant conformation and function of the cytoplasmic tail. Thus, in another alternative embodiment polypeptide C and D together comprise the polypeptide TIGSTTIINLILILVCLFCLLLVCRCTQQLRRDSDHRERAMMTMAVLSKRKGGNVGKSKRDQ IVTVSV (SEQ ID NO: 8) in which the polypeptide comprises at least one conservative amino acid substitution compared with the reference sequence SEQ ID NO: 8.

[0165] In another alternative embodiment polypeptide C and D together comprise the polypeptide according to SEQ ID NO: 47, in which the polypeptide comprises at least one conservative amino

[0166] 31acid substitution compared with the reference sequence SEQ ID NO: 47. In a preferred embodiment polypeptide C and D together comprise the polypeptide according to SEQ ID NO: 47.

[0167] In a preferred embodiment, polypeptides C and D together comprise a sequence comprising at least 90%, preferably at least 95%, most preferably 100% sequence identity with a sequence selected from SEQ ID NO:s 8 and 47.

[0168] Antigenic polypeptide (polypeptide B)

[0169] The experimental data provided herein taken as a whole show that the nucleic acid molecule of the invention is useful to produce VLPs that incorporate a variety of different antigens, for example different types of envelope (ENV) proteins and other antigens suitable for surface display, such as viral surface antigens, such as Influenza HA or human metapneumovirus glycoprotein F, as polypeptide “B” of the invention. In view of these results when using the nucleic acid molecule of the invention also other proteins than ENV proteins can effectively be incorporated as polypeptide “B” into VLPs. Suitable proteins that can be used as polypeptide “B” of the invention include proteins where the C-terminal amino acid is not embedded inside the folded protein. Glycoproteins are preferred examples of “B” polypeptides of the invention.

[0170] The present invention is a suitable platform for displaying antigens to the immune system. Thus, in principle the coding sequence for any type of protein, against which it is desired to raise an immune response, can be incorporated in the at least one nucleic acid molecule of the invention to encode polypeptide B as described herein.

[0171] It is generally believed that expression of the at least one nucleic acid molecule described herein directs the antigenic polypeptide B to dendritic cells (DCs), which present antigens to cells of the adaptive immune system. Presentation of antigens on MHC class I induces activation and proliferation of CD8+ T cells. These cytotoxic T lymphocytes (CTLs), specific for the antigenic polypeptide, infiltrate cells, such as tumour cells, and kill such cells displaying the respective antigens. Presentation of antigens on MHC class II by professional antigen presenting cells (APCs) activates CD4+ T cells, which subsequently co-activate B cells. Activated B cells that encounter the antigenic polypeptide, such as an ERV Env target protein, in the circulation or antigens displayed on cells or VLPs release antibodies specific for the antigenic polypeptide (B), such as for example an ERV Env. These antibodies are able to bind their target (antigenic) polypeptide, e.g. on diseased cells, for instance cancer cells or virus infected cells or otherwise diseased cells, inducing destruction and phagocytosis of the diseased cells. The regained immunogenicity of diseased and / or tumor cellsenables priming of a set of diverse specific T cells recognizing different disease- or tumor-associated and -specific antigens. Newly primed and expanded cytotoxic T-cells (CTLs) infiltrate the diseased or tumor tissue and kill diseased and / or malignant / tumorous cells. In this way, for example antibodies directed against viral surface antigens, such as HA or glycoprotein F, or against envelope proteins (Env) are able to act against the diseased cells. Likewise, also other antigens characteristic of certain pathologies may be expressed to stimulate antibody production and immune response against cells of such cancer pathologies. Thus, presentation of the encoded antigenic polypeptides by means of the present invention may act as vaccination, wherein the vaccination is against infectious diseases, or tumor formation and / or progression, e.g. arising in the process of senescence, or against a variety of pathogens and the development of related diseases.

[0172] Thus, in another embodiment, polypeptide B is an antigenic polypeptideof a virus or an antigenic part thereof. As mentioned above, the antigen presentation strategy disclosed herein may also be used to vaccinate against tumor formation and progression by expressing tumor antigens. In an even more preferred embodiment, polypeptide B is an antigenic part of a viral Env or other viral surface protein or a viral antigenic polypeptide that is suitable for surface expression. In the experiments presented herein, antigenic polypeptide expression and presentation was found to be particularly efficient and useful in view of expression and presentation of certain antigenic polypeptides of the exemplary respiratory viruses Influenza (A) and human metapneumovirus (hMPV).

[0173] However, in principle any antigen can be displayed via the VLP platform of the invention, that was confirmed to be effective for display of and immunisation with the exemplary antigenic polypeptides tested in the examples herein. The VLPs, on which the VLP platform of the invention is based, are formed by assembly of group-specific antigen proteins (Gag) and envelope proteins (Env), originally of viral origin. The nomenclature of the surface displayed sections of Env proteins as surface unit (SU) and ectodomain (Ecto) and their orientation within the Env protein is known in the art is for instance as displayed in Figure 1 herein. In further embodiments, the antigenic part of an Env protein is or comprises the surface unit (SU) and / or ectodomain (Ecto) of said Env protein.

[0174] Any antigenic polypeptide that may be expressed on a cell surface, may in general be encoded as antigenic polypeptide (B) by the at least one nucleic acid molecule disclosed herein. In particular the antigenic polypeptide may be any tumor antigen may, for instance a tumor-specific antigen (TSA) or a tumor-associated antigen (TAA) as defined herein. TSAs and TAAs include but are not limited to the examples defined herein.In one embodiment, polypeptide B is an antigenic polypeptide that is a type I membrane protein. In a preferred embodiment, polypeptide B is a type I membrane protein associated with a diseases selected from cancer and senescence.

[0175] It has been established in the literature that certain antigens can be targeted via a vaccination to achieve a reduction of cellular senescence. Therefore, in a further preferred embodiment, the invention provides a nucleic acid molecule according to the invention as described herein, wherein polypeptide B is an antigenic polypeptide or a part of an antigenic polypeptide selected from the group consisting of Apolipoprotein D (ApoD), Apolipoprotein B (ApoB), CD153, CD30, CD87, CD9, CTSF (Cathepsin F), EGF, GPNMB), KLRG-1, NKG2DL, a peptide of amyloid-beta (A0), a peptide of Microtubule-associated protein tau (Tau), Dipeptidyl peptidase-4 (DPP4), IL-10, Prorenin, Angiotensin I, Angiotensin II, Angiotensin type 1 receptor (AT1R), Apha-ID adrenergic receptor (ADRA1D), PCSK9, ADAMTS7, NGF, ADAMI, GLI1, alpha-synuclein, VEGFR1, VEGFR2, Grehlin, CEACAM1, CEACAM1.2, CEACAM3, CEACAM5, CEACAM6, EGFR, EphA2, FOLR1, HER2, Mucinl iso2, Mucinl iso3, Mucinl6, PAP, PSCA, PSMA (isol), PSMA (iso3), uPAR, and SAGP.

[0176] In a more preferred embodiment, polypeptide B is an antigenic polypeptide selected from the group consisting of CEACAM1, CEACAM1.2, CEACAM3, CEACAM5, CEACAM6, EGFR, EphA2, FOLR1, HER2, Mucinl iso2, Mucinl iso3, Mucinl 6, PAP, PSCA, PSMA (isol), PSMA (iso3), uPAR, SAGP, CD153, a peptide of amyloid-beta (A0), a peptide of Microtubule-associated protein tau (Tau), DPP4, and alpha-synuclein.

[0177] In a more preferred embodiment, polypeptide B is an antigenic polypeptide selected from mCEACAMl and hCEACAM5.

[0178] Carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) belong to a group of mammalian immunoglobulin-related glycoproteins. They are involved in cell-cell recognition and modulate cellular processes that range from the shaping of tissue architecture and neovascularization to the regulation of insulin homeostasis and T-cell proliferation. CEACAMs have also been identified as receptors for host-specific viruses and bacteria in mice and humans, respectively, making these proteins an interesting example of pathogen-host co-evolution. Forward and reverse genetics in the mouse now provide powerful novel models to elucidate the action of CEACAM family members in vivo.

[0179] In one embodiment, polypeptide B, i.e. the antigenic polypeptide, is an antigenic part of a tumor antigen. In another embodiment, polypeptide B is an antigenic part of a polypeptide selectedfrom TSAs and TAAs. In another embodiment, polypeptide B is an antigenic part of a polypeptide selected from the group consisting of DMART-l / MelanA (MART-1), gplOO (PMEL17), tyrosinase, TRP-1, TRP-2 MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, pi5; CEA; p53, p21, Mucl, Ras, HER-2 / neu; BCR-ABL, E2A-PRL, H4-RET, 1GH-IGK, MYL-RAR; Epstein Barr virus antigen (EBVA) and human papillomavirus (HPV) antigen E6 and E7, TSP- 180, MAGE-4, MAGE-5, MAGE- 6, RAGE, NY-ESO, pl 85erbB2, p 1 80erbB-3, c-met, nm-23H 1, PSA, TAG- 72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, beta-Catemn, CDK4, Mum-1, pl5, pl6, 43-9F, 5T4 (791Tgp72), alpha-fetoprotein (AFP), beta-HCG, BCA225, BTAA, CA125, CA15-3\CA 27.29\BCAA, CA195, CA242, CA-50, CAM43, CD68\I, CO-029, FGF-5, G250, Ga733VEpCAM, HTgp-175, M344, MA-50, MG7-Ag, M0V18, NB / 70K, NY-CO-1, RCAS1, SDCCAG16, TA-90\Mac-2 binding protein / cyclophilin C-associated protein, TAAL6, TAG72, TLP, and TPS.

[0180] In one embodiment, polypeptide B, i.e. the antigenic polypeptide, is a viral antigen.

[0181] Immunization against seasonal virus infections is particularly relevant in view of annual epidemics of for instance the common cold and influenza disease, as well as other respiratory virus infections, which affect the human population in the winter seasons particularly in temperate regions. Thus, in one embodiment, Polypeptide B is a viral antigen of a respiratory virus. While the VLP platform of the invention is suitable to present an antigen of any respiratory virus, it is preferred that the polypeptide B is an antigenic polypeptide of a respiratory virus selected from the group consisting of influenza virus, respiratory syncytial virus, parainfluenza virus, metapneumovirus, rhinovirus, coronavirus, adenovirus and bocavirus.

[0182] Negative-strand RNA viruses are a broad group of animal viruses that comprise several important human pathogens, including for instance influenza, measles, mumps, rabies, respiratory syncytial, Ebola, and hantaviruses. Thus, in one embodiment, polypeptide B is an antigenic polypeptide of a negative sense RNA virus. In a more preferred embodiment polypeptide B is an antigenic polypeptide of Orthornavirae. In an even more preferred embodiment polypeptide B is an antigenic polypeptide of Negarnaviricota. In a yet more preferred embodiment polypeptide B is an antigen of Orthomyxoviridae, Paramyxoviridae or Pneumoviridae.

[0183] One of the most significant human disease caused by orthomyxoviruses is influenza (causing the flu). Thus, in a preferred embodiment polypeptide B is an antigen of a virus of Orthomyxoviridae, wherein the virus is selected from the group consisting of Alphainfluenzavirus, Betainfluenzavirus, Gammainfluenzavirus and Deltainfluenzavirus. In an even more preferred embodiment polypeptide B is an antigen of Influenza A, and most preferably polypeptide B is Influenza A hemagglutinin (HA).Also the family of Paramyxoviridae contains a number of highly relevant pathogens that are the causative agents of several pathologies known to affect humans and animals, including human parainfluenza, measles, canine distemper, rinderpest, Nipah virus, Newcastle disease, mumps, and human respiratory syncytial virus. Thus, in a preferred embodiment, polypeptide B is an antigenic polypeptide of Paramyxoviridae, preferably wherein Paramyxoviridae are selected from the group consisting of Parainfluenza Virus 1, Parainfluenza Virus 2, Parainfluenza Virus 3 and Parainfluenza Virus 4.

[0184] Pneumoviridae are another relevant group of viral pathogens, for particular relevance for instance in lower respiratory infections, especially in young children, but also affecting other patients of all ages. Thus in one embodiment polypeptide B is an antigenic polypeptide of Pneumoviridae, preferably wherein Pneumoviridae are selected from the group consisting of human metapneumovirus (hMPV), human respiratory syncytial virus A2 (hRSV-A2) and human respiratory syncytial virus Bl (hRSV-Bl). In a preferred embodiment polypeptide B is an antigenic polypeptide hMPV or RSV. In an even more preferred embodiment polypeptide B is an antigenic polypeptide selected from hMPV glycoprotein F and RSV glycoprotein F, most preferably wherein polypeptide B is hMPV glycoprotein F. In one embodiment, polypeptide B is an antigenic polypeptide of a retrovirus. In one embodiment, polypeptide B may be an Env protein of a virus, preferably of a retrovirus. In a more preferred embodiment, polypeptide B is an antigenic polypeptide of a retrovirus selected from HIV and SIV. In an even more preferred embodiment, polypeptide B is Envelope glycoprotein gpl40 of Simian immunodeficiency virus (SIV).

[0185] The immunisation effect evoked by expressing the antigenic polypeptide B may be detected and quantified via detection of the level of immune response in a subject, which is evoked by administering the at least one nucleic acid molecule or the VLP if the invention. For instance, antigenic polypeptide specific antibodies may be quantified in blood samples from an immunized subject. Conversely, such specific antibodies can be used to label the expressed antigenic polypeptide itself to analyse its presentation on cell surfaces in both in vitro and in vivo obtained samples, for instance using FACS. Furthermore, the propagation of immune cells, such as NK and T cells, as well as both extracellular and intracellular stained markers of these activated immune cells in samples from immunized subjects, such as mice may also be analysed via FACS or other suitable methods. Suitable markers of immune cell activation will be clear to a person skilled in the art. For instance, activated T cells could be detectable via IFNy, TNFa and CD44 and NK cells could be detectable via CD56.An exemplary method to test the effectivity of immunizing and / or evoking an immune response by an antigenic polypeptide B being a tumor antigen as described above, which is expressed as part of the polypeptide construct encoded by the at least one nucleic acid molecule of the invention, is to assess the resistance to the antigen-related tumor formation in a treated subject. For instance, a tumor challenge and tumor rejection assay may be performed, wherein subjects, i.e. animals, are injected with tumor cells, for instance cells of tumor cell lines B16F10-GP or CT26 or 4T1 or murine renal carcinoma cells, engineered to express or intrinsically expressing the tumor antigen of interest and subsequently treated, i.e. therapeutically vaccinated, with the at least one nucleic acid molecule or the VLP(s) of the invention (encoding the tumor antigen of interest or an antigenic part thereof as polypeptide B). After a certain time period, for instance of 1 to 6 weeks, the subjects are analysed with regard to tumor and / or metastasis formation as well as tumor size and tumor characteristics, e.g. by tumor antigen specific staining of dissected tumors. Thus, it can be determined, whether tumor formation was reduced or rejected through the treatment with the composition of the invention and whether tumors expressed the respective tumor antigen. To test effectivity of immunisation against any tumor antigen in a tumor challenge / rej ection assay, it is understood that animals may analogously also be injected with any suitable antigen related tumor cell line and be vaccinated with a nucleic acid molecule encoding the respective tumor antigen expressed in the tumor cell line specifically related to tumor formation of said respective other tumor cell line. Possible tumor cell lines include but are not limited to breast cancer, ovarian cancer, hepatocellular cancer and melanoma cell lines.

[0186] Besides the antigenic polypeptide that may be presented via a VLP as described herein the at least one nucleic acid molecule of the present invention may also encode further, such as at least one further, antigenic polypeptides or polypeptides with other functional properties. Co-encoded moieties may then for instance experience a pull-along effect towards the cell surface, being displayed along with the presented antigenic polypeptide, or may for instance further enhance an elicited immune response. The at least one further protein may be conjugated, i.e. fused, to the antigenic polypeptide, which may enable the further protein to be secreted or displayed at the cell surface along with the antigenic polypeptide. In this case, the antigenic polypeptide and the at least one further polypeptide will be encoded on the same nucleic acid molecule.

[0187] Thus, the VLP platform of the present invention may also be useful for simultaneously presenting more than one antigen to a subject’s immune system for immunization. This has the advantage that one vaccine may provide a prophylactic effect against different diseases, acting as acombinatory therapy. Thus, in one aspect the present invention provides least one nucleic acid molecule encoding at least three polypeptides:

[0188] (1) a first polypeptide A, wherein the polypeptide A is a group-specific antigen (Gag) protein of a beta-retrovirus;

[0189] (2) a second polypeptide, wherein the second polypeptide comprises the polypeptides Bl, Cl and DI in the order Bl -Cl -DI or DI -Cl -Bl; wherein polypeptide Bl is an antigenic polypeptide, polypeptide Cl is a transmembrane domain (TMD), and polypeptide DI is a cytoplasmic tail (CT) of a viral envelope protein or fragment thereof; and

[0190] (3) a third polypeptide, wherein the third polypeptide comprises the polypeptides B2, C2 and D2 in the order B2-C2-D2 or D2-C2-B2; wherein polypeptide B2 is an antigenic polypeptide, polypeptide C2 is a transmembrane domain (TMD), and polypeptide D2 is a cytoplasmic tail (CT) of a viral envelope protein or fragment thereof, wherein said first polypeptide, said second polypeptide and said third polypeptide are encoded by the same nucleic acid molecule, wherein the polypeptides Bl and B2 are different antigenic polypeptides, preferably wherein the different antigenic polypeptides are from different pathogens.

[0191] In a preferred embodiment polypeptides Bl and B2 are from different pathogens, wherein the pathogens are Influenza (A) and RSV, preferably wherein Bl and B2 are the polypeptides Influenza A HA and RSV glycoprotein F, or an antigenic part thereof, respectively. In another preferred embodiment polypeptides Bl and B2 are from different pathogens, wherein the pathogens are hMPV and RSV, preferably wherein Bl and B2 are the polypeptides hMPV glycoprotein F and RSV glycoprotein F, or an antigenic part thereof, respectively. In another preferred embodiment polypeptides Bl and B2 are from different pathogens, wherein the pathogens are hMPV and Influenza (A), preferably wherein Bl and B2 are the polypeptides hMPV glycoprotein F and Influenza A HA, or an antigenic part thereof, respectively.

[0192] The at least one further protein may also be expressed separately, i.e. not be fused to the antigenic polypeptide. In this case, the antigenic polypeptide and the at least one further polypeptide can be encoded on the same or on separate nucleic acid molecules.

[0193] The average skilled person is aware of how to obtain fused and not fused co-expressed polypeptides which are encoded on the same nucleic acid molecule strand. In one embodiment, the further protein may be directly linked to the antigenic polypeptide, or linked to it via a linker also encoded by the nucleic acid molecule. Suitable linkers are known in the art. In one embodiment, the at least one further protein may be conjugated with the antigenic polypeptide via a linker, whereinsaid linker is for instance a suitable amino acid sequence, in particular of preferably between 1 and 30, such as between 1 and 10 amino acid residues. Preferred examples of such amino acid sequences include but are not limited to Gly-Ser linkers.

[0194] In some cases, it may be desirable to create a stronger immune response in a subject receiving treatment by the composition of the invention or to evoke immunity against a further antigen in the subject. Thus, in one embodiment, the further encoded protein may be an antigen. In one embodiment, the further encoded protein may be an adjuvant.

[0195] One suitable way of enhancing an evoked immune response may also be to encode an enzyme which is capable of producing an adjuvant substance. One non-limiting example includes encoding the enzyme cyclic GMP-AMP synthase (cGAS) by the at least one nucleic acid molecule as defined herein. cGAS plays a crucial role in the innate immune response of mammals. It is primarily responsible for detecting and responding to the presence of foreign DNA, such as that from viruses or bacteria, within the cell as a signalling enzyme. Furthermore, cGAS synthesizes the small molecule cyclic GMP-AMP (cGAMP) from ATP and GTP, which acts as a second messenger molecule that may initiate a signalling cascade ultimately leading to the production of type I interferons and other pro-inflammatory cytokines, thus activating innate immune response. Thus, encoding the enzyme by and expressing cGAS from the at least one nucleic acid molecule of the invention promotes an immune activating effect, which may enhance the immune response against the encoded antigenic polypeptide (B).

[0196] Cancer cells frequently upregulate surface receptors that promote growth and survival. These receptors constitute valid targets for intervention. One strategy involves the delivery of toxic receptor binding agents with the goal of killing those cancer cells with high receptor levels. Thus, to enforce the anti cancer effect of a treatment by use of the at least one nucleic acid molecule, the further encoded protein in one embodiment may be an agent suitable to kill cancer cells, such as for instance a protein toxin.

[0197] Group-specific antigen (Gag) (polypeptide A)

[0198] As outlined above, it was found that expression of a Group-specific antigen (Gag) of a betaretrovirus (first polypeptide A as described herein), which contributes to the formation of a VLP comprising said Gag protein enhances the surface expression of and the immune response to an antigenic polypeptide also expressed as part of said VLP. Using a Gag protein of a foreign betaretrovirus may for instance be particularly advantageous to break tolerance of the immune systemtowards certain antigenic stimulants. This may, for instance, be facilitated via the help of T-cell activity. Using a non-human endogenous beta-ERV may for instance be particularly advantageous when targeting cells affected by infectious diseases. Particular advantages of this approach include that vaccinating and / or treating a subject to display VLPs with a non-human endogenous beta-ERV Gag protein will reduce the chances of causing autoimmunity, as the non-human endogenous beta-ERV Gag presents a distinct target from those targets known in the subject’s own body. Suitable examples of non-human endogenous beta-ERV include “Intracisternal A-type Particle elements with an Envelope” (IAPE) murine endogenous retrovirus and simian retrovirus 2 (SRV2). However, any Gag protein from a non-human endogenous beta-ERV may suitably be combined, which allows for a sufficiently dense protein surface coverage upon VLP formation. Using a human beta-ERV may for instance be particularly advantageous when targeting cancer cells in anti-cancer therapy in a human subject. As described herein, it was surprisingly found that particularly the expression of a HERV-K Gag as first polypeptide A of the VLP or of a IAPE Gag as first polypeptide A of the VLP as described herein lead to an enhanced surface expression and immune response to an antigenic polypeptide also expressed as part of said VLP. Thus, especially a HERV-K or an IAPE Gag may be selected for the purpose of vaccination with the VLP platform of the invention.

[0199] Thus, in one embodiment the first polypeptide A is a Gag protein of an endogenous betaretrovirus (beta-ERV) or of a foreign beta-retrovirus. In a preferred embodiment the first polypeptide A is a Gag protein of an endogenous beta-retrovirus (beta-ERV) selected from the group consisting of human beta-ERV and non-human beta-ERV. In a more preferred embodiment the human beta-ERV is a HERV-K and the non-human beta-ERV is selected from IAPE (Intracisternal A-type Particles elements with an Envelope) murine endogenous retrovirus and simian retrovirus 2 (SRV2). In an even more preferred embodiment the non-human beta-ERV is IAPE (Intracisternal A-type Particles elements with an Envelope) murine endogenous retrovirus. In an even more preferred embodiment, the HERV-K is selected from the group consisting of HERV-K108 (=ERVK-6), ERVK-19, HERV-K115 (=ERVK-8), ERVK-9, HERV-K113, ERVK-21, ERVK-25, HERV-K102 (=ERVK-7), HERV-K101 (=ERVK-24), andHERV-KHO (=ERVK-18).

[0200] Nucleic acid elements and features

[0201] The at least one nucleic acid molecule, such as mRNA, encoding the polypeptides as defined herein, is preferably constructed so as to allow the encoded polypeptides to be expressed in vivo and presented to the immune system of a subject to elicit an immunological response in said subject.Suitable nucleic acid features and modifications to enhance in vivo expression and allow immunological presentation in a subject are known in the art.

[0202] In one embodiment, the at least one nucleic acid molecule of the invention is an RNA. In a preferred embodiment the RNA is selected from the group consisting of mRNA (messenger RNA), circular RNA and self-amplifying RNA. In a preferred embodiment the RNA is non- self-amplifying mRNA.

[0203] Circular RNA (circRNA) contains a closed-loop structure and lacks free ends, wherein the loop structure is a covalently closed by so called backsplicing. Due to the lack of free amino acid ends, this structure has the advantage of being more stable. CircRNA can be translated in a cap-independent manner and therefore the circular RNA as defined herein suitably comprises a means and / or structures of cap-independent translation initiation. For instance, this includes encoding of an IRES (internal ribosome entry site) by the at least one nucleic acid molecule of the invention.

[0204] Self-amplifying mRNA (saRNA) can independently amplify antigen encoding mRNA in a host cell, much similar as during a viral infection, which results in enhanced and sustained levels of the target protein encoded by the mRNA. Self-amplifying mRNA may have a “self-adjuvanting” innate immune response effect, which can lead to potent and long-lasting antigen-specific humoral and cellular immune responses.

[0205] The at least one nucleic acid molecule may also be a DNA. The DNA may be double-stranded or single-stranded. For instance, the polypeptides as defined herein may be encoded by a non-viral DNA vector, such as on one or more plasmids, or may be encoded on a viral DNA vector, such as an adenoviral vector (carrying double-stranded DNA) or an AAV vector (Adeno-Associated Virus, carrying single-stranded DNA).

[0206] In one embodiment, the first polypeptide A in (1) and the second polypeptide in (2) are encoded on the same nucleic acid molecule, wherein the first polypeptide A in (1) is encoded by a first open reading frame (ORF) and wherein the second polypeptide in (2) is encoded by a separate second ORF. In a preferred embodiment, the sequence of the first ORF encoding the first polypeptide in (1) and the sequence of the separate second ORF encoding the second polypeptide in (2) are connected by a sequence enabling separate translation of the first and second polypeptide, preferably wherein said sequence enabling separate translation encodes a p2A self-cleaving peptide or an internal ribosomal entry site (IRES). Further, the sequence of the first ORF and the sequence of the separate second ORF may be connected by a sequence encoding any suitable operative linker.As an alternative to separating the translation of the first and second polypeptide described herein by means of a connecting sequence connecting the encoding ORFs on a same nucleic acid molecule, the first polypeptide (A) and the second polypeptide (comprising polypeptides B, C and D) may be expressed in a separate manner by encoding these polypeptides on two separate nucleic acid molecules. Thus, in another embodiment, the first polypeptide A in (1) is encoded on a first nucleic acid molecule and the second polypeptide comprising polypeptides B, C and D in (2) is encoded on a separate second nucleic acid molecule.

[0207] Modifying nucleic acid, such as mRNA, elements, such as the 5' cap, 5'-and 3 '-untranslated regions (UTRs), the coding region, and polyadenylation tail, helps to reduce excessive nucleic acid immunogenicity and / or may improve nucleic acid stability and translational efficiency. Therefore, the at least one nucleic acid molecule of the invention may have certain functional sequence features, optimizing its properties with regard to stability, expression efficiency and tolerance in a subject. mRNA molecules may comprise an elongated oligo-A sequence or poly-A sequence at their 3’-end, i.e. a poly-A tail. The 3' poly-A facilitates nuclear export, and provides RNA stability and translational efficiency of the mRNA. Over time, the poly-A is shortened, eventually leading to the initiation of enzymatic mRNA degradation. Elongating the poly-A may therefore provide additional stability. Thus, in one embodiment, the at least one nucleic molecule comprises at least 60 adenosine nucleotides at the 3’-UTR. In a preferred embodiment, the nucleic acid molecule, such as an mRNA, may comprise at least 100 and more preferably 120 adenosine nucleotides at the 3’-UTR.

[0208] In a further embodiment, the at least one nucleic acid molecule of the invention may comprise a poly(C) (poly-cysteine) tail at the 3'-terminus of typically about 10 to 200 cytosine nucleotides, preferably about 10 to 100 cytosine nucleotides, more preferably about 20 to 70 or even more preferably about 20 to 60 cytosine nucleotides.

[0209] Codon optimization is another approach to improve gene expression by changing synonymous codons based on an organism's codon bias. Mutations are introduced into a gene of interest based on a host organism’s own codon usage bias to increase translational efficiency in said organism and thus protein expression without altering the sequence of the protein. Thus, in one embodiment, the at least one nucleic molecule is codon optimized for expression in a human. Human codon usage is known in the art, as for instance provided by GenScript Codon Usage Frequency Table(chart) Tool. The mRNA of the invention may also be codon optimized for expression in other animals, such as for instance in other mammals.In one embodiment, the at least one nucleic acid molecule comprises at least 300 nucleotides. It may further be preferred that the at least one nucleic acid molecule comprises at least 400, 500, 800, 1000, 1500, 2000, more preferably at least 3000 or most preferably at least 4000 nucleotides.

[0210] In some cases, modified nucleobases are introduced into nucleic acid sequences (e.g. into mRNA nucleic acids) to improve stability. In one embodiment, the at least one nucleic acid molecule of the invention may comprise at least one artificially modified nucleotide. In another embodiment the at least one nucleic acid molecule of the invention may comprise no artificially modified nucleotides.

[0211] In one embodiment, the at least one nucleic acid molecule or an mRNA transcribed from said at least one nucleic acid molecule of the invention may be modified and thus stabilized, by modifying, particularly by increasing, the guanosine / cytosine (G / C) content of the at least one nucleic acid molecule or of an mRNA transcribed from said at least one nucleic acid molecule compared to the G / C content of the coding region of the respective wild type (i.e. unmodified) nucleotide sequence. According to a specific embodiment at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, more preferably at least 70%, even more preferably at least 80% and most preferably at least 90%, 95% or even 100% of the substitutable codons in a coding region of the at least one nucleic acid molecule of the invention or the whole sequence of the wild type nucleic acid sequence may be substituted, thereby increasing the GC / content of said at least one nucleic acid molecule or of an mRNA transcribed from said at least one nucleic acid molecule.

[0212] In one embodiment, the at least one nucleic acid molecule of the invention may be modified by modifying, and preferably by increasing, the cytosine (C) content of the at least one nucleic acid molecule or of an mRNA sequence transcribed from said at least one nucleic acid molecule, preferably of the coding region of the nucleic acid sequence, compared to the C content of the coding region of the respective wild type (i.e. unmodified) sequence. Preferably, the at least one nucleic acid molecule or of an mRNA sequence transcribed from said at least one nucleic acid molecule may be modified such that at least 10%, 20%, 30%, 40%, 50%, 60%, 70% or 80%, or at least 90% of the theoretically possible maximum cytosine-content or even a maximum cytosine-content is achieved.

[0213] In some embodiments, the at least one nucleic acid molecule or an mRNA transcribed from said at least one nucleic acid molecule of the invention may, for instance and without limitation, include or encode at least one of the sequence elements selected from the group consisting of CAP analogue structures; suitable promoters or subgenomic promoters yielding a high translation rate; selfamplifying mRNA features, such as for instance cytomegalovirus promoter, T7 promoter orsubgenomic SFV promoter; Kozak consensus sequence (5'-CCACCATGG-3'); a spacer of 3 to 6 nucleotides between (T7) promoter sequence and Kozak sequence, if present; stabilizing and / or structural sequence elements in UTR sequences. Suitable nucleic acid, such as RNA, elements are described in the art and will be clear to a person of average skill in the relevant field. It is preferred that polypeptide expression from the at least one nucleic acid molecule of the invention or from an mRNA transcribed from said at least one nucleic acid molecule is initiated at and driven by a CMV (cytomegalovirus) promotor.

[0214] For instance, suitable CAP analogue structures may be selected from the non-limiting group consisting of Vaccinia 2'-O-Methyltransferase Cap 1, ARCA anti-reverse CAP analogue or 0-S-ARCA cap, modified ARCA (e.g. phosphothioate modified ARCA); m7GpppN, capl (methylation of the ribose of the adjacent nucleotide of m7G), cap2 (additional methylation of the ribose of the 2nd nucleotide downstream of the m7G), cap3 (additional methylation of the ribose of the 3rd nucleotide downstream of the m7G), cap4 (methylation of the ribose of the 4th nucleotide downstream of the m7G), inosine, N1 -methyl-guanosine, 2'-fluoroguanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-aminoguanosine, LNA-guanosine, and 2-azido-guanosine.

[0215] For instance, suitable stabilizing and / or structural sequence elements in UTR sequences may be based on a variant of the UTR sequence(s) of a gene, such as on a variant of the UTR(s) of an albumin gene, an a-globin gene, a B-globin gene, a tyrosine hydroxylase gene, a lipoxygenase gene, or collagen alpha gene, such as a collagen alpha 1 gene, or a part thereof. Thus, in some preferred embodiments, the mRNA of the invention may comprise a 5 ’UTR HBA1, 5 ’UTR SFV, or 5 ’UTR 7, 3 ’UTR HBB; and / or a 3 ’UTR AES mtRNRl. In another embodiment, an mRNA 5'-UTR may comprise or consist of a nucleic acid sequence, which is derived from the 5 '-UTR of a ribosomal protein Large gene or from the 5'-UTR of a vertebrate TOP gene.

[0216] Immunogenicity and surface display

[0217] The present invention provides advantageous possibilities of displaying a variety of antigenic polypeptides using co-expression with a Gag protein of a beta-retrovirus in a VLP construct comprising said antigenic polypeptide(s). It is preferred that the antigenic polypeptide B expressed as part of the VLP construct as described herein (i.e. as part of the second polypeptide described herein) is co-expressed with a HERV-K Gag protein. This combination was shown to be particularly advantageous in view of surface display of and immunization against the expressed antigenic polypeptides B in the examples herein. It was further found to be advantageous to the display andimmunisation effect to additionally include the cytoplasmic tail of a HERV-K Gag or IAPE Gag protein or part thereof as polypeptide D of the second polypeptide described herein, which comprises antigenic polypeptide B, i.e. to construct a chimeric second polypeptide comprising polypeptides B, C and D as described herein to enable and / or improve surface display and immunogenicity of the antigenic polypeptide B.

[0218] Therefore, in one embodiment the polypeptide B is displayed on the surface of the VLP. In a preferred embodiment, the antigenic polypeptide B may have an increased surface display upon expression of the second polypeptide comprising the polypeptides B, C and D and of the first polypeptide A in a cell upon these polypeptides forming virus like particles (VLPs). The surface display of the antigenic polypeptide B may be increased compared to the surface display of an antigenic polypeptide B as part of a forming VLP wherein the group-specific antigen (Gag, polypeptide A) protein is not of a beta-retrovirus. Surface display may be increased on the surface of a host cell and / or on the surface of VLPs. The surface display may be detected and quantified by a method known in the art such as immunofluorescence staining of the displayed polypeptides and quantification by flow cytometry. Surface expression of the antigenic polypeptides may also be investigated and quantified by an imaging method known in the art, with and without previous antigenic polypeptide staining, including transmission electron microscopy (TEM) as described in the examples section herein.

[0219] In another embodiment, the VLP when administered into a subject or expressed from the at least one nucleic acid molecule in a subject as a vaccine generates an immune response against polypeptide B. In a preferred embodiment, the antigenic polypeptide B may have an increased immunogenicity upon expression of the second polypeptide comprising the polypeptides B, C and D and the polypeptide A in a cell upon these polypeptides forming virus like particles (VLPs). The immunogenicity of the antigenic polypeptide B may be increased compared to the immunogenicity of an antigenic polypeptide B upon expression as part of a forming VLP, wherein the group-specific antigen (Gag, polypeptide A) protein is not of a beta-retrovirus. The increased immunogenicity may be detected by an increase of IgG formation specifically directed against the antigenic polypeptide B by a method known in the art and this described herein.

[0220] Methods to determine and quantify antigenic polypeptide surface display and immunogenicity are described in the present disclosure, for instance in but not limited to Example 3.Means of delivery

[0221] The at least one nucleic acid molecule of the invention may be delivered directly as such or as part of a suitably selected delivery vehicle.

[0222] The at least one nucleic acid molecule may be delivered as or as part of a non-viral vehicle, such as one or more plasmids. Plasmids are particularly suitable if the at least one nucleic acid molecule is DNA and particularly for transfection in cell culture. Transfection methods for transfecting with a plasmid(s) are known in the art, including for instance electroporation and calcium phosphate transfection.

[0223] The at least one nucleic acid molecule of the invention may also be delivered as “naked” nucleic acid molecule, such as “naked” RNA or DNA.

[0224] Delivery of both “naked” RNA and DNA may be enhanced from encapsulation in lipids or lipid compositions, enabling improved efficiency in lipid-based transfection of cells in vitro but also of vaccine delivery an immunogenicity of vaccines in vivo. Typically, in this context cationic lipids or lipid-like molecules are used to form lipoplexes with DNA or with RNA, to form liposomes or lipid nanoparticles. Particularly suitable embodiments of lipid encapsulation are outlined further below in the context of pharmaceutical composition components.

[0225] It may be further suitable to deliver the at least one nucleic acid molecule of the invention as part of a viral vector, particularly if the at least one nucleic acid molecule is DNA. Thus, in one aspect the invention relates to a viral vector comprising the at least one nucleic acid molecule of the invention. The viral vector may be selected from any viral vector known in the art that are suitable for transfection purposes and particularly for vaccination purposes, preferably in mammals. Examples include, but are not limited to adenovirus vectors, adeno associated virus (AAV) vectors lentivirus vectors, vesicular stomatitis virus (VSV) vectors and Modified Vaccinia Ankara (MV A) vectors.

[0226] In a preferred embodiment, the vector is an adenoviral vector. In a more preferred embodiment, the vector is a human adenoviral vector. In an even more preferred embodiment the human adenoviral vector is selected from subtype C and subtype D human adenoviral vectors. In an even more preferred embodiment, the subtype C human adenoviral vector is Ad5F35 and the subtype D human adenoviral vector is Adi 9a. In a most preferred embodiment, the vector is an Adl9a / 64 adenoviral vector. Other suitable adenoviral vectors suitable for mammal, including human, vaccination strategies are known in the art and will be clear to the skilled person.Method of production of VLPs

[0227] An exemplary manner of producing the VLP(s) of the invention is outlined in the examples of the disclosure (see e.g. example 3.3 and example 4), including transfecting cells in cell culture, such as for instance cells of a A549 cell line, with the at least one nucleic acid molecule of the invention and expressing the polypeptides encoded by the at least one nucleic acid molecule of the invention. The cultured cells will then. Allow formation of VLPs and VLP budding at the cell surface.

[0228] Thus, in one aspect, the invention relates to a method of producing the VLP of the invention comprising the step of transfecting a nucleic acid molecule according of the invention into a cell. In a preferred embodiment, the nucleic acid molecule of the invention is transfected into a cell of a A549 cell line. Further steps of producing the VLPs of the invention, such as expression and isolation, are known in the art and may be performed accordingly. Further steps may include growing the transfected or cells in appropriate culture media under controlled conditions, allowing them to express the viral proteins, wherein parameters like temperature, pH, and nutrient availability may be optimized to maximize protein expression. Further steps may include harvesting / collecting cells or cell culture supernatant comprising the VLPs, including cell lysis and other extraction methods. Further steps may include purification of the harvested material to isolate the VLPs from cellular debris and contaminants, comprising for instance (ultra-)centrifugation, filtration, chromatography (e.g. size exclusion or affinity chromatography). Purification steps may be repeated to increase purity. Further steps may include analysing obtained VLPs, for instance regarding identity, structure, and purity, comprising microscopy, dynamic light scattering, Western blotting, and / or ELISA.

[0229] The produced VLPs may be further formulated in a suitable buffer or formulation to ensure stability, delivery (upon administration to a subject) and to allow long-term storage. Formulation agents may include stabilizers, cryoprotectants, or preservatives. Further formulation and pharmaceutical composition components are outlined below.

[0230] Pharmaceutical composition

[0231] The at least one nucleic acid molecule, the VLP, or the viral vector as described herein is for instance useful in the treatment or prophylaxis of diseases. For administration to as subject, it may be suitable to incorporate these components into a pharmaceutical composition. Thus, in one aspect, the invention relates to a pharmaceutical composition comprising the at least one nucleic acid molecule, the VLP, or the viral vector of the invention.In one aspect the present invention relates to a composition comprising at least one nucleic acid molecule or a pharmaceutically acceptable salt thereof, encoding (1) a first polypeptide A, wherein the polypeptide A is a group-specific antigen (Gag) protein of a beta-retrovirus; and (2) a second polypeptide, wherein the second polypeptide comprises the polypeptides B, C and D in the order B-C-D or D-C-B; wherein polypeptide B is an antigenic polypeptide, polypeptide C is a transmembrane domain (TMD), and polypeptide D is a cytoplasmic tail (CT) of a viral envelope protein or fragment thereof, wherein said first polypeptide and second polypeptide is encoded by the same nucleic acid molecule or by two different nucleic acid molecules, wherein the composition comprises a buffering agent, buffering the composition at a pH in the range of 7 to 8. In a preferred embodiment, the pH is in the range of pH 7,3 to 7,7. In a more preferred embodiment, the pH at about pH 7,4. In an alternative preferred embodiment, the pH at about pH 7,6.

[0232] In some cases, further agents, such as excipients, may be beneficial to improve therapeutic or prophylactic effectivity and / or a subject’s tolerance when administering the composition of the invention in therapy or prophylaxis. Thus, in one aspect the invention relates to a pharmaceutical composition comprising the at least one nucleic acid molecule, the VLP, or the viral vector of the invention, wherein the pharmaceutical composition comprises a pharmaceutically acceptable excipient. In some embodiments, pharmaceutically acceptable excipients are as defined herein. In some embodiments the pharmaceutical composition may comprise at least one pharmaceutically acceptable excipient selected from the group consisting of water, sodium chloride, potassium chloride, sucrose, sodium acetate or saline.

[0233] In some embodiments, the pharmaceutical composition may comprise one or more transfection agents. Such a pharmaceutical composition may then also be particularly suitable to transfect cells and / or tissue in vitro. Transfection agents of the composition may for instance be any compounds, formulations or mixtures that enhance transport or uptake of a nucleic acid into cells, including cells of different tissues. These agents can increase the uptake of the amount of a nucleic acid, when applied in an effective amount, as defined elsewhere herein. Such effect is caused by one or more substances comprised by the transfection reagent promoting said uptake. The protein or peptide encoded by the introduced nucleic acid can then modulate, evoke or be integrated in cellular processes of the target cell. Transfection agent compositions may for instance be composed adjusted for the targeted cell type and / or the substance to be delivered, as well as depending on other parameters such as the delivery environment, i.e. in vivo or in vitro. Suitable transfection agents comprise a range of different uptake promoting substances selected from the non-limiting group consisting of: calcium phosphate; cationicpolymers, such as DEAE-dextran or polyethyleneimine (PEI); liposome forming substances or mixtures thereof, such as cationic lipids like 2,3 -dioleoyloxy- N-[2(sperminecarboxamido)ethyl]-N, N-dimethyl-l-propaniminium-trifluoroacetate (DOSPA), Dioleoy 1-3 -trimethylammonium propane (DOTMA) or dioleoyloxypropyl-trimethylammonium (DOTAP), and / or helper lipids like dioleoyl phosphatidylethanolamine (DOPE), cholesterol and polyethylene glycol (PEG)-lipid; non-liposomal agents; and dendrimers. Further preferred is a wide range of commercially available Lipofectamine mixtures (for instance from ThermoFisher Scientific). In some embodiments the transfection agent may be a transfection agent that comprises a cationic lipid and / or a cationic polymer.

[0234] The transfection agent may be a composition particularly suitable for the administration of the at least one nucleic acid molecule, e.g. an mRNA, to a subject. Such suitable compositions are known in the art. Such a suitable composition may be a composition of lipids with beneficial properties in vivo, for instance a Lipid Nanoparticle (LNP) composition. LNPs increase circulation time in the body and effectively help to deliver nucleotide sequences, e.g. mRNA, to the target site and have thus emerged as a suitable non-viral encapsulating delivery vehicle for exogenous nucleic acids such as mRNA. Formulation in LNPs can reduce adverse responses. In preferred embodiments, LNPs are used that comprise lipids known to exhibit a reduced Toll-Like-Receptor (TLR) agonism. It has been observed that reduced activation of TLR signalling plays a key role in triggering, for instance, RNA vaccine-associated innate signalling and the triggering effect is believed to be amplifiable by certain lipids used in vaccine formulations, which reduce TLR signalling (Tahtinen, S., Tong, AJ., Himmels, P. et al. IL-1 and IL- Ira are key regulators of the inflammatory response to RNA vaccines. Nat Immunol 23, 532-542 (2022)). Without wishing to be bound by theory it is predicted that the use of such lipids will increase immunogenicity, leading to a potent innate immune response to the VLP-displayed antigenic polypeptide encoded by the at least one nucleic acid molecule of the invention. In one embodiment the pharmaceutical composition comprises liposomes that comprise a cationic lipid and the at least one nucleic acid molecule of the invention. In another embodiment, the pharmaceutical composition comprises lipid nanoparticles (LNPs) that comprise the at least one nucleic acid molecule of the invention. In a preferred embodiment the at least one nucleic acid molecule is RNA.

[0235] In one embodiment the pharmaceutical composition may comprise an LNP composition selected from a liposome composition, nanostructured lipid carrier (NLCs) composition and solid lipid nanoparticle (SLNs) composition. In one embodiment the pharmaceutical composition may comprise an LNP composition comprising at least one lipid selected from the group consisting of (i) an ionizable lipid, preferably an ionizable cationic lipid, more preferably an ionizable cationic amino lipid; (ii) anon-cationic helper lipid or phospholipid, wherein the lipid is preferably neutral, more preferably wherein the lipid is DSPC; (iii) a sterol or other structural lipid, wherein the sterol is preferably cholesterol; and (iv) a PEG lipid, preferably PEG-DMG. The lipid components of the LNP composition may be used in suitable molar ratios with respect to the other lipids. The amount of ionizable lipid, preferably cationic lipid, preferably cationic amino-lipid, may range for instance from about 45 mol % to about 50 mol %. The amount of non-cationic helper lipid or phospholipid, preferably a neutral lipid, preferably DSPC, may for instance range from about 5 mol % to about 15 mol %. The amount of structural lipid, such as sterol, preferably cholesterol, may for instance range from about 30 mol % to about 45 mol %. The amount of PEG-lipid in the lipid composition of a pharmaceutical composition disclosed herein may range for instance from about 0.1 mol % to about 5 mol %. Further suitable lipid components for the formulation of the at least on nucleic acid molecule of the invention in lipid nanoparticles are known in the art and the embodiments set forth herein are examples only and are in no way limiting.

[0236] It may be desired and / or useful to further increase the efficacy or potency of the pharmaceutical composition of the invention, particularly for instance when the composition is administered to a subject or patient for prophylaxis or treatment. Among other things this can be achieved by administering an adjuvant. Adjuvants may act by a combination of various mechanisms to elicit and boost immune responses, including one or more of: a sustained release of antigen at the site of injection (depot effect), an up-regulation of (further) signalling molecules, cytokines and chemokines, cellular recruitment at the site of administration of the composition, an increased antigen uptake and presentation to antigen presenting cells (APCs), an activation and maturation of APCs, increased expression of major histocompatibility complex (MHC) class II and co-stimulatory molecules, promoting antigen transport to draining lymph nodes and activation of the inflammasome. Thus, in one embodiment, the pharmaceutical composition comprises an adjuvant.

[0237] In a preferred embodiment the adjuvant may be a cytokine and more preferably a cytokine selected from the group consisting of INFy, IL-2, IL-12, GM-CSF, IL-15, and IL-7. The adjuvant may also be introduced as a polynucleotide or as part of the at least one nucleotide of the invention, wherein the polynucleotide is configured to express one or more of the aforementioned cytokines in eukaryotic cells. The adjuvant may for instance also be cyclic GMP-AMP (cGAMP). The adjuvant may also be introduced as a polynucleotide or as part of the at least one nucleotide of the invention, wherein the polynucleotide is configured to express the enzyme cGAS (cGAMP synthetase) in eukaryotic cells, which is capable of producing cGAMP) from ATP and GTP.Medical use

[0238] The polypeptides encoded by the at least one nucleic acid molecule of the invention, which may form VLPs upon expression in the same cell, exhibit the surprising beneficial effect of greatly improving cellular surface display of an encoded antigenic polypeptide. As described, efficient surface presentation of an antigen is promotes evoking a response from the immune system through promoting contact of immune cells with the presented antigenic polypeptide. The increased surface display of the encoded antigenic polypeptide promotes an immunologic reactivity of a subject’s body against the antigenic polypeptide. Thus, a subject may be immunized with the pharmaceutical composition of the invention, herein the immunization is effective against the development or progression of a disease, such as an infectious disease caused by a virus, and / or causes the subject’s body to fight the disease, such as an infectious disease caused by a virus, through an immunologic action. The nucleic acid molecule, the VLP, the viral vector, or the pharmaceutical composition described herein above is therefore suitable to be administered in therapy or prophylaxis. The pharmaceutical composition may be administered as a treatment or vaccine, or as part of a treatment or vaccine, in order to induce a specific immune response against diseases relating to the expression of the antigenic polypeptide (B). For instance, the pharmaceutical composition may be administered as a treatment against tumor antigen expressing tumor cells, and / or to immunize a subject against the development or progression of a pathology, such as against infectious diseases, aging or senescence related diseases and / or tumors associated with or occurring as a late effect of the aforementioned pathologies.

[0239] The VLP platform of the invention is particularly useful in the prophylaxis and / or treatment of a disease selected from the group consisting of COVID-19, Crimean-Congo haemorrhagic fever, Ebola virus disease and Marburg virus disease, Lassa fever, Middle East respiratory syndrome coronavirus (MERS-CoV) and Severe Acute Respiratory Syndrome (SARS), Nipah and henipaviral diseases, Rift Valley fever and Zika.

[0240] The VLP platform of the invention is particularly useful in the prophylaxis and / or treatment of a “disease X”, wherein such “disease X” represents the knowledge that a serious international epidemic could be caused by a pathogen currently unknown to cause human disease. The versatile technology of the invention enables early cross-cutting R& D preparedness that is also relevant for an unknown “disease X”.

[0241] The skilled person can readily select an antigenic polypeptide of the above pathogens as polypeptide B in the VLP construct of the invention. The skilled person is further aware, if necessary, how to adapt such polypeptide for it to be incorporated in the VLP platform of the invention.Furthermore, in the context of a (so far unknown or not yet relevant) “disease X”, the skilled person will use their skill the art to derive a suitable antigenic polypeptide to be used in the VLP construct of the invention to provide a useful vaccination agent.

[0242] Thus, in one aspect the invention relates to the at least one nucleic acid molecule, the VLP, the viral vector or the pharmaceutical composition of the invention for use as a medicament. In another aspect, the invention related to the at least one nucleic acid molecule, the VLP, the viral vector or the pharmaceutical composition of the invention for the manufacture of a medicament.

[0243] In another aspect, the invention relates to the at least one nucleic acid molecule, the VLP, the viral vector or the pharmaceutical composition of the invention for use in the prophylaxis and / or treatment of a disease. In a further aspect, the invention relates to the use of the at least one nucleic acid molecule, the VLP, the viral vector or the pharmaceutical composition of the invention for the manufacture of a medicament for the prophylaxis and / or therapeutic treatment of a disease. In preferred embodiments of both aforementioned aspects, the use is for immunizing a subject against a disease.

[0244] In another aspect, the invention relates to a method of treatment or prophylaxis of a disease comprising administering the at least one nucleic acid molecule, the VLP, or the viral vector, or the pharmaceutical composition of the invention to a subject.

[0245] The nucleic acid molecule, the VLP, the viral vector and the pharmaceutical composition of the present invention is suitably applicable in the treatment and / or prophylaxis of wide range of pathologies, which are related to the expression of the respective antigenic polypeptide (B) as defined herein, such as encoded by the at least one nucleic acid molecule of the invention. This includes treatment and or prophylaxis of a wide range of pathologies caused by infectious agents, such as viruses.

[0246] In some preferred embodiments, the disease (referred to in the medical applications herein) is an infectious disease. In some preferred embodiments, the disease (referred to in the medical applications herein) is selected from the group consisting of wherein the infectious disease is a respiratory disease, more preferably a disease caused by a viral pathogen selected from the group consisting of influenza virus, coronavirus, respiratory syncytial virus (RSV), human metapneumovirus, adenovirus, rhinovirus, enterovirus, parainfluenza virus and parvovirus.

[0247] In some preferred embodiments, the disease (referred to in the medical applications herein) may also be selected from the group consisting of cancer, neuropathological disorders, neurodegenerative diseases, insulin deficiency, type II diabetes and type I diabetes. In a preferredembodiment, the neuropathological disorder is selected from multiple sclerosis, schizophrenia and bipolar disorder.lt is known that neurodegenerative diseases are associated with the aberrant folding of host-encoded proteins into insoluble complexes, which can lead to the formation of protein aggregates, associated for instance with Alzheimer’s disease and other tauopathies. Furthermore, it was found that the underlying molecular mechanism of protein aggregation in neurodegenerative diseases is also relevant in the development of type II diabetes, linking these two diseases, Thus, in one embodiment, the disease is a neurodegenerative disease. In a preferred embodiment the neurodegenerative disease is selected from the group consisting of Alzheimer’s disease, amyotrophic lateral sclerosis, frontotemporal lobar degeneration, cerebrospinal fluid, Progressive supranuclear palsy, frontotemporal dementia and sporadic Creutzfeldt-Jakob disease, transmissible spongiform encephalopathies. In another embodiment, the disease is type II diabetes. In the context of prophylaxis and / or treatment of the aforementioned pathologies using the VLP platform of the invention, a preferred polypeptide B may be an antigen selected from a peptide of amyloid-beta (A0) or a peptide of Microtubule-associated protein tau (Tau).

[0248] In one embodiment, the disease is cancer. In a preferred embodiment of the invention, the cancer is a cancer expressing a tumor antigen selected from the group consisting of differentiation antigens such as MART-l / MelanA (MART-1), gplOO (PMEL17), tyrosinase, TRP-1, TRP-2 and tumor-specific multilineage antigens such as MAGE-1, MAGE-3, BAGE, GAGE- 1, GAGE-2, pi 5; overexpressed (carcinogenic) embryonic antigens such as CEA; overexpressed oncogenes and mutated tumorsuppressor genes such as p53, p21, Mucl, Ras, HER-2 / neu; unique tumor antigens resulting from chromosomal translocations; such as BCR-ABL, E2A-PRL, H4-RET, 1GH-IGK, MYL-RAR; and viral antigens, such as the Epstein Barr virus antigens EBVA and the human papillomavirus (HPV) antigens E6 and E7. Other large, protein-based antigens include TSP- 180, MAGE-4, MAGE-5, MAGE- 6, RAGE, NY-ESO, pl 85erbB2, p 1 80erbB-3, c-met, nm-23H 1, PSA, TAG- 72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, beta-Catemn, CDK4, Mum-1, pl5, pl6, 43-9F, 5T4 (791Tgp72), alpha-fetoprotein (AFP), beta-HCG, BCA225, BTAA, CA125, CA15-3\CA 27.29\BCAA, CA195, CA242, CA-50, CAM43, CD68\I, CO-029, FGF-5, G250, Ga733VEpCAM, HTgp-175, M344, MA-50, MG7-Ag, M0V18, NB / 70K, NY-CO-1, RCAS1, SDCCAG16, TA-90\Mac-2 binding protein / cyclophilin C-associated protein, TAAL6, TAG72, TLP, and TPS. In an even more preferred embodiment the cancer is a cancer selected from the group consisting of bladder cancer, urothelial cell cancer, prostate cancer, germ cell cancer (seminoma), breast cancer, ovarian cancer, endometrialcancer lymphomas, melanomas, neuroblastoma, leukemia, sarcomas, colorectal cancer, testicular cancer, lung cancer, meningioma, glioblastoma, schwannoma and liver cancer.

[0249] A vaccination using the VLP platform of the invention may be useful for immunizing against an antigen associated with a cellular senescence (or organismal aging) related disease in order to alleviate or slow down cellular senescence, tissue degeneration and, organismal aging. In one aspect, the invention relates to the at least one nucleic acid molecule, the VLP, the viral vector or the pharmaceutical composition of the invention for the prevention or slowing of aging and / or cellular senescence. In another aspect, the invention relates to the pharmaceutical composition described herein for use in the manufacture of a medicament for the prevention or slowing of aging and / or cellular senescence. In some embodiments the disease is a disease related to the process of senescence. In one embodiment the invention relates to the at least one nucleic acid molecule, the VLP, the viral vector or the pharmaceutical composition of the invention for use in the prophylaxis and / or treatment against symptoms related to aging, senescence and / or diseases related to aging and / or senescence. In another embodiment, the invention relates to the pharmaceutical composition described herein for use in the manufacture of a medicament for the prophylaxis and / or treatment against symptoms related to aging, senescence and / or diseases related to aging and / or senescence. Aging as used herein may be considered as the time-related deterioration of the physiological functions involved in survival and fertility of a subject. Aging is known to comprise certain hallmarks, including (1) the causes of age-associated damage (primary hallmarks); (2) the responses to the damage (antagonistic hallmarks); and (3) the consequences of the responses and mechanisms responsible for the aging phenotype (integrative hallmarks). Senescence is a cellular response that limits the proliferation of aged or damaged cells and thus belongs to antagonistic hallmarks. On the one hand, senescence is relevant during normal development and tissue homeostasis. On the other hand senescence occurs as a stress response, which can be triggered due to damage associated with aging such as genomic instability and telomere shortening of the DNA (primary aging hallmarks). The stress response features connected to senescence, such as protease secretion and chronic tissue inflammation, which can change the cellular microenvironment within a tissue, may be associated with and can further contribute to aging and aging related diseases. For instance, the presence of senescent cells may be related to osteoarthritis, idiopathic pulmonary fibrosis, cancer, atherosclerosis, Alzheimer's disease and general aging related frailty, as well as further aging related diseases

[0250] In some embodiments, the subject who is administered the treatment and / or prophylaxis of a disease may be a mammal. In some embodiments, the subject who is administered the treatment and / orprophylaxis of a disease may be a human, a non-human primate, or a mouse. In a preferred embodiment, a non-human primate subject may be of the species macaca fascicularis. When encoding a human endogenous retrovirus derived antigenic polypeptide (B) by the at least one nucleic acid of the invention, it may be preferred that the subject is a human.

[0251] Treatment and dosing regimen

[0252] Suitable treatment and / or dosing regimens for prophylaxis and / or treatment of any of the above outlined pathologies in a subject are known in the art and will be clear to a person skilled in the art. For instance, under certain conditions, it may be advantageous to treat a patient using a prime-boost regimen. Thus, the use in the prophylaxis and / or treatment of cancer may for instance comprise the step of priming a subject with the composition of the invention and then boosting the subject later on with the same or even with a pharmaceutical composition comprising different or additional components such as viral vectors, mRNA, VLPs, or other modalities. The priming and / or boosting may also be performed in multiple dosing steps, for instance by boosting the subject repeatedly.

[0253] Thus, in one embodiment the invention relates to the pharmaceutical composition or the method of treatment described herein, wherein (i) the pharmaceutical composition is administered in a first dose; and (ii) the pharmaceutical composition is administered in a second dose, wherein the second dose is administered about 3 weeks after administration of the first dose. A suitable subject to be administered the foregoing doses according to the outlined time schedule may be a mammal, preferably a mouse.

[0254] In yet another embodiment, the invention relates to the pharmaceutical composition or the method defined herein, wherein(i) the pharmaceutical composition is administered in a first dose; and (ii) the pharmaceutical composition is administered in a second dose; wherein the second dose is administered about 25 to 35 days after administration of the first dose, and preferably about 28 days after administration of the first dose.

[0255] In yet another embodiment, the invention relates to the pharmaceutical composition or the method defined herein, wherein (i) the pharmaceutical composition is administered in a first dose; and (ii) the pharmaceutical composition is administered in a second dose,

[0256] wherein the pharmaceutical composition comprises a viral vector as defined herein, wherein the serotype of the vector to be administered in the pharmaceutical composition for the first dose is different from the serotype of the vector to be administered in the pharmaceutical composition for the second dose.In yet another embodiment, the invention relates to the pharmaceutical composition or the method defined herein, wherein (i) the pharmaceutical composition is administered in a first dose; and (ii) the pharmaceutical composition is administered in a second dose,

[0257] wherein the pharmaceutical composition administered in the first and / or the second dose comprises an mRNA encoding a first and a second polypeptide as defined herein viral vector as defined herein. In another embodiment the pharmaceutical composition administered in the first or the second dose comprises an mRNA encoding a first and a second polypeptide as defined herein, and the pharmaceutical composition administered in the respective other dose does not comprise said mRNA. In a preferred embodiment the pharmaceutical composition administered in the first and the second dose comprises an mRNA encoding a first and a second polypeptide as defined herein.

[0258] After said first and second dose additional doses can be administered. Thus, a further embodiment of the invention relates to the pharmaceutical composition or the method defined herein, wherein (i) the pharmaceutical composition is administered in a first dose; and (ii) the pharmaceutical composition is administered in a second dose; and optionally (iii) the pharmaceutical composition is administered in at least one further dose;

[0259] wherein the pharmaceutical composition administered in the first, the second and optionally the at least one further dose comprises an mRNA encoding a first and a second polypeptide as defined herein viral vector as defined herein. Preferably, the first and second dose are administered in a time of about 1 to 2 months time.

[0260] A suitable subject to be administered the foregoing doses according to the outlined time schedule may be a mammal, preferably a primate. The primate may for instance be a non-human primate, more preferably a non-human primate of the species macaca fascicularis.

[0261] Preferably the at least one nucleic acid molecule, the VLP, a viral vector, an mRNA, or the pharmaceutical composition of the invention is administered to a subject to be treated in an effective amount.

[0262] In one embodiment, the amount of viral like particles administered to the subject is in the range of from 2 x 1010to 5 x 1011viral like particles per dose. In one embodiment, the amount of viral particles administered to the subject is in the range of 106to 109infectious units. In one embodiment, the amount of viral particles administered to the subject is in the range of 106to 109virus like particles. In a preferred embodiment, the amount is about 1 x 1011viral like particles per dose. In another preferred embodiment, an mRNA to be administered to a subject is provided at a dose of between 5-100 pg. In a further embodiment, the pharmaceutical composition is administered via anadministration route selected from the group consisting of intramuscular (i.m.), intraperitoneal (i.p.), subcutaneous (s.c.), intracranial, intracerebral, intraspinal space, intracerebroventricular space, intraspinal, intrapleural and intratumoral administration, administration to any body cavity comprising a tumor or comprising an epithelial lining comprising a tumor and administration by inhalation, preferably via an administration route selected from intramuscular and subcutaneous administration. These administration routes may for instance be particularly suitable when a dose in the range of 2 x 1010to 5 x 1011viral like particles per dose is administered. In a preferred embodiment the pharmaceutical composition is administered via an administration route selected from the group consisting of intramuscular (i.m.), subcutaneous (s.c.), intravenous, and administration by inhalation. In an even more preferred embodiment the pharmaceutical composition is administered via an administration route selected from intramuscular and subcutaneous administration.

[0263] In one embodiment, the pharmaceutical composition elicits local immunity. Local immunity as used herein is acquired as immunity to an antigen upon immunization, manifested by an organ or a tissue, as a whole or in part. Thus, the organ or tissue exhibits a locally increased resistance without the (systemic) participation of the organism as a whole.

[0264] In one embodiment, the pharmaceutical composition elicits systemic immunity. It is preferred that when the pharmaceutical composition is used for vaccination against an infectious disease, such as a disease caused by an infectious virus, that the elicited immunity is systemic.

[0265] In one embodiment, the pharmaceutical composition elicits local immunity in the central nervous system (CNS). In another preferred embodiment, the pharmaceutical composition is administered via an administration route selected from the group consisting of intracranial, intracerebral, intraspinal, intracerebroventricular administration, preferably wherein the pharmaceutical composition elicits local immunity in the central nervous system (CNS). Eliciting a local immunity in the central nervous system (CNS) may be particularly suitable for the prophylaxis and / or treatment of neurodegenerative, neuropathological diseases and / or cancer of the brain or spinal cord, as well as of infectious diseases specifically affecting the brain and / or spinal cord, as the CNS can be difficult to reach for antibodies or infiltrating immune cells without such local immunity.

[0266] Intracranial, intracerebral, intracerebroventricular and intraspinal administration of a pharmaceutical composition described herein may be particularly useful if the central nervous system, such as the brain is targeted as a region of immunity development. It in this way a pharmaceutical composition of the inventions or its components do not have to pass the blood brain barrier, as would be the case if the composition of the invention was administered elsewhere in the body, such assystemically. This is particularly interesting since usually only lipophilic, positively-charged molecules with a low molecular weight (e.g. less than about 400 to 600 Da) can cross the blood brain barrier. Furthermore, also most immune cells circulating through the body are limited from entry into the CNS over the blood brain barrier, as well as antibodies produced by the body’s adaptive immunity mechanisms, which usually do not pass the blood brain barrier. Thus, direct administration into the CNS and local eliciting of immunity therein is a suitable method to nevertheless gain immunity in this area. In yet another embodiment, the amount of viral like particles administered to the subject is in the range of 2 x 1011to 5 x 1012viral like particles per dose. In a preferred embodiment, the amount is about 1 x 1012viral like particles per dose. In another embodiment, the pharmaceutical composition is administered via an administration route selected from the group consisting of intraperitoneal (i.p.), intrapleural and intratumoral administration, administration by inhalation and administration to any body cavity comprising a tumor or comprising an epithelial lining comprising a tumor. These administration routes may for instance be particularly suitable when a dose in the range of 2 x 1011to 5 x 1012viral like particles per dose is administered.

[0267] It is preferred that the at least one nucleic acid molecule of the invention is used as a genetic vaccine, in particular in the prophylaxis and / or treatment of a disease, such as cancer. Alternatively, the nucleic acid molecule can also be used to produce VLPs, in particular HERV-K Gag comprising VLPs in vitro. The resulting VLPs can then be used in immunotherapy, in particular in the prophylaxis and / or treatment of a disease, preferably cancer, by administering the VLPs directly, for instance as part of a pharmaceutical composition, to a subject. It is understood that also in this context a cancer to be treated may preferably be a cancer expressing an ERV Env protein, which or part of which is encoded as antigenic polypeptide (B) by the at least one nucleic acid molecule of the invention.

[0268] In one embodiment of the at least one nucleic acid molecule of the invention, the encoded polypeptides A, B, C and D comprise the following amino acid sequences: (a) Polypeptide A comprises a sequence comprising at least 90%, more preferably at least 95%, and most preferably 100%, sequence identity with SEQ ID NO: 1 or 13; Polypeptide B comprises a sequence comprising at least 90%, more preferably at least 95%, and most preferably 100%, sequence identity with SEQ ID NO:s 64, 67, 71, 73 or 75, and preferably with SEQ ID NO: 64 or 67; and Polypeptides C and D together comprise a sequence comprising at least 90%, more preferably at least 95%, and most preferably 100%, sequence identity with SEQ ID NO: 8 or 47.

[0269] In one embodiment of the at least one nucleic acid molecule of the invention, the encoded polypeptides A, B, C and D comprise the following amino acid sequences: (b) Polypeptide Acomprises a sequence comprising at least 90%, more preferably at least 95%, and most preferably 100%, sequence identity with SEQ ID NO: 1 or 13; and Polypeptide B comprises a sequence comprising at least 90%, more preferably at least 95%, and most preferably 100%, sequence identity with SEQ ID NO:s 64, 67, 71, 73 or 75, and preferably with SEQ ID NO: 64 or 67; and Polypeptides C and D together comprise a sequence comprising at least 90%, more preferably at least 95%, and most preferably 100%, sequence identity with SEQ ID NO: 77, 78, 80, 82 or 84, and preferably wherein polypeptide B comprises a sequence comprising at least 95% with SEQ ID NO: 64 and polypeptides C and D together comprise a sequence comprising at least 95% sequence identity with SEQ ID NO: 77.

[0270] In a preferred embodiment the polypeptides A, B, C and D comprise the following amino acid sequences: Polypeptide A comprises SEQ ID NO: 1, polypeptide B comprises one of SEQ ID NO:s 64 or 71, and polypeptides C and D together comprise one of SEQ ID NO:s 8.

[0271] In one embodiment the polypeptides A, B, C and D comprise the following amino acid sequences: Polypeptide A comprises SEQ ID NO: 13, polypeptide B comprises one of SEQ ID NO:s 67, 73 or 75, and polypeptides C and D together comprise one of SEQ ID NO:s 47.

[0272] In an even more preferred embodiment the polypeptides A, B, C and D comprise the following amino acid sequences: Polypeptide A comprises SEQ ID NO: 1, polypeptide B comprises one of SEQ ID NO:s 64, and polypeptides C and D together comprise one of SEQ ID NO:s 8.

[0273] In another more preferred embodiment the polypeptides A, B, C and D comprise the following amino acid sequences: Polypeptide A comprises SEQ ID NO: 13, polypeptide B comprises one of SEQ ID NO:s 67, and polypeptides C and D together comprise one of SEQ ID NO:s 47.

[0274] In one particularly preferred embodiment, Polypeptide A comprises SEQ ID NO: 13; Polypeptide B comprises SEQ ID NO:s 67 and Polypeptides C and D together comprise SEQ ID NO: 47.

[0275] In one particularly preferred embodiment, Polypeptide A comprises SEQ ID NO: 1, polypeptide B comprises a sequence comprising at least 95% with SEQ ID NO: 64 and polypeptides C and D together comprise a sequence comprising at least 95% sequence identity with SEQ ID NO: 77.

[0276] Options outlined above correspond to polypeptide constructs described herein:

[0277] - HERV-K-Gag-P2A-InfluenzaA-HAEcD-HERV-K-EnvTMD-cT and HERV-K-Gag-P2A- InfluenzaA-HA,

[0278] IAPE-Dl-Gag-P2A-hMPV-F-proteinECD-IAPE-Dl-EnvTMD-cT and IAPE-Dl-Gag-P2A-hMPV- F-protein,- HERV-K-Gag-P2A-SIV-gpl40-HERV-K-EnvTMD-cT and HERV-K-Gag-P2A-SIV-gpl40, - IAPE-D 1 -Gag-P2A-mCEAC AMI ECD-IAPE-D 1 -EnvTMD-cT and IAPE-D 1 -Gag-P2A- mCEACAMl, and

[0279] - IAPE-D 1 -Gag-P2A-hCEAC A 5ECD-I APE-D 1 -EnvTMD-cT and IAPE-D 1 -Gag-P2A- hCEACAM5.

[0280] In a further embodiment of the at least one nucleic acid molecule of the invention the encoded polypeptides A, B, C and D comprise the following amino acid sequences:

[0281] Polypeptide A comprises SEQ ID NO: 1, polypeptides B comprises SEQ ID NO: 64 and polypeptides C and D together comprise SEQ ID NO: 8; or

[0282] Polypeptide A comprises SEQ ID NO: 1 and polypeptides B, C and D together comprise SEQ ID NO: 64; or

[0283] Polypeptide A comprises SEQ ID NO: 1, polypeptides B comprises SEQ ID NO: 71 and polypeptides C and D together comprise SEQ ID NO: 8; or

[0284] Polypeptide A comprises SEQ ID NO: 1, and polypeptides B, C and D together comprise SEQ ID NO: 71;wherein said first and second polypeptide are encoded by the same nucleic acid molecule and wherein said first and second polypeptide are linked to each other via a p2A amino acid sequence according to SEQ ID NO: 3. In a further embodiment of the at least one nucleic acid molecule of the invention the encoded polypeptides A, B, C and D comprise the following amino acid sequences:

[0285] Polypeptide A comprises SEQ ID NO: 13, polypeptides B comprises SEQ ID NO: 67 and polypeptides C and D together comprise SEQ ID NO: 47; or

[0286] Polypeptide A comprises SEQ ID NO: 13 and polypeptides B, C and D together comprise SEQ ID NO: 67; or

[0287] Polypeptide A comprises SEQ ID NO: 13, polypeptides B comprises SEQ ID NO: 73 and polypeptides C and D together comprise SEQ ID NO: 47; or

[0288] Polypeptide A comprises SEQ ID NO: 13, and polypeptides B, C and D together comprise SEQ ID NO: 73; or

[0289] Polypeptide A comprises SEQ ID NO: 13, polypeptides B comprises SEQ ID NO: 75 and polypeptides C and D together comprise SEQ ID NO: 47; or

[0290] Polypeptide A comprises SEQ ID NO: 13, and polypeptides B, C and D together comprise SEQ ID NO: 75;wherein said first and second polypeptide are encoded by the same nucleic acid molecule and wherein said first and second polypeptide are linked to each other via a p2A amino acid sequence according to SEQ ID NO: 3.

[0291] In a further embodiment of the at least one nucleic acid molecule of the invention the encoded polypeptides A, B, C and D comprise the following amino acid sequences:

[0292] Polypeptide A comprises SEQ ID NO: 1, polypeptides B comprises SEQ ID NO: 64 and polypeptides C and D together comprise SEQ ID NO: 8; or

[0293] Polypeptide A comprises SEQ ID NO: 1 and polypeptides B, C and D together comprise SEQ ID NO: 64; or

[0294] Polypeptide A comprises SEQ ID NO: 1, polypeptides B comprises SEQ ID NO: 71 and polypeptides C and D together comprise SEQ ID NO: 8; or

[0295] Polypeptide A comprises SEQ ID NO: 1, and polypeptides B, C and D together comprise SEQ ID NO: 71;

[0296] wherein said second polypeptide comprises the polypeptides B, C and D in the order B-C-D, wherein said first and second polypeptide are encoded by the same nucleic acid molecule and wherein said first and second polypeptide are linked to each other via a GSG-linker according to SEQ ID NO: 2 and a p2A amino acid sequence according to SEQ ID NO: 3.

[0297] In a further embodiment of the at least one nucleic acid molecule of the invention the encoded polypeptides A, B, C and D comprise the following amino acid sequences:

[0298] Polypeptide A comprises SEQ ID NO: 13, polypeptides B comprises SEQ ID NO: 67 and polypeptides C and D together comprise SEQ ID NO: 47; or

[0299] Polypeptide A comprises SEQ ID NO: 13 and polypeptides B, C and D together comprise SEQ ID NO: 67; or

[0300] Polypeptide A comprises SEQ ID NO: 13, polypeptides B comprises SEQ ID NO: 73 and polypeptides C and D together comprise SEQ ID NO: 47; or

[0301] Polypeptide A comprises SEQ ID NO: 13, and polypeptides B, C and D together comprise SEQ ID NO: 73; or

[0302] Polypeptide A comprises SEQ ID NO: 13, polypeptides B comprises SEQ ID NO: 75 and polypeptides C and D together comprise SEQ ID NO: 47; or

[0303] Polypeptide A comprises SEQ ID NO: 13, and polypeptides B, C and D together comprise SEQ ID NO: 75;wherein said second polypeptide comprises the polypeptides B, C and D in the order B-C-D, wherein said first and second polypeptide are encoded by the same nucleic acid molecule and wherein said first and second polypeptide are linked to each other via a GSG-linker according to SEQ ID NO: 2 and a p2A amino acid sequence according to SEQ ID NO: 3.

[0304] In a particularly preferred embodiment, the present invention refers to at least one nucleic acid molecule encoding (1) a first polypeptide A, wherein the polypeptide A is a group-specific antigen (Gag) protein of a beta-retrovirus, preferably of a human endogenous retrovirus K (HERV-K); and (2) a second polypeptide, wherein the second polypeptide comprises the polypeptides B, C and D in the order B-C-D or D-C-B, and preferably in the order B-C-D; wherein polypeptide B is an antigenic polypeptide, preferably an antigenic polypeptide of a virus or an immunogenic part thereof, more preferably of a respiratory virus (even more preferably of Influenza, most preferably wherein polypeptide B is an Influenza A hemagglutinin (HA) polypeptide); wherein polypeptide C is a transmembrane domain (TMD), and polypeptide D is a cytoplasmic tail (CT) of a viral envelope protein or fragment thereof, preferably wherein polypeptide C and polypeptide D comprise the transmembrane domain and the cytoplasmic tail of human endogenous retrovirus K (HERV-K) Env protein or a part thereof; wherein said first polypeptide and said second polypeptide is encoded by the same nucleic acid molecule, wherein the native genomic structure connecting the first and the second polypeptide has been replaced by an operative linker, wherein the linker preferably is p2A; and wherein the nucleic acid molecule encodes a CMV promotor upstream of the sequence section encoding the first and the second polypeptides. The nucleic acid molecule may be comprised by an adenoviral vector, and preferably by an Adi 9a adenoviral vector, most preferably an Adl9a / 64 adenoviral vector.

[0305] It is preferred that the nucleic acid molecule continuously encodes amino acid sequences of SEQ ID NO:s 1, 2, 3, 64 and 8, wherein SEQ ID NO: 1 represents the amino acid sequence of the first polypeptide A (Gag), wherein SEQ ID NO: 64 represents the amino acid sequence of antigenic polypeptide B and wherein SEQ ID NO: 8 represents the amino acid sequence of polypeptides C and D together, i.e. of transmembrane domain and cytoplasmic tail. In this embodiment, SEQ ID NO:s 2 and 3 represent a GSG-linker peptide and a P2A cleavable linker enabling a separate translation of the first and the second polypeptide described herein.

[0306] In another particularly preferred embodiment, the present invention refers to at least one nucleic acid molecule encoding (1) a first polypeptide A, wherein the polypeptide A is a group-specific antigen (Gag) protein of a beta-retrovirus, preferably of IAPE (Intracisternal A-type Particle elements with an Envelope) murine endogenous retrovirus; and (2) a second polypeptide, wherein the second polypeptide comprises the polypeptides B, C and D in the order B-C-D or D-C-B, and preferably in the order B-C-D; wherein polypeptide B is an antigenic polypeptide, preferably an antigenic polypeptide of a virus or an immunogenic part thereof, more preferably of a respiratory virus (even more preferably of hMPV, most preferably wherein polypeptide B is an hMPV glycoprotein F polypeptide); wherein polypeptide C is a transmembrane domain (TMD), and polypeptide D is a cytoplasmic tail (CT) of a viral envelope protein or fragment thereof, preferably wherein polypeptide C and polypeptide D comprise the transmembrane domain and the cytoplasmic tail of IAPE murine endogenous retrovirus Env protein or a part thereof; wherein said first polypeptide and said second polypeptide is encoded by the same nucleic acid molecule, wherein the native genomic structure connecting the first and the second polypeptide has been replaced by an operative linker, wherein the linker preferably is p2A; and wherein the nucleic acid molecule encodes a CMV promotor upstream of the sequence section encoding the first and the second polypeptides. The nucleic acid molecule may be comprised by an adenoviral vector, and preferably by an Ad 19a adenoviral vector, most preferably an Adl9a / 64 adenoviral vector.

[0307] It is preferred that the nucleic acid molecule continuously encodes amino acid sequences of SEQ ID NO:s 1, 2, 3, 64 and 8, wherein SEQ ID NO: 1 represents the amino acid sequence of the first polypeptide A (Gag), wherein SEQ ID NO: 64 represents the amino acid sequence of antigenic polypeptide B and wherein SEQ ID NO: 8 represents the amino acid sequence of polypeptides C and D together, i.e. of transmembrane domain and cytoplasmic tail. In this embodiment, SEQ ID NO:s 2 and 3 represent a GSG-linker peptide and a P2A cleavable linker enabling a separate translation of the first and the second polypeptide described herein.

[0308] In a further embodiment the invention relates to a nucleic acid molecule encoding at least one polypeptide, wherein the polypeptide comprises

[0309] (1) a first polypeptide A, wherein the polypeptide A is a group-specific antigen (Gag) protein of HERV-K; and

[0310] (2) a second polypeptide, wherein the second polypeptide comprises the polypeptides B, C and D in the order B-C-D or D-C-B; wherein

[0311] polypeptide B is Influenza A HA,

[0312] polypeptide C is the transmembrane domain (TMD) of HERV-K Env, andpolypeptide D is the cytoplasmic tail (CT) of HERV-K Env,

[0313] wherein said first polypeptide and second polypeptide is encoded by the same nucleic acid molecule or by two different nucleic acid molecules;

[0314] wherein the nucleic acid molecule is mRNA.

[0315] In a preferred embodiment the invention relates to a nucleic acid molecule encoding at least one polypeptide, wherein the polypeptide comprises

[0316] (1) a first polypeptide A is HERV-K Gag according to SEQ ID NO: 1; and

[0317] (2) a second polypeptide, wherein the second polypeptide comprises the polypeptides B, C and D in the order B-C-D or D-C-B; wherein

[0318] polypeptide B is Influenza A HA according to SEQ ID NO: 64,

[0319] polypeptide C is the transmembrane domain (TMD) of HERV-K Env, and polypeptide D is the cytoplasmic tail (CT) of HERV-K Env,

[0320] wherein polypeptide C and polypeptide D together have a sequence according to SEQ ID NO: 8; wherein said first polypeptide and second polypeptide is encoded by the same nucleic acid molecule or by two different nucleic acid molecules and wherein the nucleic acid molecule is preferably mRNA.

[0321] In a further embodiment the invention relates to a nucleic acid molecule encoding at least one polypeptide, wherein the polypeptide comprises

[0322] (1) a first polypeptide A, wherein the polypeptide A is group-specific antigen (Gag) protein of IAPE; and

[0323] (2) a second polypeptide, wherein the second polypeptide comprises the polypeptides B, C and D in the order B-C-D or D-C-B; wherein

[0324] polypeptide B is hMPV glycoprotein F,

[0325] polypeptide C is the transmembrane domain (TMD) of IAPE Env, and polypeptide D is the cytoplasmic tail (CT) of IAPE Env,

[0326] wherein said first polypeptide and second polypeptide is encoded by the same nucleic acid molecule or by two different nucleic acid molecules. In a preferred embodiment the invention relates to a nucleic acid molecule encoding at least one polypeptide, wherein the polypeptide comprises (1) a first polypeptide A, wherein the polypeptide A is a group-specific antigen (Gag) protein of IAPE according to SEQ ID NO: 13; and

[0327] (2) a second polypeptide, wherein the second polypeptide comprises the polypeptides B, C and D in the order B-C-D or D-C-B; whereinpolypeptide B is hMPV glycoprotein F according to SEQ ID NO: 67, polypeptide C is the transmembrane domain (TMD) of IAPE Env, and polypeptide D is the cytoplasmic tail (CT) of IAPE Env,

[0328] wherein polypeptide C and polypeptide D together have a sequence according to SEQ ID NO: 47; wherein said first polypeptide and second polypeptide is encoded by the same nucleic acid molecule or by two different nucleic acid molecules.

[0329] In some embodiments of any embodiment described herein, the nucleic acid molecule of the invention is an mRNA molecule in which at least one T nucleotide is replaced with ml'P (Nl- methylpseudouridine) and wherein the mRNA molecule encodes

[0330] (1) a first polypeptide A, wherein the polypeptide A is a group-specific antigen (Gag) protein of a beta-retrovirus; and

[0331] (2) a second polypeptide, wherein the second polypeptide comprises the polypeptides B, C and D preferably in the order B-C-D; wherein

[0332] polypeptide B is an antigenic polypeptide,

[0333] polypeptide C is a transmembrane domain (TMD), and

[0334] polypeptide D is a cytoplasmic tail (CT) of a viral envelope protein or fragment thereof, wherein polypeptides A, B, C and D are selected as defined in the embodiments herein.

[0335] In a further aspect the invention provides

[0336] as aspect (X): a method of treatment or prophylaxis of a disease comprising administering to a human subject a at least a first and a second dose; wherein the first and second dose each comprises a first and second composition that each comprises a polynucleotide, respectively; and

[0337] as aspect (Y): a first and second composition each comprising a polynucleotide for use in the treatment or prophylaxis of a disease by administering to a human subject said first and second composition as a first and second dose.

[0338] In the aspects (X) and (Y) mentioned above, said polynucleotide encodes the following polypeptides (A), (B), (C) and (D) as set out in the table below and wherein said first and second composition comprises a adenovirus vector as set out in the table below:embodiment First Second A B C D composition composition

[0339] 1 Ad5F35 Adi 9a HERV-K Gag Influenza A T ransmembrane C-terminus vector vector hemagglutinin domain of of HERV-K (HA) HERV-K ENV ENV 2 Ad5F35 Adi 9a HERV-K Gag Envelope T ransmembrane C-terminus vector vector glycoprotein domain of of HERV-K gpl40 of Simian HERV-K ENV ENV immunodeficiency

[0340] virus (SIV)

[0341] 3 Ad5 Adi 9a IAPE-D1 Gag hMPV T ransmembrane C-terminus glycoprotein F domain of of IAPE-D1

[0342] IAPE-D1ENV ENV 4 Ad5 Adi 9a IAPE-D1 Gag murine T ransmembrane C-terminus Carcinoembryonic domain of of IAPE-D1 antigen-related IAPE-D1ENV ENV cell adhesion

[0343] molecule 1

[0344] (CEACAM1)

[0345] 5 Ad5 Adi 9a IAPE-D1 Gag human T ransmembrane C-terminus Carcinoembryonic domain of of IAPE-D1 antigen-related IAPE-D1 ENV ENV cell adhesion

[0346] molecule 5

[0347] (CEACAM5)

[0348]

[0349] In the embodiments listed above the time between the administration of said first and second dose is preferably between 3 weeks and 2 months.

[0350] In a further aspect the invention provides an mRNA or DNA molecule encoding

[0351] (1) a first polypeptide A; and

[0352] (2) a second polypeptide, wherein the second polypeptide comprises the polypeptides B, C and D preferably in the order B-C-D,

[0353] wherein the components A, C and D correspond to embodiment 1-1 or 1-2 as listed in the table below, wherein component B is or comprises an antigenic polypeptide of a virus causing an infectious disease or a tumor antigen, preferably an antigenic polypeptide from a pathogen selected the group consisting of a negative sense RNA virus, preferably an antigen of Orthornavirae, more preferably an antigen ofNegarnaviricota, even more preferably an antigen of Orthomyxoviridae, Paramyxoviridae or Pneumoviridae, and preferably wherein the Orthomyxoviridae are selected from the group consisting of Alphainfluenzavirus, Betainfluenzavirus, Gammainfluenzavirus and Deltainfluenzavirus, and preferably wherein the Paramyxoviridae are selected from the group consisting of Parainfluenza Virus 1, Parainfluenza Virus 2, Parainfluenza Virus 3, Parainfluenza Virus 4, and preferably wherein the Pneumoviridae are selected from the group consisting of human metapneumovirus (HMPV), human respiratory syncytial virus A2 (HRSV-A2) and human respiratory syncytial virus B1 (HRSV-B1), most preferably wherein component B comprises glycoprotein F of hMPV or HA of Influenza A: Embodiment A C D

[0354] 1-1 Gag protein from transmembrane domain of the ENV cytoplasmic tail of the ENV protein of HERV-K protein of HERV-K HERV-K

[0355] 1-2 Gag protein from transmembrane domain of the ENV cytoplasmic tail of the ENV protein of IAPE-D1 virus protein of the IAPE-D1 virus the IAPE-D1 virus

[0356]

[0357] In a further aspect the invention provides an mRNA or DNA molecule encoding

[0358] (1) a first polypeptide A; and

[0359] (2) a second polypeptide, wherein the second polypeptide comprises the polypeptides B, C and D preferably in the order B-C-D,

[0360] wherein the components A, C and D correspond any of the embodiments 2-1 through 2-8 as listed in the table below, wherein component B is or comprises an antigenic polypeptide of a virus causing an infectious disease or a tumor antigen, preferably an antigenic polypeptide from a pathogen selected the group consisting of a negative sense RNA virus, preferably an antigen of Orthornavirae, more preferably an antigen of Negarnaviricota, even more preferably an antigen of Orthomyxoviridae, Paramyxoviridae or Pneumoviridae, and preferably wherein the Orthomyxoviridae are selected from the group consisting of Alphainfluenzavirus, Betainfluenzavirus, Gammainfluenzavirus and Deltainfluenzavirus, and preferably wherein the Paramyxoviridae are selected from the group consisting of Parainfluenza Virus 1, Parainfluenza Virus 2, Parainfluenza Virus 3, Parainfluenza Virus 4, and preferably wherein the Pneumoviridae are selected from the group consisting of human metapneumovirus (hMPV), human respiratory syncytial virus A2 (hRSV-A2) and human respiratory syncytial virus Bl (hRSV-Bl), most preferably wherein component B comprises glycoprotein F of hMPV or HA of Influenza A:Embodiment A C D

[0361] 2-1 Gag protein from HERV-K transmembrane domain of the cytoplasmic tail of the ENV with at least 95% sequence ENV protein of HERV-K protein of HERV-K identity with SEQ ID No: 1

[0362] 2-2 Gag protein from HERV-K transmembrane domain of the cytoplasmic tail of the ENV with at least 95% sequence ENV protein of HERV-K protein of HERV-K identity with SEQ ID No: 1 C and D together having at least 95% sequence identity with SEQ ID No: 8

[0363] 2-3 Gag protein from IAPE-D1 transmembrane domain of the cytoplasmic tail of the ENV with at least 95% sequence ENV protein of the IAPE-D1 virus protein of the IAPE-D1 virus identity with SEQ ID No: 13

[0364] 2-4 Gag protein from IAPE-D1 transmembrane domain of the cytoplasmic tail of the ENV with at least 95% sequence ENV protein of the IAPE-D1 virus protein of the IAPE-D1 virus identity with SEQ ID No: 13 C and D together having at least 95% sequence identity with SEQ ID No: 47

[0365] 2-5 Gag protein from HERV-K transmembrane domain of the cytoplasmic tail of the ENV having 100% sequence ENV protein of HERV-K protein of HERV-K identity with SEQ ID No: 1

[0366] 2-6 Gag protein from HERV-K transmembrane domain of the cytoplasmic tail of the ENV having 100% sequence ENV protein of HERV-K protein of HERV-K identity with SEQ ID No: 1 C and D together having 100% sequence identity with SEQ ID No: 8 2-7 Gag protein from IAPE-D1 transmembrane domain of the cytoplasmic tail of the ENV having 100% sequence ENV protein of IAPE-D1 virus protein of IAPE-D 1 virus identity with SEQ ID No: 13

[0367] 2-8 Gag protein from IAPE-D1 transmembrane domain of the cytoplasmic tail of the ENV having 100% sequence ENV protein of IAPE-D1 virus protein of IAPE-D 1 virus identity with SEQ ID No: 13 C and D together having 100% sequence identity with SEQ ID No: 47

[0368]

[0369] In a further aspect the invention provides an mRNA or DNA molecule encoding

[0370] (1) a first polypeptide A; and

[0371] (2) a second polypeptide, wherein the second polypeptide comprises the polypeptides B, C and D preferably in the order B-C-D,

[0372] wherein the components A, B, C and D correspond any of the embodiments 3-1 through 3-5 as listed in the table below:Embodiment A B c D

[0373] 3-1 Gag protein from Influenza A transmembrane domain of cytoplasmic tail of HERV-K hemagglutinin the ENV protein of the ENV protein of (HA)ECD HERV-K HERV-K

[0374] 3-2 Gag protein from Envelope transmembrane domain of cytoplasmic tail of HERV-K glycoprotein gpl40 of the ENV protein of the ENV protein of simian HERV-K HERV-K immunodeficiency

[0375] virus (SIV)

[0376] 3-3 Gag protein from hMPV glycoprotein transmembrane domain of cytoplasmic tail of IAPE-D1 virus FECD the ENV protein of from the ENV protein of IAPE-D1 virus from IAPE-D1 virus 3-4 Gag protein from murine transmembrane domain of cytoplasmic tail of IAPE-D1 virus Carcinoembryonic the ENV protein of from the ENV protein of antigen-related cell IAPE-D1 virus from IAPE-D1 virus adhesion molecule 1

[0377] (CEACAMI)ECD

[0378] 3-5 Gag protein from human transmembrane domain of cytoplasmic tail of IAPE-D1 virus Carcinoembryonic the ENV protein of from the ENV protein of antigen-related cell IAPE-D1 virus from IAPE-D1 virus adhesion molecule 5

[0379] (CEACAM5)ECD

[0380]

[0381] In preferred embodiments of the embodiments listed above the mRNA and / or DNA molecule is formulated as a lipid nanoparticle.

[0382] In a further aspect the invention provides an mRNA or DNA molecule encoding

[0383] (1) a first polypeptide A; and

[0384] (2) a second polypeptide, wherein the second polypeptide comprises the polypeptides B, C and D preferably in the order B-C-D,

[0385] wherein the components A, B, C and D correspond any of the embodiments 4-1 through 4-5 as listed in the table below:

[0386] Embodiment A B C D

[0387] 4-1 Gag protein from Influenza A hemagglutinin transmembrane domain cytoplasmic tail of HERV-K with at (HA)ECD with at least 95% of the ENV protein of the ENV protein of least 95% sequence HERV-K HERV-K

[0388]

[0389] identity with SEQ ID sequence identity with C and D together having at least 95% sequence No: 1 SEQ ID No: 64 identity with SEQ ID No: 8

[0390] 4-2 Gag protein from Envelope glycoprotein transmembrane domain cytoplasmic tail of HERV-K with at gpl40 of simian of the ENV protein of the ENV protein of least 95% sequence immunodeficiency virus HERV-K HERV-K identity with SEQ ID (SIV) with at least 95% C and D together having at least 95% sequence No: 1 sequence identity with identity with SEQ ID No: 8

[0391] SEQ ID No: 71

[0392] 4-3 Gag protein from hMPV glycoprotein FECD transmembrane domain cytoplasmic tail of IAPE-D1 with at with at least 95% sequence of the ENV protein of the ENV protein of least 95% sequence identity with SEQ ID No: from IAPE-D1 virus from IAPE-D1 virus identity with SEQ ID 67 C and D together having at least 95% sequence No: 13 identity with SEQ ID No: 47 4-4 Gag protein from murine Carcinoembryonic transmembrane domain cytoplasmic tail of IAPE-D1 with at antigen-related cell of the ENV protein of the ENV protein of least 95% sequence adhesion molecule 1 from IAPE-D1 virus from IAPE-D1 virus identity with SEQ ID (CEACAMI)ECD with at C and D together having at least 95% sequence No: 13 least 95% sequence identity with SEQ ID No: 47

[0393] identity with SEQ ID No:

[0394] 73

[0395] 4-5 Gag protein from human transmembrane domain cytoplasmic tail of IAPE-D1 with at C arcinoembry onic of the ENV protein of the ENV protein of least 95% sequence antigen-related cell from IAPE-D1 virus from IAPE-D1 virus identity with SEQ ID adhesion molecule 5 C and D together having at least 95% sequence No: 13 (CEACAM5)ECD with at identity with SEQ ID No: 47

[0396] least 95% sequence

[0397] identity with SEQ ID No:

[0398] 75

[0399]

[0400] In a further aspect the invention provides an mRNA or DNA molecule encoding

[0401] (1) a first polypeptide A; and

[0402] (2) a second polypeptide, wherein the second polypeptide comprises the polypeptides B, C and D preferably in the order B-C-D,

[0403] wherein the components A, B, C and D correspond any of the embodiments 5-1 through 5-5 as listed in the table below:Embodiment A B C D

[0404] 5-1 Gag protein from Influenza A transmembrane domain cytoplasmic tail of HERV-K with 100% hemagglutinin (HA) of the ENV protein of the ENV protein of sequence identity with with 100% sequence HERV-K HERV-K

[0405] SEQ ID No: 1 identity with SEQ ID C and D together having 100% sequence identity No: 64 with SEQ ID No: 8

[0406] 5-2 Gag protein from Envelope glycoprotein transmembrane domain cytoplasmic tail of HERV-K with 100% gpl40 of simian of the ENV protein of the ENV protein of sequence identity with immunodeficiency virus HERV-K HERV-K

[0407] SEQ ID No: 1 (SIV) with 100% C and D together having 100% sequence identity sequence identity with with SEQ ID No: 8

[0408] SEQ ID No: 71

[0409] 5-3 Gag protein from IAPE- hMPV glycoprotein F transmembrane domain cytoplasmic tail of D1 with 100% sequence with 100% sequence of the ENV protein of the ENV protein of identity with SEQ ID identity with SEQ ID from IAPE-D1 virus from IAPE-D1 virus No: 13 No: 67 C and D together having 100% sequence identity with SEQ ID No: 47

[0410] 5-4 Gag protein from IAPE- murine transmembrane domain cytoplasmic tail of D1 with 100% sequence C arcinoembry onic of the ENV protein of the ENV protein of identity with SEQ ID antigen-related cell from IAPE-D1 virus from IAPE-D1 virus No: 13 adhesion molecule 1 C and D together having 100% sequence identity (CEACAM1) with with SEQ ID No: 47

[0411] 100% sequence identity

[0412] with SEQ ID No: 73

[0413] 5-5 Gag protein from IAPE- human transmembrane domain cytoplasmic tail of D1 with 100% sequence C arcinoembry onic of the ENV protein of the ENV protein of identity with SEQ ID antigen-related cell from IAPE-D1 virus from IAPE-D1 virus No: 13 adhesion molecule 5 C and D together having 100% sequence identity (CEACAM5) with with SEQ ID No: 47

[0414] 100% sequence identity

[0415] with SEQ ID No: 75

[0416]

[0417] BRIEF DESCRIPTION OF THE FIGURES

[0418] Figure 1: Schematic overview of nucleotide constructs exemplarily tested in vaccination studies.

[0419] A) Schematic representation of the position / association of VLP forming polypeptide components Gagand Env and in / at the cellular or VLP membrane. Env is a membrane integral protein with intracellular and extracellular polypeptide components and comprises a membrane integral transmembrane domain. Gag (c.f. polypeptide A of the present disclosure) is typically situated at the interior side of the biological membrane and may associate with the cytoplasmic C-terminal end of the Env. Env may be divided into certain subsections: surface subunit (SU) and transmembrane subunit. Transmembrane subunit consists of three domains; an ectodomain (Ecto) with extracellular orientation, i.e. being displayed on the cell / VEP surface, which comprise or represent the antigenic polypeptide B of the present disclosure; a transmembrane domain (TMD, c.f. polypeptide C of the present disclosure) spanning the cellular membrane; and an intracellularly oriented cytoplasmic tail (CT, c.f. polypeptide D of the present disclosure). B) The common design of all constructs encodes a beta-retroviral Gag sequence followed by a self-cleavable P2A peptide and a retroviral Env sequence in a nucleotide vector downstream of a CMV promoter: The first construct (HERV-W Gag HERV-W Env) depicts HERV-W Env encoded with a native HERV-W Gag sequence. Gag proteins have been associated with the formation of virus-like-particles (Komurian-Pradel F, Paranhos-Baccala G, Bedin F, Ounanian-Paraz A, Sodoyer M, Ott C, Rajoharison A, Garcia E, Mallet F, Mandrand B, Perron H. Molecular cloning and characterization of MSRV-related sequences associated with retrovirus-like particles. Virology. 1999 Jul 20;260(l):l-9.). The second construct (HERV-K Gag HERV-W Env) depicts HERV-W Env encoded with a HERV-K Gag that is a known VLP particle inducer. The third construct (HERV-K GagTMD-cr HERV-W Env) depicts HERV-K Gag co-expressed with a chimeric HERV-W / K Env where the sequence of the extracellular portion of HERV-W comprises the sequence of the HERV-K Env transmembrane domain and cytoplasmic tail. The 4thconstruct (HERV-K Gag HERV-K Env) depicts a HERV-K Gag and a HERV-K Env co-expressed. The 5thconstruct (JAPE Gag JAPE Env) depicts an IAPE-D1 Gag protein co-encoded with an IAPE-D1 Env protein, demonstrating other possible beta-retroviral constructs.

[0420] Figure 2: A) Shown is the mean fluorescence intensity of staining for an anti-HA tag bound to various HA-tagged HERV envelope constructs. The results demonstrate that human endogenous retrovirus envelope proteins of HERV-W, HERV-H, HERV-K, HERV-FRD and HERV-3.1 variants are efficiently and similarly expressed, all of them comprising functional TMD-CT domains, and expression efficiency of these proteins does not benefit from a Vesicular Stomatitis Virus (VSV) cytoplasmic tail and transmembrane domain (VSVTMD-CT) substitution. In particular the HERV-W envelope is already efficiently expressed on its own, but expression is significantly reduced upon substitution with VSVTMD-CT. It is therefore particularly surprising that this HERV-W envelopeprotein is even better expressed when fused to the cytoplasmic tail of HERV-K envelope protein compared to the expression of HERV-W envelope protein alone. This is even more surprising since the experiment shows, that the HERV-K envelope protein was much less expressed than HERV-W and thus it would not be apparent that HERV-W expression could benefit from HERV-K’ s cytoplasmic tail. B) Schematic depiction of tested constructs.

[0421] Figure 3: (A) Vaccination scheme, BALB / c mice were vaccinated with an Adl9a / 64 vector via subcutaneous administration and then end-bled on days 14 or 28 (left). Blood serum samples were analysed for immune response to the vaccination, i.e. the formation of antibodies using a mouse renal adenocarcinoma cell line RENCA model (right) was assessed. (B) Serum IgG response to the vaccination with different constructs as indicated, quantified on days 14 or 28 after vaccination by measuring mean fluorescence intensity (MFI) indicative of the amount of detected bound IgG. Anti-HERV-W Env response by IgG antibody formation is improved by vaccination with a construct comprising HERV-K Gag and is even further improved in a construct, wherein the HERV-W Env comprises the C-terminal and transmembrane region of HERV-K Env. Data analysed in a 2- way ANOVA multiple comparison. (C) Serum IgG response to vaccination with the different constructs as in B) but with end-bleed only on day 28 and serum samples being tested by cell-based ELISA at 1:50 (left) and 1:150 dilution (right) of serum at an OD of 450 nm. The results again show HERV-W Env comprising the C-terminal and transmembrane domain of HERV-K Env elicit much higher IgG antibody response than the reference constructs. Data was analysed in a 2-way ANOVA multiple comparison. (D) Surface expression of HERV-W-Env on A549 cells from different constructs as indicated, quantified by measuring mean fluorescence intensity (MFI) indicative of the amount of detected HERV-W Env displayed on live cells. Anti-HERV-W Env display on live cells is improved with a construct comprising HERV-K Gag (instead of for instance HERV-W’ s own Gag) and is even further improved with a construct, wherein the HERV-W Env comprises the C-terminal and transmembrane region of HERV-K Env. These data are surprising since incorporating cytoplasmic tails of retroviral envelope proteins has been associated with less VLP incorporation, while a truncation of the cytoplasmic domain improved VLP incorporation (Zingler K, Littman DR. Truncation of the cytoplasmic domain of the simian immunodeficiency virus envelope glycoprotein increases env incorporation into particles and fusogenicity and infectivity. J Virol. 1993 May; 67(5):2824-31). In the present experiment, particularly incorporation of the full-length HERV-K cytoplasmic tail is shown to increase an antibody response against the attached HERV-W Env, even compared to an Env protein expressing a native HERV-W Env (c.f Figure 2).Figure 4: Transmission electron microscopy (TEM) of chimeric VLPs (bottom) and the assumed structural scheme of the chimeric construct located in the cell membrane. (A) Expression of a VLP comprising HERV-K-Env and HERV-K-Gag as a positive control reference results in VLPs displaying a multitude of Env proteins on its surface (left). Expression of a construct coding for a VLP comprising HERV-W-Env and HERV-W-Gag results in no formation of VLPs at all (right). (B) Expression of a VLP comprising HERV-W-Env and HERV-K-Gag results in a VLP displaying a few HERV-W-Env proteins on its surface (left). Expression of a VLP comprising HERV-W-Env, which comprises the transmembrane and C-terminal domain (cytoplasmic tail) of a HERV-K-Env, and HERV-K-Gag results in a much-improved display of a number of HERV-W-Env proteins on the VLP surface (right). These data confirm that a HERV-K TMD-CT construct augments the incorporation of chimeric Env into HERV-K Gag based VLPs. This finding is surprising since earlier findings regarding lentiviruses and regarding HERV-K envelope proteins grafted onto lentiviral particles showed that the elimination of the HERV-K Env cytoplasmatic tail lead to increased virion incorporation (Hanke K, Kramer P, Seeher S, Beimforde N, Kurth R, Bannert N. Reconstitution of the ancestral glycoprotein of human endogenous retrovirus k and modulation of its functional activity by truncation of the cytoplasmic domain. J Virol. 2009 Dec; 83(24): 12790-800. Epub 2009 Oct 7.).

[0422] Figure 5: A VLP comprising HERV-K Gag and HERV-K Env induces a potent and rapid antibody response in cynomolgus macaques, the serum of which comprise antibodies that can be used in staining of human cancer cells expressing HERV-K Env on their surface. Animals were immunized and bled later on, serum was analyzed using ELISA on stained human cancer cells. (A) Vaccination schedule. Non-human primates (NHPs) (Macaca fascicularis) were vaccinated with an Adl9a / 64 vector via intramuscular administration on day 0 and day 90. Blood serum samples were obtained on days 0, 30, 60, 90, 120 and 150. (B) Serum IgG response to HERV-K Env proteins induced by the vaccine. ELISA plates were coated with the transmembrane subunit (TM) or surface subunit (SU) of the HERV-K Env protein. HERV-K specific antibodies in NHP blood serum from different time points (days 0, 30, 60, 90, 120 and 150) were detected by ELISA using a serum dilution of 1:100. Each line represents one monkey. Serum antibody responses were induced after the first immunization and slowly decline after day 30. Responses increased after day 90 by a second administration of the Adl9a / 64 vector. (C) Animals’ serum IgG responses to HERV-K expressing breast cancer cells. MDA-MB-231 cells were incubated with NHP blood serum followed by an APC-coupled secondary anti-IgG antibody. X-axis shows amount of MDA-MB-231 cell-binding antibodies and Y axis shows the number of cells detected with a given discrete fluorescence value. Fluorescence of cells wasdetected by flow cytometry. Serum from day 150 (60 days after boost) showed increased levels of MDA-MB-231 cell-binding antibodies compared to native serum and serum from day 30. Results are shown for one of three monkeys. Samples were run in duplicates.

[0423] The displayed data shows that a single immunization with HERV-K Gag and HERV-K Env rapidly induces stable antibody responses in cynomolgus macaques, despite the existence of an endogenous simian ERV-K homologues with high homology to HERV-K in these animals. The antibodies induced are rapidly detectable, stable and mature into a response capable of staining human cancer cells, a correlate of protection in responses to immunotherapies.

[0424] Figure 6: Staining of transduced THP1 cells with rat serum comprising IAPE-D1 or HERV-K directed antibodies. Serum was isolated from rats immunized against IAPE-D1 VLP (comprising IAPE-D1 Gag and Env) (Adl9a / 64 vector + protein) or HERV-K VLP (comprising HERV-K Gag and Env) (Adl9a / 64 + Ad5 vectors), i.e. serum contains rat anti-IAPE-Dl VLP and rat anti-HERV-K VLP antibodies. THP1 cells were transduced to express IAPE-D1 or HERV-K VLP on their surface using respective Adl9a / 64 vectors. Transduced THP1 cells were stained by incubating with the respective rat serum followed by a secondary anti-rat IgG AF647 antibody and samples were analysed by flow cytometry. The graph shows the mean fluorescence intensity (MFI) of AF647 indicating the amount of rat antibody bound to THP1 cell surface. Antibodies in serum from IAPE-D1 VLP vaccinated rats bound strongly to IAPE-D1 VLP transduced THP1 cells compared to serum from nonimmunized control rats, for which no binding was observed. The detected MFI signal for IAPE was even higher than the MFI signal observed for HERV-K VLP rat serum with antibodies binding to HERV-K VLP transduced THP1 cells. Neither IAPE-D1 VLP antibodies nor HERV-K VLP antibodies in respective serum bound unspecifically to untransduced THP1 cells, i.e. cells not displaying these VLPs. These effects are surprising since while HERV-K is known to be capable of forming VLPs and known to be associated with immunogenicity in human cancers, some other ERVs, such as MERV34 and HERV-3 (Ng Kevin W et al.; Antibodies against endogenous retroviruses promote lung cancer immunotherapy. Nature. 2023 Apr;616(7957):563-573.), are known to not be particularly associated with immunogenicity in humans. These data suggest that beta-retroviral VLPs in general are capable and suitable to induce antibody responses via virus-vectored VLPs - not only HERV-K. They may thus be used to break endogenous retroviral tolerance in NHPs (see also figure 5) and in humans.

[0425] Figure 7: Staining of HEK293 cells for surface expression of HERV-W Env subunit, 24 hours post transfection. HEK293 cells were transfected with DNA plasmids as indicated on the x-axis. Non-transfected HEK293 cells (“No DNA”) or transfected cells with an “irrelevant” plasmid (“Neg. Ctrl DNA”) were used as negative controls. Transfected HEK293 cells were stained with a rabbit antihuman HERV polyclonal antibody (PA5-22819) as described in the example section and analysed by flow cytometry. Each sample was assessed in duplicates and the experiment was performed twice. The graph shows the mean fluorescence intensity of HERV-W Env subunit expressed on the surface of HEK293 cells indicated on the y-axis. Cells were gated following this order: Single cells, HEK293 cells, live cells. Each bar includes the duplicate values of the two independent experiments with the standard error of the mean (SEM). In comparison to the plasmid expressing HERV-W Gag and Env (W-Gag W-Env), the replacement of HERV-W Gag by HERV-K Gag (K-Gag W-Env) increased the surface expression of HERV-W Env. A similar increase was observed for K-Gag W-Env that comprises the TMD and CT domains of HERV-K Env, wherein the cytoplasmic tail of HERV-K Env is truncated. A significant increase in surface expression was observed when the CT was either truncated by 6 C-terminal amino acids (K-GagTMD-cT-6aatruno W-Env) or by 30 C-terminal amino acids (K-GagTMD-cT-30aatrunc W-Env). The increase was even more enhanced with a truncation of the cytoplasmic tail of HERV-K Env (included as part of HERV-W Env) by 20 C-terminal amino acids (K-GagTMD-CT-20aatrunc W-Env). Still, as discussed for C-terminal amino acid exchanges in the cytoplasmic tail of HERV-K Env herein in the following, the incorporation of the 6 C-terminal amino acid residues of the HERV-K Env cytoplasmic tail was also found to be beneficial for the surface expression of HERV-W Env. These findings indicate that removal of a largely lipophilic peptide section in the range from the 7th amino acid counted from the C-terminus of the cytoplasmic tail of HERV-K Env to the 20th amino acid counted from the C-terminus of the cytoplasmic tail of HERV-K Env may provide a particularly improved (HERV-W) Env surface expression. The amino acid sequence from amino acid 7 to 20 counted from the C-terminus of the cytoplasmic tail of HERV-K Env on the other hand may not be supportive and could possibly have an inhibitory effect, particularly if the cytoplasmic tail does not comprise the 6 C-terminal amino acids of HERV-K Env CT at the C-terminus. Truncating the cytoplasmic tail further again lowers expression, indicating that the Env surface expression also benefits from the peptide section in the range from the 21 st amino acid counted from the C-terminus of the cytoplasmic tail of HERV-K Env to the 30th amino acid counted from the C-terminus of the cytoplasmic tail of HERV-K Env.

[0426] Results were also obtained with partial cytoplasmic tail substitutions of HERV-W Env, wherein C-terminal residues in the cytoplasmic tail (CT) domain of a HERV-K Gag HERV-W Env construct (K-Gag W-Env) were exchanged with C-terminal HERV-K Env cytoplasmic tail residues. Here, anincrease of HERV-W Env expression on the surface of HEK293 cells was observed for an exchange of at least the last 6 C-terminal amino acid residues of the cytoplasmic tail of HERV-W Env with the corresponding 6 C-terminal amino acid residues in the cytoplasmic tail of HERV-K Env (K-Gag W-Enveaa-exoh). This increase was maintained with a corresponding further exchange of the last 20 C-terminal amino acid residues with the corresponding residues of HERV-K Env (K-Gag W-Env20aa-exoh). A complete replacement of the C-terminus of the full cytoplasmic tail domain of HERV-W Env with the corresponding full cytoplasmic tail of HERV-K Env in turn diminished the surface expression of HERV-W Env (K-Gag W-Envcr-exoh). These findings suggest that for a construct comprising HERV-K Gag and an Env protein of interest, the last 6 C-terminal amino acid residues of HERV-K Env cytoplasmic tail may advantageously be included to replace the last 6 C-terminal amino acids of the cytoplasmic tail of the Env of interest to obtain a beneficial effect on Env surface expression. The addition of the full HERV-K Env cytoplasmic tail to replace the cytoplasmic tail of HERV-W Env without also including the corresponding HERV-K transmembrane domain (TMD) may be less beneficial. Without wishing to be bound by theory, this may be due to disturbance of interactions of the cytoplasmic tail with the transmembrane domain and membrane proximal residues.

[0427] Figure 8: Staining of HEK293 cells for surface expression of a selection of ERV Env subunit, 24 hours post transfection. HEK293 cells were transfected with DNA plasmids as indicated above each FACS results panel (A) or under the histogram (B), respectively. Transfected HEK293 cells were stained with the primary antibody as described in the method section and analysed by flow cytometry.

[0428] (A) shows the representative dot plots from flow cytometry illustrating the percentage of transfected HEK293 cells expressing the respective ERV-Env on their surface. Cells were gated following this order: single cells, HEK293 cells, live cells and ERV Env expression (as indicated in the y-axis of the dot plot). The x-axis of the dot plot corresponds to forward scatter signal (in this case measuring GFP fluorescence intensity indicative of cell size, which is not of relevance for the present assessment). It was observed that the replacement of the TMD-CT of the respective ERV with the TMD-CT of HERV-K (K-Gag TMD-CT Env) increased the percentage of transfected HEK293 cells expressing on their surface MER34-Env (K-Gag TMD-CT MER34-Env), FRD-Env (K-Gag TMD-CT FRD-Env) or MSRV-Env (K-GagTMD-CT MSRV-Env), respectively, in comparison to their respective controls, wherein the TMD-CT domain of the ERV was not replaced by that of HERV-K Env (K-Gag MER34-Env, K-Gag FRD-Env, K-Gag MSRV-Env). (B) shows a representative overlay histogram from flow cytometry data illustrating the expression level of ERV3.1 Env on the surface of transfected HEK293 cells with the construct K-GagTMD-CT ERV3.1 -Env (white area under the curve), wherein the TMD-CT of the ERV3.1 was replaced with the TMD-CT of HERV-K. The area under the curve histogram is the transfected HEK293 cells stained with the secondary antibody only. Here cells were gated following this order: single cells, HEK293 cells, live cells and ERV3.1 Env expression (x-axis). In the case of the ERV3.1 Env subunit, no WT membrane anchored protein could be identified and compared at all due to mutation, but the expression was confirmed when ERV3.1 Env was expressed with the TMD-CT of HERV-K Env.

[0429] Figure 9: BALB / c mice received one immunization of one of the Adl9a / 64 vectors indicated on the x-axis of the graph. Blood was collected on day 0 before vaccination and on days 14 and 28 post vaccination. Blood serum samples were analysed by ELISA, for their ability to bind HERV-H Env protein. The graph shows the EC50 serum dilution values (y-axis) from blood serum collected at day 14 and 28 post vaccination (x-axis). Statistical analysis was performed by GraphPad Prism 8 using ANOVA test with Brown-Forsythe and Welch tests. P values (p), * means p<0.05, ** meaning p<0.01. Mice vaccinated with a construct of HERV-K Gag and HERV-H Env, wherein the TMD-CT domain of HERV-H Env was replaced with the TMD-CT domain of HERV-K Env (K-GagTMD-CT H-Env) induced significantly higher antibody responses in the vaccinated BALB / c mice (as a higher serum dilution is required to reach 50% of binding (EC50)) against the HERV-H protein compared to the vaccine comprising only H-Env and compared to the vaccine comprising HERV-K Gag HERV-H-Env (without modified TMD-CT) on day 28. A significantly higher antibody response in the BALB / c mice vaccinated with K-GagTMD-CT H-Env was also observed compared to HERV-H Env on day 14 post immunization. Furthermore, already the vaccination with HERV-K Gag HERV-H-Env induced higher antibody responses in mice than vaccination with HERV-Env alone.

[0430] Figure 10: Vaccination schedule for immunization of mice. BALB / c mice were vaccinated on day 0 with an Ad5F35 vector encoding either IAPE-D1 Gag with murine endogenous retrovirus MM9 Env, wherein MM9 Env comprises the TMD-CT domain of IAPE-D1 Env (Ad5F35-IAPE-GagTMD-CT MM9-Env) or IAPE-D1 Gag with MM9 Env, wherein MM9 Env comprises the TMD-CT domain of IAPE-D1 Env including an ISD (immune suppressive domain) mutation (Ad5F35-IAPE-GagTMD-CT MM9-EnvISDmut) or with an Ad5 vector encoding either MelARV Gag with MelARV Env (Ad5-MelARV-Gag MelARV-Env) or MelARV Gag with MelARV Env including an ISD mutation (Ad5-MelARV-Gag MelARV-EnvISDmut). The mice were boosted on day 27 with an Adl9a / 64 vector encoding the same transgene as in the first immunization IAPE-D1 Gag with MM9 Env, wherein MM9 Env comprises the TMD-CT domain of IAPE-D1 Env (Adl9a-IAPE-GagTMD-cr MM9-Env) or IAPE-D1 Gag with MM9 Env, wherein MM9 Env comprises the TMD-CT domain of IAPE-D1 Envincluding an ISD mutation (Adl9a-IAPE-GagTMD-cT MM9-EnvISDmut) or with an Adi 9a vector encoding either MelARV Gag with MelARV Env (Adl9a-MelARV-Gag MelARV-Env) or MelARV Gag with MelARV Env including an ISD mutation (Adl9a-MelARV-Gag MelARV-EnvISDmut). Blood serum samples were harvested on day 0 and day 37 (DO, D37) and analysed for immune responses induced by the vaccine.

[0431] Figure 11: Serum IgG response to the vaccination with different constructs as indicated, analysed on day 37 (10 days post boost vaccination, D37) by ELISA (see vaccination schedule in Figure 10). Comparison is performed against previously published immunization vectors (Neukirch 2019 PMID: 30858929 and Daradoumis 2023 PMID: 37112906) encoding MelARV in Ad5 vectors. In naive mice Ad5 and Ad5F35 are similar (see cited references) with a trend towards superiority of Ad5 at lower vector doses. The MelARV and MM9 are both endogenous murine leukemia viruses. They are genetically closely related, however not fully identical (99.4% Env protein sequence identity). In view of their high sequence identity, these viruses and use of their proteins for vaccination allow a direct comparison. MelARV surface unit protein was used in the ELISA, wherein it was found that the tested vaccine approach was able to induce a stronger immune response against a genetically slightly more distant antigen, MM9 Env, than the MelARV vaccine tested against its own Env (SU) sequence (MelARV-Gag combined with MelARV-Env). As shown in figure 11, MM9 Env encoding vaccines induced higher antibody responses against the surface protein domain of the Env protein gp70 compared to the MelARV vaccines in the mice.

[0432] Figure 12: Vaccination schedule for immunization of mice. BALB / c mice were vaccinated on day 0 (DO) with 5 pg of mRNA vectors with N1 -Methylpseudouridine modification (Lipid nanoparticle with ALC0315 formulated in PBS / sucrose) encoding either HERV-K Gag with HERV-H Env including an ISDmut (mutated immune suppressive domain) (mRNA construct K-Gag H-EnvISDmut) or encoding HERV-K Gag with HERV-H Env, wherein HERV-H Env comprises the TMD-CT domain of HERV-K Env including an ISD mutation (mRNA K-GagTMD-cr H-EnvISDmut). For immunizations all mice were injected intramuscularly in the right muscle tibialis. Blood was collected on day 0 before vaccination (DO) and on day 12 (DI 2, termination) post vaccination. Serum was isolated at DO and D12 and used for ELISA.

[0433] Figure 13: Serum IgG response to the vaccination with different constructs as indicated, analysed on day 12 (12 days post vaccination, D12 - vaccination schedule according to Figure 12) by ELISA.The results show that mRNA encoding VLP constructs with HERV-H Env comprising the C-terminal and transmembrane domain of HERV-K Env elicit an increased IgG antibody response to the HERV envelope protein in the immunized mice compared to the reference construct, wherein HERV-H Env does not comprise the C-terminal and transmembrane domain of HERV-K Env. Statistical differences between the vaccination groups were calculated in Graph Pad Prism V. 8 using Mann-Whitney nonparametric test with significance levels defined as * = p<0.05.

[0434] Figure 14: A) Vaccination schedule for immunization of mice. BALB / c mice were vaccinated on day 0 (DO) and day 21 (D21) with 25 pg of DNA vector encoding either IAPE-D1 Gag with hMPV F-protein (ECD = extracellular domain), wherein the hMPV F-protein comprised the TMD-CT of IAPE-D1 Env (construct IAPE-Dl-Gag-P2A-hMPV-F-proteinECD-IAPE-Dl-EnvTMD-cT), or encoding IAPE-D1 Gag with hMPV F-protein (construct IAPE-Dl-Gag-P2A-hMPV-F-protein). For immunizations all mice were injected subcutaneously in the right lower limb. Blood was collected on day 0 before vaccination (DO) and on day 43 (D43). Serum harvested on DO and D43 was isolated and used for ELISA. B) Serum IgG response to the vaccination with different constructs as indicated, was analysed on day 0 and day 43 by ELISA. The results show that DNA encoding VLP constructs with hMPV F-protein comprising the cytoplasmic tail and transmembrane domain of IAPE-D1 Env (IAPE-D 1 -Gag-P2A-hMPV-F-proteinEcD-I APE-D 1 -EUVTMD-CT) elicit an increased IgG antibody response to the hMPV F-protein in the immunized mice compared to the reference construct, wherein F-protein does not comprise the cytoplasmic tail and transmembrane domain of IAPE-D1 Env (construct IAPE-D1 -Gag-P2A-hMPV-F-protein).

[0435] Figure 15: A) Vaccination schedule for immunization of mice. BALB / c mice were vaccinated on day 0 (DO) and day 21 (D21) with 25 pg of DNA vector encoding either HERV-K Gag with Influenza A HA protein, wherein the Influenza A HA protein comprised the TMD-CT of HERV-K Env (construct HERV-K-Gag-P2A-InfluenzaA-HAECD-HERV-K-EnvTMD-cT), or encoding either HERV-K Gag with Influenza A HA protein (construct HERV-K-Gag-P2A-InfluenzaA-HA). For immunizations all mice were injected subcutaneously in the right lower limb. Blood was collected on day 0 before vaccination (DO) and on day 42 (D42). Serum harvested on DO and D42 was isolated and used for ELISA. B) Serum IgG response to the vaccination with different constructs as indicated, was analysed on day 0 and day 42 by ELISA. The results show that DNA encoding VLP constructs with Influenza A HA comprising the cytoplasmic tail and transmembrane domain of HERV-K Env(construct HERV-K-Gag-P2A-InfluenzaA-HAECD- HERV-K-EUVTMD-CT) elicit a similar IgG antibody response to the Influenza A HA protein in the immunized mice compared to the reference construct, wherein Influenza A HA protein does not comprise the cytoplasmic tail and transmembrane domain of HERV-K Env (construct HERV-K-Gag-P2A-InfluenzaA-HA).

[0436] EXAMPLES

[0437] EXAMPLES - support of working principle (development of system for HERV Env display)

[0438] EXAMPLE 1: Adenoviral vaccine designs

[0439] HERV-W Env and Gag sequences used in vaccines and vaccination constructs herein are included in the sequences displayed herein (see Tables 1-8) and exemplary constructs are schematically depicted in Figure 1. Gag and Env sequences herein were encoded separated by a self-cleavable P2A peptide sequence and polypeptide expression was controlled by a CMV promoter comprising a Tet operator sequence. Sequences were assembled by commercial gene synthesis followed by subcloning into adenoviral shuttle plasmids pO6A19 for recombineering into hAdl9a / 64 BAC (bacterial artificial chromosome) plasmids containing El and E3 deleted genomes (deletion rendering the virus replication incompetent) of hAdl9a / 64 (human adenoviral vector 19a / 64). The obtained inserts in recombined backbone were then amplified in e-coli, purified, linearized and transfected into HEK293 derived cells. HEK293 cells used herein expressed a Tet repressor protein that reduces expression of transgenes harbouring a Tet operator sequence in their upstream CMV promoter. Viruses were amplified in these HEK293 derived cells, purified and tested for infectivity. The methods used herein above are as described in detail in Ragonnaud et al. 2017 and 2018 (Ragonnaud E, Pedersen AG, Holst PJ. Breadth of T Cell Responses After Immunization with Adenovirus Vectors Encoding Ancestral Antigens or Polyvalent Papillomavirus Antigens. Scand J Immunol. 2017 Mar;85(3):182-190; Ragonnaud E, Schroedel S, Mariya S, Iskandriati D, Pamungkas J, Fougeroux C, Daradoumis J, Thomsen AR, Neukirch L, Ruzsics Z, Salomon M, Thirion C, Holst PJ. Replication deficient human adenovirus vector serotype 19a / 64: Immunogenicity in mice and female cynomolgus macaques. Vaccine. 2018 Oct l;36(41):6212-6222).

[0440] The expression vectors used in the viral transduction examples were encoded into replicationdeficient human adenoviral vectors type 19a / 64 (hAdl9a / 64). The negative control vaccine (Neg. Ctrl vaccine) consisted of the same adenoviral vector but without any encoded antigens.EXAMPLE 2: Surface expression of different HA-tagged HERV Envelope proteins Data in this experiment was obtained in duplicates with 3 biological replicates each. Reagent compositions of reagents with specific names are outlined below. HEK293-T cells were seeded in 6-well plates with 400000 cells per well suspended in 2 ml DMEM-K and incubated over night at 37 °C. The following day a transfection mix of 660pl DMEM-0 with 2.75 pg DNA and 8.25 pl PEI (DNA: PEI ratio = 1:3) was prepared by mixing DMEM-0 and DNA, then adding PEI, vortexing at low speed for 5 s and incubating the transfection mix at room temperature (RT, about 21 °C) for 10 min. Medium on cells was changed to 600 pl DMEM-0 and 600 pl transfection mix transferred to each well, followed by 5-6 hours of incubation before a media change to 2 ml DMEM-K per well and incubation for 48. Encoding constructs to be expressed in the cells were as outlined in Figure 2B, wherein VSVTMD-CT indicates that the transmembrane domain and cytoplasmic tail of VSV (vesicular stomatitis virus) was (in some constructs) fused to the Ecto and surface unit domains of the tested envelope proteins.

[0441] Cells were then detached and suspended into the supernatant by pipetting and then transferred into FACS tubes, which were then stored on ice or at 4 °C. Cells were washed with PBS by centrifuging for 5 min at 300 g, supernatant being discarded before washing the pellet with 1 ml PBS, and then again centrifuging for 5 min at 500 g and discarding of supernatant. Cells were then washed again with 1 mL of FACS buffer before centrifugation for 5 min at 500 g, and discarding of supernatant. Fixation / Permeabilization of cells was performed by resuspending the cell pellet in 250 pl Cytofix / Cytoperm, a cell fixation / permeabilization kit, followed by incubation on ice for 20 min, then centrifugation for 5 min at 500 g and discarding of the supernatant.

[0442] Primary antibody staining was prepared for by washing cells twice, each time by resuspending in 0.5 ml Perm / Wash washing buffer, centrifuging for 5 min at 500 g and discarding of supernatant before initiating the staining by resuspension of cells in 100 pl antibody solution with anti -HA antibody diluted in a ratio of 1:150 and then incubation on ice for 25 min. After incubation, 0.5 ml Perm / Wash washing buffer was added, cells were centrifuged for 5 min at 500 g before discarding of supernatant and washing twice in Perm / Wash buffer as outlined above. Secondary antibody staining was performed following the same steps as for primary antibody staining, including post-staining washing steps except that rat anti-mouse IgGl / PE diluted 1: 500 in 3.5 ml Perm / Wash instead of anti-HA antibody was added. For quantification samples were resuspended in 500 pl FACS buffer before measuring in a flow cytometer at low flow speed. Results are shown in Figure 2 A.Reagent compositions:

[0443] - FACS-Buffer: 1 % inactivated FCS (incubate FCS at 56 °C for 30 min), 0.1% NaN3, 2 rnM EDTA, 500 ml PBS

[0444] - Cytofix / Cytoperm: 2g PF A, 0.5 g Saponin, Ad up to 50 ml PBS

[0445] - Perm / Wash: 0.1 % Saponin in PBS

[0446] - Primary antibody: Anti-HA tag antibody (Sino Biological #100028-MM15)

[0447] - Secondary antibody: rat anti-mouse IgGl PE (BD Bioscience)

[0448] EXAMPLE 3

[0449] 3.1 Animal procedures and serum isolation

[0450] All animal procedures were performed in accordance with the national guidelines and the experimental procedures were approved by the National Animal Experimental Inspectorate (Dyreforsogstilsynet). Female BALB / c mice were obtained at 6-8 weeks of age from Envigo and housed at the Panum Institute, University of Copenhagen, for at least one week before conducting any experiments.

[0451] For vaccination, mice were anaesthetised with isoflurane and then vaccinated subcutaneously (s.c.) in the lower right limb with 30 pL of the respective vaccine containing 2E+07 infectious units in IxPBS buffer. Animals were vaccinated with constructs as outlined in Figure 1, wherein the constructs encoded Gag and Env of the virus W-HERV, or Gag of K-HERV and Env of W-HERV, or Gag of K-HERV and Env of W-HERV, wherein the W-HERV Env comprised the transmembrane domain (TMD) and the cytoplasmic tail (CT) of the Env protein of K-HERV. Before and at the end of immunization studies, blood samples were taken by bleeding from the cheek of the animals. The vaccination schedule was as depicted in Figure 3 A: vaccination of mice on day 0 and end bleeding mice after 14 or after 28 days. Serum was isolated from the obtained blood samples by two consecutive centrifugation steps at 800 g for 8 min at 8°C. At the end of the immunization studies, mice were euthanized by cervical dislocation.

[0452] 3.2 Evaluation of HERV-W Env-specific antibody responses by flow cytometry

[0453] HERV-W Env-specific antibody responses were evaluated using RenCa cells modified to stably express HERV-W Env on their cell surface, as described in Skandorff et al 2023 (Skandorff, I.; Ragonnaud, E.; Gille, J.; Andersson, A.-M.; Schrodel, S.; Duvnjak, L.; Turner, L.; Thirion, C.; Wagner, R.; Holst, P. J. Human Adl9a / 64 HERV-W Vaccines Uncover Immunosuppression Domain-Dependent T-Cell Response Differences in Inbred Mice. Int. J. Mol. Sci. 2023, 24, 9972). Serum from pre- and end-bleeds of mice was diluted at 1:20 and added in duplicates to the HERV-W Env+ (expressing) RenCa cells. Hereafter, cells were stained by adding secondary PE goat anti-mouse IgG antibody (405307, BioLegend®, dilution 1:100) and eBioscience™ Fixable Viability Dye eFlour™ 780 (65-0865, Invitrogen™, 1:1000) for 30 min at 4°C to detect the bound secondary antibodies. Finally, the cells were fixated in 1% PFA for 15min at room temperature (about 21 °C). Flow cytometry was performed on an LSRFortessa™ 3 -laser flow cytometer, and the data were analysed using FlowJo™ vlO and GraphPad Prism 9. The mean fluorescence intensity for the different administered vaccination constructs on end bleed days 14 and 28 is depicted in Figure 3 B. The graph shows the mean of pre-bleeds from each mouse subtracted from the mean end-bleed measurements for each individual mouse. Statistical differences between the vaccination groups were calculated using two-way ANOVA multiple comparisons with significance levels defined as * = p<0.05, ** = p<0.01, *** =p<0.001.

[0454] 3.3 Evaluation of HERV-W Env-specific antibody responses by cell-based ELISA

[0455] HERV-W Env-specific antibody responses were also evaluated using cell-based ELISA, where wells were coated with RenCa cells modified to stably express HERV-W Env on their cell surface as described previously in Skandorff et al. 2023 (Skandorff, I.; Ragonnaud, E.; Gille, J.; Andersson, A -M.; Schrodel, S.; Duvnjak, L.; Turner, L.; Thirion, C.; Wagner, R.; Holst, P. J. Human Adl9a / 64 HERV-W Vaccines Uncover Immunosuppression Domain-Dependent T-Cell Response Differences in Inbred Mice. Int. J. Mol. Sci. 2023, 24, 9972). HERV-W Env+ RenCa cells were seeded in MaxiSorp flat-bottom plates (10394751; Thermo Scientific™, Waltham, MA, USA) for 2 h at 37°C and 5% CO2 prior to fixation with 4% PFA. Plates were washed five times and blocked overnight at 4°C in a blocking buffer (0.05% BSA buffer, 2.07% NaCl, 0.05% Tween-20 in PBS). After another round of washing, serum from pre- and end-bleeds was diluted 1:50 or 1:150 in blocking buffer and added to the wells in duplicates followed by incubation for 1 h at room temperature. Hereafter, wells were washed and incubated in secondary horseradish peroxidase (HRP) conjugated polyclonal goat anti-mouse IgG antibody (P0260, Dako Glostrup, Denmark; 1:2000) diluted in blocking buffer for 1 h at room temperature. Cell-based antibody response was detected using TMB PLUS2 solution (4395A, Kem-En-Tec Diagnostics, Taastrup, Denmark) and the colorimetric reaction of HRP was stopped using 0.2M H2SO4after 8 min. The absorbance at 450 nm was measured using the EnVision plate reader (Perkin Elmer, Waltham, MA, USA).The colorimetric signal from serum binding to the HERV-W Env expressing RenCa cells 28 days after immunization detected at an OD of 450 nm is depicted in Figure 3 C. The graph shows the mean of pre-bleeds from each mouse subtracted from the mean end-bleed measurements for each individual mouse. Statistical differences between the vaccination groups were calculated using two-way ANOVA multiple comparisons with significance levels defined as * = p<0.05, ** = p<0.01, *** = p<0.001, **** =p<0.0001.

[0456] 3.4 Evaluation of HERV-W Env improved surface display

[0457] Surface expression of HERV-W-Env on A549 cells, in which HERV Env was expressed from different constructs as indicated above, was tested by transfecting A549 cells with the described Env encoding constructs and measuring mean fluorescence intensity (MFI) after staining of HERV-W Env on the cell surface, which is indicative of the amount of detected HERV-W Env displayed on the live cells. For this, A549 cells were transduced with the different hAdl9a / 64-vectored vaccines at a multiplicity of infection (MOI) of 50. Tweenty-four hours after transduction, cells were stained with HERV-W Env surface subunit polyclonal antibody (rabbit anti-human HERV polyclonal antibody, PA5-22819; Invitrogen™, Waltham, MA, USA) at 15 pg / mL in FACS buffer (PBS with 1% BSA, 0.1% NaN3) for Ih at 4°C. Next, cells were stained with the secondary PE donkey anti-rabbit IgG antibody (406421; BioLegend®, San Diego, CA, USA; 1:100) and eBioscience™ Fixable Viability Dye eFlour™ 780 (65-0865; Invitrogen™, Waltham, MA, USA; 1:1000) for 30min at 4°C. Finally, the cells were fixated in 1% paraformaldehyde (PF A) for 15min at 4°C, and flow cytometry was run on the LSRFortessa™ 3-laser cell analyser (BD Biosciences, Franklin Lakes, NJ, USA). The flow cytometry data analysis was performed using FlowJo™ vlO analysis software. The results of the geometric mean fluorescence intensity (MFI) are depicted in Figure 3 D.

[0458] EXAMPLE 4: Transmission electron microscopy of ultrathin sections for virus-like particle (VLP) imaging

[0459] A549 cells were seeded and transduced and fixated as outlined in Example 3.3 above. Transmission electron microscopy (TEM) was performed on the fixated cells as described in Daradoumis et al., 2023 and Skandorff et al. 2023 (Daradoumis, J.; et al. An Endogenous Retrovirus Vaccine Encoding an Envelope with a Mutated Immunosuppressive Domain in Combination with Anti-PDl Treatment Eradicates Established Tumours in Mice. Viruses 2023, 15, 926; Skandorff, I.; Ragonnaud, E.; Gille, J.; Andersson, A.-M.; Schrodel, S.; Duvnjak, L.; Turner, L.; Thirion, C.;Wagner, R.; Holst, P. J. Human Adl9a / 64 HERV-W Vaccines Uncover Immunosuppression Domain-Dependent T-Cell Response Differences in Inbred Mice. Int. J. Mol. Sci. 2023, 24, 9972) by the Core Facility for Integrated Microscopy at the University of Copenhagen. In brief, A549 cells were seeded on Thermanox coverslips (150067, Thermo Scientific™) in 24-well plates, pre-coated with poly-L-lysine. In this, the cells were transduced with 50 MOI (Multiplicity of infection; number of viral particles present relative to host cells) of the respective construct and incubated for 24h before fixation with 2% glutaraldehyde in 0.05M sodium phosphate buffer (pH 7.2). Coverslips with seeded cells were rinsed with 0.15 M phosphate Buffer (pH 7.2) and post-fixed in 1% OsO4 in 0.12 M sodium phosphate buffer (pH 7.2) for 2h. The coverslips were then dehydrated in graded series of ethanol, transferred to propylene oxide, and finally embedded in EPON epoxy resin. The coverslips were then removed from the wells of the 24-well plate and ultrathin sections of ~60nm were cut using a Leica UC7 microtome (Leica Microsystems). Sections were collected on copper grids with Formvar (thermoplastic resins polyvinyl formats) supporting membranes and stained with uranyl acetate and lead citrate. The sections were finally examined with a Philips CM 100 Transmission electron microscope (Philips), operated at an accelerating voltage of 80 kV. Images were captured with an OSIS Veleta digital slow scan 2k x 2k CCD camera and the ITEM software package. Electron microscopic images of the detected forming VLPs are displayed along with supposed schematic representation of formed VLPs in Figure 4 A and B.

[0460] EXAMPLE 5

[0461] 5.1 Serum samples from non-human primates

[0462] Female cynomolgus macaques (Macaca fascicularis) subjected to this experiment were between 3 and 10 years old with a weight of from 3 to 5 kg. All experimental procedures were approved by the primate research center at Bogor Agricultural University IACUC (Animal Care and Use Committee). Three animals were selected based on the absence of tuberculosis, simian T-cell leukaemia virus and simian retrovirus infections. The selected animals were acclimatized for a month before the start of the trial. Animal handling procedures (blood collection, vaccination) were performed under anaesthesia (administration of ketamine and xylazine). Animals were housed in separate cages. They were given an enrichment environment and were fed with monkey chow (Charoen Pokpand, Thailand) twice a day. For each procedure, the animals were monitored for pain, distress, body temperature and weight.The animals were prime immunized on day (D) 0 (DO) by intramuscular (i.m.) injection of 2E+09 infectious units (IFU) of the adenoviral vector construct Adl9a(II)-(TetO)-CMV-ISDmut_coHERV-K-P2TS followed by a booster immunization of the same dose on D90. Blood samples were taken on DO, D30, D60, D90, D120 and DI 50 (see also vaccination and sample schedule depicted in Figure 5 A) and serum was isolated by centrifugation at 2500rpm for 15min (Beckman GS 6R).

[0463] 5.2 ELISA

[0464] MaxiSorp (NUNC) flat bottom plates were coated with proteins of the HERV-K Env (envelope protein) transmembrane subunit (TM) or HERV-K surface subunit (SU) at 2 pg / mL in PBS overnight at 4 °C (for orientation of units, see Figure 1 A). Plates were washed three times with Wash buffer (PBS + 354 mM NaCl + 0.1% Tween20, pH 7.2) and blocked for 1 h at RT (about 21 °C) using Blocking buffer (PBS + 354 mM NaCl + 5 g / L BSA + 0.05% Tween20, pH 7.2). After removing the blocking buffer, 100 pL / well cynomolgus macaque serum samples (obtained as described above) were added at a dilution of 1: 100 in blocking buffer, followed by 1 h incubation at RT. Plates were washed three times with washing buffer and horseradish peroxidase (HRP)-conjugated polyclonal anti-human / monkey IgG secondary antibody (Dako, P0214) was added at a dilution of 1:2000 in blocking buffer for 1 h of incubation at RT. After three washing steps, 50 pL of 3, 3', 5, 5'-tetramethylbenzidine (TMB) PLUS 2 chromogenic substrate for horseradish peroxidase (Kem-en-tec, 4395 A) was added, and the colorimetric reaction was stopped after 8 min with 50 pL 0.2 M H2SO4. Colour intensity in each well was determined by absorbance (OD) quantification at a wavelength of 450 nm (SpectraMax Microplate Reader). The background measured for the sample obtained on day 0 (DO) was subtracted in each sample of the respective animal. The results are shown in Figure 5 B.

[0465] 5.3 Cell surface staining

[0466] To analyse tumor-specific antibodies, epithelial human breast cancer cell line MDA-MB-231 cells were resuspended and incubated (30min, RT) with cynomolgus macaque blood serum, obtained as outlined above, diluted at 1:50 in FACS buffer (PBS + 1% BSA + 0.1% NaN3). MDA-MB-231 cell is a breast cancer cell line expressing HERV-K and particularly its envelope protein on the cancer cell surface. After washing, cells were resuspended and incubated (30min, 4 °C) in FACS buffer containing Phycoerythrin (PE)-labelled secondary antibody against Rhesus monkey IgG (Abeam; abl 25852) at a dilution of 1:100. Cell-surface bound serum antibodies were detected by flowcytometry in a BD LSR II Flow Cytometer via fluorescence quantification of the PE-label. Results are shown in Figure 5 C.

[0467] EXAMPLE 6

[0468] 6.1 Rat immunization

[0469] Female Wistar outbred rats at 6-8 weeks of age were obtained from Charles River. The rats were allowed to acclimatize for one week prior to the initiation of an experiment. All experiments were performed according to national guidelines and experimental protocols approved by the national animal experiments inspectorate (Dyreforsogstilsynet).

[0470] Obtaining IAPE rat serum: In the present context, IAPE rat serum is rat serum comprising IAPE antigen directed antibodies. 2 rats were immunized on day 0 (DO) with 2E+08 IFU of the adenoviral vector construct Adl9-IAPE-Dl in lOOpL PBS by subcutaneous (s.c.) injection into the right lower limb. Adl9-IAPE-Dl expresses the Gag and Env proteins of the murine endogenous retrovirus (ERV) IAPE-D1 under a strong CMV promoter, while Gag and Env are separated by a self-cleavable peptide p2A (see also amino acid sequences of this construct separately displayed in Table 6). On day 43 (D 43) rats were boosted with protein solutions of 50 pg IAPE-D1 Gag protein and 50pg IAPE-D1 Env protein in 500pL PBS + Incomplete Freunds Adjuvant (1:1). The reagent was administered s.c. with lOOpL in the neck and 200pL in each flank. Blood samples were taken on day 77 (D77) by heart puncture, and serum was isolated by centrifugation at 800g for 8min. Serum from both rats were pooled.

[0471] Obtaining HERV-K rat serum: In the present context, HERV-K rat serum is rat serum comprising HERV-K antigen directed antibodies. 6 rats were immunized on day 0 (DO) with 2E+08 IFU of the adenoviral vector construct Adl9-HERV-K_ISDmut in lOOpL PBS injected s.c. into the right lower limb. Adl9-HERV-K_ISDmut expresses the Gag and Env proteins of the human endogenous retrovirus (HERV) HERV-K under a strong CMV promoter, while Gag and Env are separated by a self-cleavable peptide p2A. On day 28 (D28), rats were boosted with 2E+08 of Ad5-HERV-K ISDmut. On day 55 (D55), serum samples were obtained as described for the IAPE rat serum above.

[0472] Obtaining control rat serum: Rats were immunized and treated as described for obtaining the HERV-K rat serum above, but using empty Adi 9 and Ad5 vectors, i.e. not encoding the Gag and Env proteins.6.2 Cell line

[0473] THP1 cells (human leukaemia monocytic cell line) were cultured in RPMI1640 supplemented with 10% heat- inactivated FBS, 2 mM L-glutamine, 25 mM HEPES, 100 pg / mL Normocin, and Pen / Strep. Cells were maintained at 37°C with 5% CO2 in a humidified atmosphere.

[0474] For generation of THP1 cells expressing IAPE-D1 or HERV-K (both Gag and Env of the respective virus expressed), the transduction enhancer LentiBOOST (Sirion Biotech) was added as an adjuvant to the cell culture medium according to manufacturer’s instruction for improving transduction efficiency. Cells were transduced with vector constructs Adl9-IAPE-Dl or Adl9-HERV-K at a multiplicity of infection (MOI) of 100. Transduced cells were incubated for 48h under standard cell culture conditions. As a negative control, cells were transduced with empty Adi 9 vectors, as described above.

[0475] 6.3 Cell surface staining

[0476] To analyse antigen-specific antibodies (directed against HERV-K or IAPE proteins) in serum obtained from IAPE or HERV-K immunized rats, transduced THP1 cells expressing IAPE-D1 or HERV-K or control cells (without said expression) were resuspended and treated with TruStain FcX FC receptor blocking solution (BioLegend, 422301) in a 1:20 dilution in FACS buffer (PBS + 1% BSA + 0.1% NaN3) for 10 min at RT (about 21°C) to avoid unspecific antibody binding. Rat serum was added to the cells to a final dilution of 1:50 in FACS buffer and incubated for 20 min at 4°C. After washing, cells were resuspended and incubated (20 min, 4 °C) in FACS buffer containing viability dye eFluor780 (Thermo Fisher; 65-0865-14) at a dilution of 1: 1000 (staining only alive cells) and with AF647-conjugated secondary antibody against rat IgG (BioLegend; 405416) at a dilution of 1:100. Cells were washed and fixed in 1% paraformaldehyde (PF A). Cell surface bound serum antibodies (directed against IAPE or HERV-K proteins) were detected by flow cytometry in a BD LSRFortessa III Cytometer, detecting the AF647 labelled secondary antibody. Results of MFI (mean fluorescence intensity) quantification are shown in Figure 6.

[0477] EXAMPLE 7

[0478] HEK293 cells were transfected with plasmids encoding either of the following constructs (“W” indicating “HERV-W” and “K” indicating “HERV-K”): W-Gag W-Env, K-Gag W-Env, K-Gag W-EnV6aa-exch, K-Gag W-EnV20aa-exoh, K-Gag W-EnVCT-exoh, K-GagTMD-CT-6aatruno W-EnV, K-GagTMD-CT-20aatruno W-Env, K-GagTMD-cT-30aatruno W-Env and a control plasmid (Neg. Ctrl DNA). Sequences of the respective constructs are outlined in table 9 herein. The transfection was performed using PEI and Opti-MEM (11058021, Gibco) in complete DMEM media without pen / strep. Transfection with PEI and Opti-MEM was in the ratio DNA: PEI of 1:3 and the ratio DNA: Opti-MEM was 1: 100, meaning 3pg DNA to 9pL (Img / mL) PEI to 300pL Opti-MEM were used. Cells were incubated for 24 h prior to surface staining. Cells were first stained with the primary antibody which was 15pg / mL of rabbit anti-human HERV polyclonal antibody (PA5-22819) directed against the HERV-W Env surface subunit. The primary antibodies were incubated in FACS buffer consisting of IxPBS with 1% BSA and 0.1% NaN3, for Ih at 4°C. Subsequently, cells were stained with secondary PE donkey anti-rabbit IgG antibody (406421, BioLegend®, 1: 100) and eBioscience™ Fixable Viability Dye eFlour™ 780 (65-0865, Invitrogen™, 1:1000) for 30min at 4°C in FACS buffer. Next, cells were fixated in 1% PFA for 15min at 4°C and flow cytometry was run on the LSRFortessa™ 3-laser. The results of the quantified mean fluorescence identity indicative of the amount of W-Env surface display are shown in Figure 7.

[0479] EXAMPLE 8

[0480] HEK293 cells transfected with plasmids encoding either of the following constructs (“MER34”, “FRD” and “MSRV” indicating “HERV-MER34”, “HERV-FRD” and “HERV-MSRV”, respectively and “K” indicating “HERV-K”): K-Gag MER34-Env, K-GagTMD-CT MER34-Env, K-Gag FRD-Env, K-GagTMD-CT FRD-Env, K-Gag MSRV-Env, K-GagTMD-CT MSRV-Env and K-GagTMD-CT ERV3.1-Env. Sequences of the respective constructs are outlined in tables 10 and 11 herein. The transfection was performed using PEI and Opti-MEM (11058021, Gibco) in complete DMEM media without pen / strep. Transfection with PEI and Opti-MEM was in the ratio DNA: PEI of 1:3 and the ratio of DNA: Opti-MEM was 1:100. Cells were incubated for 24h prior to surface staining.

[0481] Cells were first stained with the primary antibody which was either 1 Opg / mL of rabbit anti-human ERVMER34-1 polyclonal antibody (PA5-52836) for the ERV-MER34-1 Env surface subunit, or 30pg / mL of rabbit anti-human HERV-FRD polyclonal antibody (PA5-44470) for the HERV-FRD Env surface subunit, or 30pg / mL of rabbit anti-human HERV polyclonal antibody (PA5-95785) for the HERV-MSRV Env surface subunit, or lOpg / mL of rabbit anti-human ERV3.1 polyclonal antibody (PA5-82579) for the HERV-ERV3.1 Env surface subunit. The primary antibodies were incubated in FACS buffer consisting of IxPBS with 1% BSA and 0.1% NaNs, for 1 h at 4°C. Subsequently, cells were stained with secondary PE donkey anti-rabbit IgG antibody (406421, BioLegend®, 1:100) andeBioscience™ Fixable Viability Dye eFlour™ 780 (65-0865, Invitrogen™, 1: 1000) for 30min at 4°C. Next, cells were fixated in 1% PFA for 15 min at 4°C and flow cytometry was run on the LSRFortessa™ 3 -laser. The results regarding the detected respective amount of cells displaying the respective Envelope protein on their surface is depicted in Figure 8.

[0482] EXAMPLE 9

[0483] BALB / c mice were vaccinated on day 0 with an Adl9a / 64 vector encoding either a HERV-H Env subunit (H-Env), HERV-K Gag with HERV-H Env (K-Gag H-Env), or HERV-K Gag with HERV-H Env, wherein HERV-H Env comprises the TMD-CT domain of HERV-K Env (K-GagTMD-CT H-Env). Sequences of the respective constructs are outlined in tables 12 and 12 herein. The dose was 2x107infectious units (IFU) per mouse and doses were injected subcutaneously in the lower limb. Blood was collected on day 0 before vaccination, on day 14 (full-bleed) and on day 28 (full-bleed) post vaccination. Serum was isolated and used for ELISA. Wells of a Maxisorp Nunc 96-well plate (Thermo Fisher, #442404) were coated with 0.25 pg of recombinant soluble HERV-H Env protein overnight at 4 °C. The following day the wells were washed with washing buffer (PBS containing 0.1 % Tween-20) and then blocked with PBS containing 0.1 % Tween-20 and 0.5 % BSA for 2 hours at room temperature (about 21 °C). Afterwards, the wells were washed again and then incubated with BALB / c mouse pre-bleed serum (day 0) at a 1:50 dilution or the respective end-bleed serum (day 14 or day 28) in a four-fold serial dilution, starting at 1: 50, for 1 h at room temperature. Next, wells were washed and subsequently incubated with an anti-mouse IgG / HRP (P0448, Dako) at a 1:2000 dilution for 1 h at room temperature. Antibodies were detected using TMB and 1 M H2SO4as stop-solution. Absorbance was measured at 450 nm using an ELISA plate reader. The results regarding dilution of samples required to achieve a value of EC50 of antibody binding for the samples from respectively tested vaccinations on days 14 and 28 after vaccination are shown in Figure 9.

[0484] EXAMPLE 10

[0485] Viral vectors were produced as in Example 1. For Ad5 and Ad5F35 vectors the sequences were subcloned into adenoviral shuttle plasmids pO6A5 for recombineering into hAd5 and hAd5F35 BAC (bacterial artificial chromosome) respectively, plasmids containing El and E3 deleted genomes (deletion rendering the virus replication incompetent) of hAd5 (human adenoviral vector 5). BALB / c mice (4 groups of 5 mice) were vaccinated on day 0 with an Ad5F35 vector encoding antigens that can assemble into the form of VLPs, i.e. either IAPE-D1 Gag with murine endogenous retrovirusMM9 Env, wherein MM9 Env comprises the TMD-CT domain of IAPE-D1 (IAPE D subfamily copy 1 PMID: 18256233) Env (Ad5F35-IAPE-GagTMD-cTMM9-Env) or IAPE-D1 Gag with MM9 Env, wherein MM9 Env comprises the TMD-CT domain of IAPE-D1 Env including an ISD (immune suppressive domain) mutation (Ad5F35-IAPE-GagTMD-cT MM9-EnvISDmut) or with an Ad5 vector encoding either MelARV Gag with MelARV Env (Ad5-MelARV-Gag MelARV-Env) or MelARV Gag with MelARV Env including the ISD mutation (Ad5-MelARV-Gag MelARV-EnvISDmut). Amino acid sequences of the respective constructs are outlined in tables 14 and 15. Each group of mice received, respectively, a second immunization at day 27 with adenovector Adl9a / 64 encoding the same VLP encoding constructs as in the first immunization (DO), i.e. IAPE-D1 Gag with MM9 Env, wherein MM9 Env comprises the TMD-CT domain of IAPE-D1 Env (Adl9a-IAPE-GagTMD-cr MM9-Env) or IAPE-D1 Gag with MM9 Env, wherein MM9 Env comprises the TMD-CT domain of IAPE-D1 Env including the ISD mutation (Adl9a-IAPE-GagTMD-cr MM9-EnvISDmut) or with an Adi 9a vector encoding either MelARV Gag with MelARV Env (Adl9a-MelARV-Gag MelARV-Env) or MelARV Gag with MelARV Env including the ISD mutation (Adl9a-MelARV-Gag MelARV-EnvISDmut). All immunizations were injected at a dose of 2x108infectious units (IFU) per mouse and doses were injected subcutaneously in the lower limb. Blood was collected on day 0 before vaccination, and on day 37 (termination) post vaccination. The experimental outline is shown in Figure 10. Serum that was isolated at DO and D37 was assessed in ELISA. Wells of a Maxisorp Nunc 96-well plates were coated with 0.2 pg of recombinant soluble MelARV Env gp70 protein (surface protein domain of the Env protein) overnight at 4 °C. The following day the wells were washed with washing buffer (PBS containing 0.1 % Tween-20, 2.07% NaCl) and then blocked with PBS containing 0.05 % Tween-20 and 0.5 % BSA for 1 h at room temperature (about 21 °C). Afterwards, the wells were washed again and then incubated with BALB / c mouse serum taken on DO at a 1:20 dilution or taken on D37 in a two-fold serial dilution, starting at 1:20, for 1 h at room temperature (about 21 °C). Next, wells were washed and subsequently incubated with an anti-mouse IgG / HRP (P0260, Dako) at a 1:2000 dilution for 1 h at room temperature (about 21 °C). Antibodies were detected using TMB Plus and 0.2 M H2SO4as stop-solution. Absorbance was measured at a wavelength of 450 nm using an ELISA plate reader. To determine the endpoint titers (D37), the mean absorbance value plus five standard deviations for the average of the DO naive serum samples was first calculated (sample background). The endpoint titers (D37) were then determined from the x-axis intercept of the samples dilution curves at the previously calculated sample background. The results are shown in Figure 11.EXAMPLE 11

[0486] BALB / c mice (2 groups of 5 mice) were vaccinated on day 0 with an mRNA vector with Nl-Methylpseudouridine modification (formulated as Lipid nanoparticle with ALC0315 in PBS / sucrose), wherein the mRNA vector encodes constructs that can assemble into VLPs, i.e. either HERV-K Gag with HERV-H Env including an ISDmut (mutated immune suppressive domain) (mRNA K-Gag H-EnvISDmut) or HERV-K Gag with HERV-H Env including an ISDmut, wherein HERV-H Env comprises the TMD-CT domain of HERV-K Env (mRNA K-GagTMD-cr H-EnvISDmut) (provided by Genscript). Sequences of the respective constructs are outlined in tables 16, 17 (amino acid sequences), 18 and 19 (nucleic acid sequences). Immunizations of mice was conducted by injecting them at a dose of 5 pg per mouse and doses were injected intramuscularly in the right muscle tibialis. Blood was collected on day 0 (DO) before vaccination and on day 12 (DI 2) (termination) post vaccination. Serum was isolated from the blood samples taken at DO and DI 2 and used for ELISA. The experimental schedule is shown in Figure 12. Wells of a Maxisorp Nunc 96-well plate were coated with 0.2 pg of recombinant soluble HERV-H Env Con62 (HERV-H provirus PMID: 11162811) surface unit protein over night at 4 °C. The following day the wells were washed with washing buffer (PBS containing 0.1 % Tween-20, 2.07% NaCl) and then blocked with PBS containing 0.05 % Tween-20 and 0.5 % BSA for 1 h at room temperature (about 21 °C). Afterwards, the wells were washed again and then incubated with BALB / c mouse serum from DO and D12 at a 1:20 dilution, for 1 h at room temperature (about 21 °C). Next, wells were washed and subsequently incubated with an antimouse IgG / HRP (P0260, Dako) at a 1:2000 dilution for 1 h at room temperature (about 21 °C). Antibodies were detected using TMB Plus (3, 3', 5, 5'-tetramethylbenzidine) and 0.2 M H2SO4as stopsolution. Absorbance was measured at a wavelength of 450 nm using an ELISA plate reader. The results are shown in Figure 13.

[0487] EXAMPLES - support of universal applicability of VLP platform: extension to other antigens

[0488] EXAMPLE 12: DNA vectors

[0489] The DNA vectors pO6A19 encoding codon optimized HERV-K Gag, and Influenza A HA extracellular domain with the transmembrane domain and cytoplasmic tail of HERV-K Env (see sequences in table 20) (construct HERV-K-Gag-P2A-InfluenzaA-HAECD-HERV-K-EnvTMD-cT) or HERV-K GAG and Influenza A hemagglutinin (HA) (see sequences in table 21) (construct HERV-K-Gag-P2A-InfluenzaA-HA), or IAPE-D1 Gag, and hMPV F-protein extracellular domain with the transmembrane domain and cytoplasmic tail of IAPE-D1 Env (see sequences in table 24) (construct IAPE-Dl-Gag-P2A-hMPV-F-proteinECD-IAPE-Dl-EnvTMD-cT), or IAPE-D1 Gag and hMPV F-protein (see sequences in table 25) (construct IAPE-Dl-Gag-P2A-hMPV-F-protein), were synthesized by GenScript. Targeted genes to be expressed were preceded by a strong cytomegalovirus promoter and tetracycline operator (TetO) sites and were followed by the SV40 polyadenylation signal. The HERV-K GAG gene or IAPE-D1 Gag gene, respectively, was linked to Influenza A HA or hMPV F-protein, respectively, via a Glycine / Serine / Glycine (GSG) linker followed by a selfcleaving porcine tescho virus- 1 2A peptide (P2A).

[0490] EXAMPLE 13: Animal procedures and serum isolation

[0491] All animal procedures were performed in accordance with the national guidelines and the experimental procedures were approved by the National Animal Experimental Inspectorate (Dyreforsogstilsynet). Female BALB / c mice were obtained at 6-8 weeks of age from Envigo and housed at the Panum Institute, University of Copenhagen, for at least one week before conducting any experiments.

[0492] For vaccination, mice were anaesthetised with isoflurane and then vaccinated subcutaneously (s.c.) in the lower right limb with 30 pL of the respective vaccine containing 25 pg DNA in IxPBS buffer. Animals were vaccinated with constructs as outlined in Figure 1A and Figure 2A, wherein the constructs encoded:

[0493] either Gag and the transmembrane and cytoplasmic tail (TMD-CT) of IAPE-D1 and the extracellular domain of F-protein of hMPV, or Gag of IAPE-D1 and F-protein of hMPV, or Gag of HERV-K and the transmembrane and cytoplasmic tail (TMD-CT) of HERV-K and the extracellular domain of HA- protein of Influenza A, or Gag of HERV-K and the extracellular domain of HA-protein of Influenza A.

[0494] Before the beginning and at the end of immunization studies, blood samples were taken by bleeding from the cheek of the animals. The vaccination schedule was as depicted in Figure 14A and Figure 15A: vaccination of mice on day 0 and end bleeding mice at day 43 (Figure 14A) or day 42 (Figure 15A). Serum was isolated from the obtained blood samples by two consecutive centrifugation steps at 800 g for 8 min at 8°C. At the end of the immunization studies, mice were euthanized by cervical dislocation.EXAMPLE 14: ELISA

[0495] MaxiSorp (NUNC) flat bottom plates were coated with hMPV F-protein (Post Fusion F-Protein, MyBioSource) or HA protein (Influenza A H1N1 (A / Brisbane / 59 / 2007) Hemagglutinin / HA Protein extracellular domain with His Tag, Nordic Biosite) at 2 pg / mL in PBS overnight at 4 °C. It is noted that for ELISA for measurement of antibody responses to hMPV F-protein, ELISA plates could equally be coated with viral lysates of hMPV. Plates were washed three times with Wash buffer (PBS + 354 mM NaCl + 0.1% Tween20, pH 7.2) and blocked for 1 h at RT (about 21 °C) using blocking buffer (PBS + 354 mM NaCl + 5 g / L BSA + 0.05% Tween20, pH 7.2). After removing the blocking buffer, and washing the plates three times, 100 pL / well serum samples were added at a dilution of 1:25 and diluted in a 3 -fold dilution series in blocking buffer, followed by 1 h incubation at RT. Plates were washed three times with washing buffer and horseradish peroxidase (HRP)-conjugated polyclonal anti-mouse / IgG secondary antibody (Dako, P0260) was added at a dilution of 1:2000 in blocking buffer for 1 h of incubation at RT. After three washing steps, 50 pL of 3, 3', 5, 5'-tetramethylbenzidine (TMB) PLUS 2 chromogenic substrate for horseradish peroxidase (Kem-en-tec, 4395 A) was added, and the colorimetric reaction was stopped after 10-11 min with 50 pL 0.2 M H2SO4. Colour intensity in each well was determined by absorbance (OD) quantification at a wavelength of 450 nm (SpectraMax Microplate Reader). The background measured for the sample obtained on non-immunized mice was subtracted in each sample.

[0496] EXAMPLE 15: Blood collection for PBMC isolation

[0497] To obtain blood samples, approximately 10% of the total blood volume can be taken from mice by puncturing the facial vein with a Goldenrod lancet.

[0498] For full bleed cardiac puncture, mice undergo full isoflurane anaesthesia. Straight after, mice are placed upward with a facial mask which continuously supplied isoflurane, and the cardiac puncture is performed using a G27 needle connected to a 1 mL syringe. Approximately, 800-1000 pL is collected, and the mice are subsequently euthanized by cervical dislocation.

[0499] Blood samples are collected into EDTA collection tubes (microvette 500 K3E - Sarstedt 20.1341.100) and stored at 4°C until PBMC isolation.

[0500] EXAMPLE 16: PBMC isolation

[0501] Blood samples from EDTA collection tubes are transferred into 15ml tubes following by red blood lysis with ACK lysing buffer for 5min at room temperature. After red blood cell lysis, the tubes arecentrifuged at 300g for 5 min and the cell pellets are resuspended in complete RPMI (cRPMI, RPMI 1640 GlutaMAX medium, supplemented with 10% FBS, 1 mM sodium pyruvate, 1% (v / v) penicillinstreptomycin).

[0502] EXAMPLE 17: Splenocyte suspension

[0503] Once mice are euthanized, spleens are removed aseptically and transferred to cRPMI. Single-cell suspensions are obtained by pressing the spleens through a fine mesh (mesh size 70 pm), followed by centrifugation and wash, before resuspension in cRPMI. For tetramer staining, splenocytes are subjected to red blood cell lysis with ACK lysing buffer for 4-5 min at room temperature.

[0504] Example 18: Cellular immune responses

[0505] Tetramer staining: Mouse PBMCs and splenocytes without red blood cells are first incubated with the relevant tetramer. Typically 0.08pg to 0.25 pg of the relevant tetramers in FACS buffer (1% BSA, 0.1% NaN3) with 50nM of Desatinib are added to 400000 cells at 37°C, 5% CO2 for 15min in the dark (tetramer mix). Surface antibodies (CD8b-BV510, CD4-PE-Cy7, B220-PerCP-Cy5.5, CD44-FITC) and viability dye (Efluor780) in FACS Buffer with 50nM of Desatinib are then added on top the tetramer mix for 20min at 4°C in the dark. Cells are washed and fixed with 1% of paraformaldehyde (PF A) for 20min at 4°C in the dark.

[0506] Intracellular cytokine staining (ICS): 2-3x106splenocytes (without red blood cell lysis) are incubated with relevant peptides, which may be a peptide pool or specific peptides of the encoded antigens used for immunization, to stimulate cytokine production for 5 hours on 37°C, 5% CO2 in the presence of 4 pM monensin. A negative control (peptide control for measurement of background signal) for each mouse is incubated in cRPMI with monensin only.

[0507] Frequencies of epitope-specific CD8+ T cells are then determined by surface and intracellular cytokine staining (ICS). For surface staining, cells are stained with antibodies directed against CD8-BV421, CD4-PE-Cy7, B220-PerCP-Cy5.5, CD44-FITC in FACS buffer for 20 mm at 4°C in the dark. Cells are washed and fixed with 1% of paraformaldehyde (PF A) for 20 min at 4°C in the dark. Following washing, cells are stained with intracellular antibodies directed against IFN- y (APC) and TNF- a (PE) in 0.5% Saponin for 20 min at 4°C in the dark.

[0508] Fixed samples from tetramer staining and ICS are run on either Fortessa 3, 5, or X20 flow cytometers and analysed using FlowJo software VI 0.7.1.Example 19: mRNA immunization

[0509] As another immunization vehicle, mice can be immunized with an mRNA vector with Nl-Methylpseudouridine modification (e.g. formulated as Lipid nanoparticle comprising ALC0315 in PBS / sucrose), wherein the mRNA vector encodes constructs that can assemble into VLPs, i.e. wherein the mRNA vector encodes a Betaretroviral Gag, an intermittent a P2A self-cleavable linker and a surface antigen that has the transmembrane domain (TMD) from the same Betaretrovirus from which the Gag originates. For instance the mRNA vectors encode the same constructs as described for the DNA vectors (see example 12), but they can also encode other Betaretroviral Gag and surface antigen combinations.

[0510] Immunization of mice is conducted by injecting them at a dose of 5 pg per mouse and doses are injected intramuscularly, e.g. in the right muscle tibialis. Mice can be boosted on day 28. Blood is collected e.g. on day 0 before vaccination, on day 28 and day 56. Serum can be isolated from the blood samples taken at day 0, day 28 and day 56 and can be used for ELISA as described in Example 14 for detection of antibody responses. T cell responses can be analysed at day 14-28 post prime vaccination (that took place on day 0) or at day 10-12 post boost vaccination (that took place on day 28) by either tetramer staining or intracellular staining (as described in Example 18).

[0511] Example 20: Adenovirus immunization

[0512] DNA shuttle vectors (pO6A19) as described in Example 12 encoding the same or different antigens can be used for recombineering into hAdl9a / 64 BAC (bacterial artificial chromosome), a plasmid containing a viral genome, wherein El and E3 are deleted (deletion rendering the virus replication incompetent), the viral genome being of hAdl9a / 64 (human adenoviral vector 19 / 64a). A different shuttle (pO6A5) can be used for recombineering into hAd5 or hAd5F35 (bacterial artificial chromosome), respectively, which are plasmids containing viral genomes, wherein El and E3 are deleted (deletion rendering the virus replication incompetent), the viral genomes being of hAd5 (human adenoviral vector 5) and of hAd5F35 (human adenoviral vector 5F35). Mice can be vaccinated on day 0 with an Adl9a / 64 vector (2x108 infectious units (IFU)) and boosted on day 28 with and Ad5F35 or Ad5 adenovirus vector (2x107 IFU). The described adenovirus vectors can be formulated for vaccination in a formulation comprising Tris 10 mM; L-His 10 mM; NaCl 75 mM; Sucrose 5%; MgC12 ImM; EDTA 0,1 mM; Polysorbate 800,02% w / v; EtOH 0,5% v / v; pH 7,4 (A195 buffer); or a formulation comprising 10 mM Tris; 10 mM Histidine; 35 mM NaCl; 5% Sucrose; 1 mM MgCh; 0,01% Polysorbate 80; 0,1 mM EDTA; 0,5% EtOH; 60 mM Glutamate; 224 mM Alanine; pH 7,6 (D6buffer). For immunization all mice are injected at doses as mentioned above and doses are injected subcutaneously in the lower limb. Blood is collected on day 0 before vaccination, and on day 28 and day 56 for analysis of antibody responses. T cell responses can be analysed at day 14-28 post prime vaccination (that took place on day 0) or at day 10-12 post boost vaccination (that took place on day 28), e.g. by tetramer staining (Example 18) or intracellular staining (Example 18).

[0513] Example 21: Evaluation of antibody responses by flow cytometry

[0514] Antibody responses towards a specific antigen can be evaluated using a cell line expressing that antigen. As an example, the CT26 cell line can be used to express Ceacaml / CD66a an antigen of interest on its surface (see McLeod, Robbie L., et al. " Characterization of murine CEACAM1 in vivo reveals low expression on CD8+ T cells and no tumor growth modulating activity by anti-CEACAMl mAb CC1" Oncotarget 9.77 (2018): 34459). Serum from mice immunized as in Example 22 below (with a construct express the Ceacaml antigen) and isolated as in Example 13 can be used to detect Ceacaml responses, wherein Ceacam 1 is expressed on the CT26 cells. Serum of mice previously vaccinated with the construct expressing Ceacam 1 as the antigen of interest is diluted and added to the cells. Hereafter, cells and antibodies of the serum bound thereto can be stained by adding secondary anti-mouse IgG antibody conjugated to a selected fluorophore, and a viability dye eFlour for 30 min at 4°C to detect the bound antibodies. Finally, the cells can be fixated in 1% PFA for 15 min at room temperature (about 21 °C). Flow cytometry is then performed on a flow cytometer, and the data is analysed using FlowJo™ software and GraphPad Prism. Thereby, the proportion of antibodies directed against Ceacaml, which are bound to the respective amount of CT26 cells, can be detected to quantify the elicited antibody response in the mouse.

[0515] Example 22:

[0516] As another example murine Ceacaml or human Ceacam5 can be targeted via vaccination using the above described VLP expression approach of previous examples. The betaretroviral IAPE-D1 Gag can be encoded in a plasmid with Ceacaml or Ceacam5 (without its propeptide) as antigen, which antigen includes the transmembrane domain and cytoplasmic tail of IAPE-D1 Env (envelope protein). IAPE-D1 Gag gene can be linked to murine Ceacaml and human Ceacam5 respectively, via a Glycine / Serine / Glycine (GSG) linker followed by a self-cleaving porcine teschovirus- 1 2A peptide (P2A). The two constructs can be used for immunization as DNA as in example 13, as mRNA vectors as in example 19 or as adenoviral vectors as in example 20. Serum can be isolated as in example 13and can be used to analyse responses to murine Ceacaml as expressed on CT26 cells or human Ceacam5 as expressed on a human colorectal cancer cell line.

[0517] Example 23:

[0518] As a further example the Envelope glycoprotein of Simian Immunodeficiency Virus (SIV) can be targeted to raise robust responses against a difficult target antigen, i.e. a target antigen against which raising an immune response is rather challenging. The betaretroviral HERV-K Gag can be encoded in a plasmid along with a SIV Envelope protein gpl40, which includes the transmembrane domain and cytoplasmic tail of HERV-K Env. The HERV-K Gag gene is linked to murine SIV gpl40 respectively, via a Glycine / Serine / Glycine (GSG) linker followed by a self-cleaving porcine teschovirus-1 2A peptide (P2A). Upon expression these protein components will form a VLP (as described above). The construct can be used for immunization in the form of DNA as described in example 13, as mRNA vectors as described in example 19 or as adenoviral vectors as in example 20. Serum can be isolated as in example 13 and can be used to analyse responses against SIV Envelope in ELISA as in Example 14.

[0519] Example 24:

[0520] As a further example the F (Fusion) protein of RSV can be encoded in an mRNA vector together with IAPE-D1 Gag and IAPE-D1 Env TMD-CT analogously to a construct as described herein, and either used to vaccinate mice as described in example 19 or be administered together with an mRNA construct encoding IAPE-Dl-Gag-P2A-hMPV-F-proteinECD-IAPE-Dl-EnvTMD-cT as part of a combinatory therapy / prophylaxis against both RSV and hMPV infection. Immune responses against the F protein can be detected by ELISA as described in Example 14 or by determining neutralization using a commercial kit.

[0521] Example 25:

[0522] The chimeric VLP platform of the invention as well as nucleic acids encoding corresponding VLP constructs are useful in the prophylaxis of infectious diseases in patients. For instance the constructs encoding hMPV, RSV and / or Influenza (A) antigens may be used in a vaccination regimen that is applied before the start of epidemic seasons, which are common for these pathogenic viruses such as hMPV, RSV and Influenza (A). The constructs of the invention can be administered directly as VLPs but are preferably administered in the form of VLP encoding nucleic acid molecules. Such nucleicacid molecules can be administered in the form of a DNA vaccine, as adenoviral vector vaccine, in the form of an mRNA vaccine, or as another nucleotide vector. Commonly, the nucleic acid molecule of any form is formulated in a formulation that is suitable for vaccination. The vaccine can be formulated in a formulation comprising Tris lOmM; L-His 10 mM; NaCl 75 mM; Sucrose 5%; MgC12 ImM; EDTA 0,1 mM; Polysorbate 80 0,02% w / v; EtOH 0,5% v / v; pH 7,4 (A195 buffer). Alternatively, the vaccine can be formulated in a formulation comprising 10 mM Tris; 10 mM Histidine; 35 mM NaCl; 5% Sucrose; 1 mM MgCh; 0,01% Polysorbate 80; 0,1 mM EDTA; 0,5% EtOH; 60 mM Glutamate; 224 mM Alanine; pH 7,6 (D6 buffer). The vaccine would be administered as one or two vaccine shots with 3-4 weeks apart between the vaccine shots or potentially more time in between the vaccine shots.

[0523] Many further antigens besides the (non ERV) antigens described herein can be displayed using the VLP vaccination platform described above to induce an immune response against the respective antigen in a vaccinated subject. Particularly a chimeric variant of the VLP platform, which comprises a Betaretroviral Gag protein and an antigen that includes as one part the transmembrane and cytoplasmic domain of a Betaretroviral Env (envelope) protein from the same betaretrovirus as the Gag protein, can be universally applied to induce an effective immune response and even to express and display antigens that are either challenging to be effectively displayed or that are challenging to evoke an immune response against or both.

[0524] Table 1: Amino acid sequences of HERV-K Gag HERV-K Env

[0525] SEQ Description Sequence

[0526] ID NO

[0527] 1 HERV-K Gag MGQTKSKIKSKYASYLSFIKILLKRGGVKVSTKNLIKLFQIIEQFCPWFPEQGTLD LKDWKRI GKE LKQAGRKGNI I PLTVWNDWAI I KAALE PFQTEEDSVSVSDAPGS CI IDCNENTRKKSQKETEGLHCEYVAEPVMAQSTQNVDYNQLQEVIYPETLKLEGKGP ELVGPSESKPRGTSPLPAGQVPVTLQPQKQVKENKTQPPVAYQYWPPAELQYRPPP ESQYGYPGMPPAPQGRAPYPQPPTRRLNPTAPPSRQGSELHEIIDKSRKEGDTEAW QFPVTLE PMP PGE GAQE GE PPTVEAR YKS FS I KMLKDMKE GVKQY GPNS PYMRT LL DS I AHGHRLI PYDWE I LAKSSLS PSQFLQFKTWWI DGVQEQVRRNRAANPPVNI DA DQLLGIGQNWSTISQQALMQNEAIEQVRAICLRAWEKIQDPGSTCPSFNTVRQGSK EPYPDFVARLQDVAQKSIADEKARKVIVELMAYENANPECQSAIKPLKGKVPAGSD VISEYVKACDGIGGAMHKAMLMAQAITGVVLGGQVRTFGGKCYNCGQIGHLKKNCP VLNKQNITIQATTTGREPPDLCPRCKKGKHWASQCRSKFDKNGQPLSGNEQRGQPQ APQQTGAFPIQPFVPQGFQGQQPPLSQVFQGISQLPQYNNCPPPQAAVQQ

[0528] 2 GSG-linker GSG

[0529] 3 P2A peptide ATNFSLLKQAGDVEENPGP

[0530] 4 HERV-K Env MNPSEMQRKAPPRRRRHRNRAPLTHKMNKMVTSEEQMKLPSTKKAEPPTWAQLKKL

[0531]

[0532] [Underlined = TQLATKYLENTKVTQTPESMLLAALMIVSMWSLPMPAGAAAANYTYWAYVPFPPLimmune IRAVTWMDNPIEVYVNDSVWVPGPIDDRCPAKPEEEGMMINISIGYRYPPICLGRA suppressive PGCLMPAVQNWLVEVPTVSPISRFTYHMVSGMSLRPRVNYLQDFSYQRSLKFRPKG domain] KPCPKEIPKESKNTEVLVWEECVANSAVILQNNEFGTIIDWAPRGQFYHNCSGQTQ SCPSAQVSPAVDSDLTESLDKHKHKKLQSFYPWEWGEKGISTPRPKIVSPVSGPEH PELWRLTVASHHIRIWSGNQTLETRDRKPFYTVDLNSSLTVPLQSCVKPPYMLVVG NIVIKPDSQTITCENCRLLTCIDSTFNWQHRILLVRAREGVWIPVSMDRPWEASPS VHILTEVLKGVLNRSKRFIFTLIAVIMGLIAVTATAAVAGVALHSSVQSVNFVNDW QKNSTRLWNSQSSIDQKLANQINDLRQTVIWMGDRLMSLEHRFQLQCDWNTSDFCI TPQIYNESEHHWDMVRRHLQGREDNLTLDISKLKEQIFEASKAHLNLVPGTEAIAG VADGLANLNPVTWVKTIGSTTIINLILILVCLFCLLLVCRCTQQLRRDSDHRERAM

[0533]

[0534] MTMAVLSKRKGGNVGKSKRDQIVTVSV

[0535] Table 2: Amino acid sequences of HERV-W Gag HERV-W-Env

[0536] SEQ Description Sequence

[0537] ID NO

[0538] 5 HERV-W Gag MGNVPPEAKMPLERILENWDQCDTQTLRKKRFIFFCSTAWPQYPLQGRETWLPEGS INYNIILQLDLFCRKEGKWSEVPYVQTFFSLRDNSQLCKKCGLCPTGSPQSPPPYP SVPS PT PSSTNKDPPLTQTVQKE IDKGVNNE PKSANI PRLCPLQAVRGGE FGPARV PVPFSLSDLKQIKIDLGKFSDNPDGYIDVLQGLGQSFDLTWRDIMLLLNQTLTPNE RSAAVTAAREFGDLWYLSQANNRMTTEERTTPTGQQAVPSVDPHWDTESEHGDWCH KHLLTCVLEGLRKTRKKPMNYSMMSTITQGKEENLTAFLDRLREALRKHTSLSPDS IEGQLILKDKFITQSAADIRKNFKSLPLGSEQNLETLLNLATSVFYNRDQEEQAE

[0539] 2 GSG-linker GSG

[0540] 3 P2A peptide ATNFSLLKQAGDVEENPGP

[0541] 6 HERV-W Env MALPYHIFLFTVLLPSFTLTAPPPCRCMTSSSPYQEFLWRMQRPGNIDAPSYRSLS (Syncytin-1) KGTPTFTAHTHMPRNCYHSATLCMHANTHYWTGKMINPSCPGGLGVTVCWTYFTQT [italics = GMSDGGGVQDQAREKHVKEVISQLTRVHGTSSPYKGLDLSKLHETLRTHTRLVSLF T ransmembrane NTTLTGLHEVSAQNPTNCWICLPLNFRPYVSI PVPEQWNNFSTEINTTSVLVGPLV domain + SNLEITHTSNLTCVKFSNTTYTTNSQCIRWVTPPTQIVCLPSGIFFVCGTSAYRCL cytoplasmic NGSSESMCFLSFLVPPMTIYTEQDLYSYVISKPRNKRVPILPFVIGAGVLGALGTG tail]; IGGITTSTQFYYKLSQELNGDMERVADSLVTLQDQLNSLAAVVLQNRRALDLLTAE [Underlined = RGGTCLFLGEECCYYVNQSGIVTEKVKEIRDRIQRRAEELRNTGPWGLLSQ WMPWI immune LPFLGPLAAIILLLLFGPCIFNLLVNFVSSRIEAVKLQMEPKMQSKTKIYRRPLDR suppressive PASPRSDVNDIKGTPPEEISAAQPLLRPNSAGSS

[0542]

[0543] domain]

[0544] Table 3: Amino acid sequences of HERV-K Gag HERV-W Env

[0545] SEQ Description Sequence

[0546] ID NO

[0547] 1 HERV-K Gag MGQTKSKIKSKYASYLSFIKILLKRGGVKVSTKNLIKLFQIIEQFCPWFPEQGTLD LKDWKRI GKE LKQAGRKGNI I PLTVWNDWAI I KAALE PFQTEEDSVSVSDAPGS CI IDCNENTRKKSQKETEGLHCEYVAEPVMAQSTQNVDYNQLQEVIYPETLKLEGKGP ELVGPSESKPRGTSPLPAGQVPVTLQPQKQVKENKTQPPVAYQYWPPAELQYRPPP ESQYGYPGMPPAPQGRAPYPQPPTRRLNPTAPPSRQGSELHEIIDKSRKEGDTEAW QFPVTLE PMP PGE GAQE GE PPTVEAR YKS FS I KMLKDMKE GVKQY GPNS PYMRT LL DS I AHGHRLI PYDWE I LAKSSLS PSQFLQFKTWWI DGVQEQVRRNRAANPPVNI DA DQLLGIGQNWSTISQQALMQNEAIEQVRAICLRAWEKIQDPGSTCPSFNTVRQGSK EPYPDFVARLQDVAQKSIADEKARKVIVELMAYENANPECQSAIKPLKGKVPAGSD VISEYVKACDGIGGAMHKAMLMAQAITGVVLGGQVRTFGGKCYNCGQIGHLKKNCP VLNKQNITIQATTTGREPPDLCPRCKKGKHWASQCRSKFDKNGQPLSGNEQRGQPQ

[0548]

[0549] APQQTGAFPIQPFVPQGFQGQQPPLSQVFQGISQLPQYNNCPPPQAAVQQ2 GSG-linker GSG

[0550] 3 P2A peptide ATNFSLLKQAGDVEENPGP

[0551] 6 HERV-W Env MALPYHIFLFTVLLPSFTLTAPPPCRCMTSSSPYQEFLWRMQRPGNIDAPSYRSLS (Syncytin-1) KGTPTFTAHTHMPRNCYHSATLCMHANTHYWTGKMINPSCPGGLGVTVCWTYFTQT [italics = GMSDGGGVQDQAREKHVKEVISQLTRVHGTSSPYKGLDLSKLHETLRTHTRLVSLF T ransmembrane NTTLTGLHEVSAQNPTNCWICLPLNFRPYVSI PVPEQWNNFSTEINTTSVLVGPLV domain + SNLEITHTSNLTCVKFSNTTYTTNSQCIRWVTPPTQIVCLPSGIFFVCGTSAYRCL cytoplasmic NGSSESMCFLSFLVPPMTIYTEQDLYSYVISKPRNKRVPILPFVIGAGVLGALGTG tail]; IGGITTSTQFYYKLSQELNGDMERVADSLVTLQDQLNSLAAVVLQNRRALDLLTAE [Underlined = RGGTCLFLGEECCYYVNQSGIVTEKVKEIRDRIQRRAEELRNTGPWGLLSQ WMPWI immune LPFLGPLAAIILLLLFGPCIFNLLVNFVSSRIEAVKLQMEPKMQSKTKIYRRPLDR suppressive PASPRSDVNDIKGTPPEEISAAQPLLRPNSAGSS

[0552]

[0553] domain]

[0554] Table 4: Amino acid sequences of HERV-K Gag TMD-CT HERV-W Env

[0555] SEQ Description Sequence

[0556] ID NO

[0557] 1 HERV-K Gag MGQTKSKIKSKYASYLSFIKILLKRGGVKVSTKNLIKLFQIIEQFCPWFPEQGTLD LKDWKRI GKE LKQAGRKGNI I PLTVWNDWAI I KAALE PFQTEEDSVSVSDAPGS CI IDCNENTRKKSQKETEGLHCEYVAEPVMAQSTQNVDYNQLQEVIYPETLKLEGKGP ELVGPSESKPRGTSPLPAGQVPVTLQPQKQVKENKTQPPVAYQYWPPAELQYRPPP ESQYGYPGMPPAPQGRAPYPQPPTRRLNPTAPPSRQGSELHEIIDKSRKEGDTEAW QFPVTLE PMP PGE GAQE GE PPTVEAR YKS FS I KMLKDMKE GVKQY GPNS PYMRT LL DS I AHGHRLI PYDWE I LAKSSLS PSQFLQFKTWWI DGVQEQVRRNRAANPPVNI DA DQLLGIGQNWSTISQQALMQNEAIEQVRAICLRAWEKIQDPGSTCPSFNTVRQGSK EPYPDFVARLQDVAQKSIADEKARKVIVELMAYENANPECQSAIKPLKGKVPAGSD VISEYVKACDGIGGAMHKAMLMAQAITGVVLGGQVRTFGGKCYNCGQIGHLKKNCP VLNKQNITIQATTTGREPPDLCPRCKKGKHWASQCRSKFDKNGQPLSGNEQRGQPQ APQQTGAFPIQPFVPQGFQGQQPPLSQVFQGISQLPQYNNCPPPQAAVQQ

[0558] 2 GSG-linker GSG

[0559] 3 P2A peptide ATNFSLLKQAGDVEENPGP

[0560] 7 HERV-W Env MALPYHIFLFTVLLPSFTLTAPPPCRCMTSSSPYQEFLWRMQRPGNIDAPSYRSLS surface subunit KGTPTFTAHTHMPRNCYHSATLCMHANTHYWTGKMINPSCPGGLGVTVCWTYFTQT and ectodomain GMSDGGGVQDQAREKHVKEVISQLTRVHGTSSPYKGLDLSKLHETLRTHTRLVSLF of NTTLTGLHEVSAQNPTNCWICLPLNFRPYVSI PVPEQWNNFSTEINTTSVLVGPLV transmembrane SNLEITHTSNLTCVKFSNTTYTTNSQCIRWVTPPTQIVCLPSGIFFVCGTSAYRCL subunit NGSSESMCFLSFLVPPMTIYTEQDLYSYVISKPRNKRVPILPFVIGAGVLGALGTG [Underlined = IGGITTSTQFYYKLSQELNGDMERVADSLVTLQDQLNSLAAVVLQNRRALDLLTAE immune RGGTCLFLGEECCYYVNQSGIVTEKVKEIRDRIQRRAEELRNTGPWGLLSQ suppressive

[0561] domain]

[0562] 8 HERV-K Env TTGSTTTTNLThThVChFCUjhVCRCTQQLRRDSDHRERAMMTMAVLSKRKGGNVG transmembrane KSKRDQIVTVSV

[0563] domain (TMD,

[0564] bold) and

[0565] cytoplasmic tail

[0566]

[0567] (CT, italics)Table 5: Exemplary cytoplasmic tail (CT) sequences or sequence sections 8 HERV-K Env TTGSTTTTNLTLTHVC'LFCIjL'L'VCRCTQQLRRDSDHRERAMMTMAVLSKRKGGNVG transmembrane KSKRDQIVTVSV

[0568] domain (TMD,

[0569] bold) and

[0570] cytoplasmic tail

[0571] (CT, italics)

[0572] 9 HERV-K Env IVTVSV

[0573] cytoplasmic tail

[0574] (CT), terminal 6

[0575] amino acids

[0576] 10 HERV-K Env QIVTVSV

[0577] cytoplasmic tail

[0578] (CT), terminal 7

[0579] amino acids

[0580] 11 HERV-K Env KRKGGNVGKSKRDQIVTVSV

[0581] cytoplasmic tail

[0582] (CT), terminal

[0583] 20 amino acids

[0584] 12 HERV-K Env AVLSKRKGGNVGKSKRDQIVTVSV

[0585] cytoplasmic tail

[0586] (CT), terminal

[0587] 24 amino acids

[0588] 27 HERV-K Env RAMMTMAVLSKRKGGNVGKSKRDQIVTVSV

[0589] cytoplasmic tail

[0590] (CT), terminal

[0591] 30 amino acids

[0592] 28 HERV-K Env RCTQQLRRDSDHRERAMMTMAVLSKRKGGNVGKSKRDQIVTVSV cytoplasmic tail

[0593] (CT)

[0594] 29 HERV-K Env TIGSTTIINLILILVCLFCLLLVCJJCTQQLKRDSDHREKAMMTMAVLSKRKGGWVG transmembrane KSKRDQ

[0595] domain (TMD,

[0596] bold) and

[0597] cytoplasmic tail

[0598] (CT, italics): 6

[0599] C -terminal

[0600] amino acids of

[0601] deleted

[0602] 30 HERV-K Env TIGSTTIINLILILVCLFCLLLVCJJCTQQLKRDSDHREKAMMTMAVLS transmembrane

[0603] domain (TMD,

[0604] bold) and

[0605] cytoplasmic tail

[0606] (CT, italics): 20

[0607] C -terminal

[0608] amino acids of

[0609] deleted

[0610] 31 HERV-K Env TIGSTTIINLILILVCLFCLLLVCRCTQQLRRCSCHRE

[0611] transmembrane

[0612] domain (TMD,

[0613] bold) and

[0614] cytoplasmic tail

[0615] (CT, italics): 30

[0616]

[0617] C -terminalamino acids of

[0618]

[0619] deleted

[0620] Table 6: Amino acid sequences of lAPE(-Dl) Gag lAPE(-Dl) Env (lAPE(-Dl) construct) SEQ Description Sequence

[0621] ID NO

[0622] 13 IAPE Gag MGNSHSVTTALHSVLKQREIKVSTRTLETFIKEIERISPWYACSGSLTLSSWEKLR EDLAKEQQNGKLKAGTMPLWKLVRSCLEDERCRPAIITGQAILEEAQDSMAETEWC ERLGAPKKENVHKVKSPSRDLESEEVKNLRISPQGEKKEKEKVQKRKSLYPVKELE ALKLDSLKADELSSSEEEESHYEAAHYKKERYHPEERRVKKSEKKLKVTGDTEQAT SGPSSLNTPPPYVEKFYSDSFLSKEEKKKLYQAFPVFEAADGGRVHAPVEYTQIKE LAE S VRNY GV SAN FTISQIER FAT LAMT P GDWQTT VKAAL PNMGQ YME WKALWHDA S QT QAKVNAT AE GNQRNWT FE L LT GQ GQ Y ANNQTN YDWGAYNQ I S TAAI KAWKALS KKGESGRYLTKIIQNPQESFSDFVARMTEAAGRIFGDSEQAMPLVEQLIYEQATQE CRAAITPRKSKGLQDWLRVCRELGGPLSNAGLAAAILQGQRHSDTNSNNRVCYNCG KPGHMRRDCQALVKGKVLGLCTRCGKGYHRASECRSIKDVRGRFIQSGPQAERSED NSKNEFLGPRSQGPKTYGI PAHNRRTLQSTEL

[0623] 2 GSG-linker GSG

[0624] 3 P2A peptide ATNFSLLKQAGDVEENPGP

[0625] 14 IAPE Env MTPRWIPWKLPLILMLPLWIWVPSIFGEYRWAILSAFPKPMPVRHNTAVFPKFFTT [Underlined = NKTLGLPYLPFDPIWAPLGEKRSLRERGSLCFQIYELGGCIRLTSQALGMFFKYRG immune GWKITQDTSNRDITLTNRTFWHEATWVNGTFLPPNFSDKERPNQPKMAPHCSLED suppressive EGLILPWSDCQSSVTRWADQSKTFSFSPNMIDDPEQKYVMKKGLFIQDFRMHPFHK domain] WVLCGINGSCTDLNPLVFLQGGAAGKAIFNGISKFAQFHQVLLPDRTIYQNSTTEI T GFNKT L I KQTNY L PT PVCVYT P FL F I LS NGS FE S CTNE T CWMS Q CWS LKWAS RAM LAKI PRWVPVPVE T PS T I T LHRQKRD FGI TAAWVAMAASAAAATAAGIAMATS VQ SSTTVEQLSSSVAEAIDQHSVLSAQLKGGLMIVNQRIDLVEERLEILLQLAQLGCD KKSVALCITSVQYENWTHAANLSKELSLFLTGNWSEGFDEKLEALRTAVMTINSTR VDPSLIDGIKGLSSWMSSAFSHFKEWVGVGLFGATLCCGLVFLLWLVCKLRSQQKR

[0626]

[0627] DKVVI AQALAAI E QGT S PE IWLS I LKN

[0628] Table 7: Amino acid sequences of HA (hemagglutinin) tagged endogenous retrovirus (ERV) envelope proteins

[0629] SEQ Description Sequence

[0630] ID NO

[0631] 15 (GS)2 linker + GSGS YPYDVPDYA

[0632] HA

[0633] (hemagglutinin)

[0634] tag

[0635] attachable as tag

[0636] to each of the

[0637] below Env

[0638] amino acid

[0639]

[0640] sequencesERV3.1 MLGMNMLLITLFLLLPLSMLKGEPWEGCLHCTHTTWSGNIMTKTLLYHTYYECAGT Envl92C CLGTCTHNQTTYSVCDPGRGQPYVCYDPKSSPGIWFEIHVGSKEGDLLNQTKVFPS GKDWSLYFDVCQIVSMGSLFPVIFSSMEYYSSCHKNRYAHPACSTDSPVTTCWDC TTWSTNQQSLGPIMLTKIPLEPDCKTSTCNSVNLTILEPDQPIWTTGLKAPLGARV SGEEIGPGAYVYLYIIKKTRTRSTQQFRVFESFYEHVNQKLPEPPPLASNLFAQLA ENIASSLHVASCYVCGGMNMGDQWPWEARELMPQDNFTLTASSLEPAPSSQSIWFL KTSIIGKFCIARWGKAFTDPVGELTCLGQQYYNETLGKTLWRGKSNNSESPHPSPF SRFPSLNHSWYQLEAPNTWQAPSGLYWICGPQAYRQLPAKWSGACVLGTIRPSFFL MPLKQGEALGYPIYDETKRKSKRGITIGDWKDSEWPPERIIQYYGPATWAEDGMWG YRTPVYMLNRI IRLQAVLE I ITNETAGALNLLAQQATKMRNVI YQNRLALDYLLAQ EEGVCGKFSLTNCCLELDDEGKVIKEITAKIQKLAHIPVQTWKGASPDSLFRGWFS SLGGFKTLVQIVLAILGVCLILPCLLPLIVKNIQTAIEALVDRQTTTRLMALTKY ERV3.1 MLGMNMLLITLFLLLPLSMLKGEPWEGCLHCTHTTWSGNIMTKTLLYHTYYECAGT Envl92Y CLGTCTHNQTTYSVCDPGRGQPYVCYDPKSSPGIWFEIHVGSKEGDLLNQTKVFPS GKDWSLYFDVCQIVSMGSLFPVIFSSMEYYSSCHKNRYAHPACSTDSPVTTCWDC TTWSTNQQSLGPIMLTKIPLEPDYKTSTCNSVNLTILEPDQPIWTTGLKAPLGARV SGEEIGPGAYVYLYIIKKTRTRSTQQFRVFESFYEHVNQKLPEPPPLASNLFAQLA ENIASSLHVASCYVCGGMNMGDQWPWEARELMPQDNFTLTASSLEPAPSSQSIWFL KTSIIGKFCIARWGKAFTDPVGELTCLGQQYYNETLGKTLWRGKSNNSESPHPSPF SRFPSLNHSWYQLEAPNTWQAPSGLYWICGPQAYRQLPAKWSGACVLGTIRPSFFL MPLKQGEALGYPIYDETKRKSKRGITIGDWKDSEWPPERIIQYYGPATWAEDGMWG YRTPVYMLNRI IRLQAVLE I ITNETAGALNLLAQQATKMRNVI YQNRLALDYLLAQ EEGVCGKFSLTNCCLELDDEGKVIKEITAKIQKLAHIPVQTWKGASPDSLFRGWFS SLGGFKTLVQIVLAILGVCLILPCLLPLIVKNIQTAIEALVDRQTTTRLMALTKY HERV-FRD MGLLLLVLILTPSLAAYRHPDFPLLEKAQQLLQSTGSPYSTNCWLCTSSSTETPGT Env AYPASPREWTSIEAELHISYRWDPNLKGLMRPANSLLSTVKQDFPDIRQKPPIFGP IFTNINLMGIAPICVMAKRKNGTNVGTLPSTVCNVTFTVDSNQQTYQTYTHNQFRH QPRFPKPPNITFPQGTLLDKSSRFCQGRPSSCSTRNFWFRPADYNQCLQISNLSST AEWVLLDQTRNSLFWENKTKGANQSQTPCVQVLAGMTIATSYLGISAVSEFFGTSL TPLFHFHISTCLKTQGAFYICGQSIHQCLPSNWTGTCTIGYVTPDIFIAPGNLSLP IPIYGNSPLPRVRRAIHFIPLLAGLGILAGTGTGIAGITKASLTYSQLSKEIANNI DTMAKALTTMQEQIDSLAAWLQNRRGLDMLTAAQGGICLALDEKCCFWVNQSGKV QDNIRQLLNQASSLRERATQGWLNWEGTWKWFSWVLPLTGPLVSLLLLLLFGPCLL NLITQFVSSRLQAIKLQTNLSAGRHPRNIQESPF HERV-H Env MIFAGKAPSNTSTLMKFYSLLLYSLLFSFPFLCHPLPLPSYLHHTINLTHSLLAAS NPSLVNNCWLCISLSSSAYTAVPAVQTDWATSPISLHLRTSFNSPHLYPPEELIYF LDRSSKTSPDISHQQAAALLRTYLKNLSPYINSTPPIFGPLTTQTTIPVAAPLCIS WQRPTGIPLGNLSPSRCSFTLHLRSPTTNINETIGAFQLHITDKPSINTDKLKNIS SNYCLGRHLPCISLHPWLSSPCSSDSPPRPSSCLLIPSPENNSERLLVDTRRFLIH HENRTFPSTQLPHQSPLQPLTAAALAGSLGVWVQDTPFSTPSHLFTLHLQFCLAQG LFFLCGSSTYMCLPANWTGTCTLVFLTPKIQFANGTEELPVPLMTPTQQKRVIPLI PLMVGLGLSASTVALGTGIAGISTSVMTFRSLSNDFSASITDISQTLSVLQAQVDS LAAWLQNRRGLDLLTAEKGGLCIFLNEECCFYLNQSGLVYDNIKKLKDRAQKLAN QASNYAEPPWALSNWMSWVLPIVSPLIPIFLLLLFGPCIFRLVSQFIQNRIQAITN HSIRQMFLLTSPQYHPLPQDLPSA HERV-W Env MALPYHIFLFTVLLPSFTLTAPPPCRCMTSSSPYQEFLWRMQRPGNIDAPSYRSLS KGTPTFTAHTHMPRNCYHSATLCMHANTHYWTGKMINPSCPGGLGVTVCWTYFTQT GMSDGGGVQDQAREKHVKEVISQLTRVHGTSSP...

Claims

CLAIMS1. At least one nucleic acid molecule encoding(1) a first polypeptide A, wherein the polypeptide A is a group-specific antigen (Gag) protein of a beta-retrovirus; and(2) a second polypeptide, wherein the second polypeptide comprises the polypeptides B, C and D in the order B-C-D or D-C-B; whereinpolypeptide B is an antigenic polypeptide, wherein the antigenic polypeptide is not an endogenous retrovirus (ERV) envelope protein,polypeptide C is a transmembrane domain (TMD), andpolypeptide D is a cytoplasmic tail (CT) of a viral envelope protein or fragment thereof, wherein said first polypeptide and second polypeptide is encoded by the same nucleic acid molecule or by two different nucleic acid molecules.

2. At least one nucleic acid molecule encoding(1) a first polypeptide A, wherein the polypeptide A is a group-specific antigen (Gag) protein of a beta-retrovirus; and(2) a second polypeptide, wherein the second polypeptide comprises the polypeptides B, C and D in the order B-C-D or D-C-B; whereinpolypeptide B is an antigenic polypeptide of a virus causing an infectious disease, polypeptide C is a transmembrane domain (TMD), andpolypeptide D is a cytoplasmic tail (CT) of a viral envelope protein or fragment thereof, wherein said first polypeptide and second polypeptide is encoded by the same nucleic acid molecule or by two different nucleic acid molecules.

3. At least one nucleic acid molecule encoding(1) a first polypeptide A, wherein the polypeptide A is a group-specific antigen (Gag) protein of a beta-retrovirus; and(2) a second polypeptide, wherein the second polypeptide comprises the polypeptides B, C and D in the order B-C-D or D-C-B; whereinpolypeptide B is an antigenic polypeptide of a respiratory virus, preferably a respiratory virus selected from the group consisting of influenza virus, respiratory syncytial virus,parainfluenza virus, metapneumovirus, rhinovirus, coronavirus, adenovirus and bocavirus,polypeptide C is a transmembrane domain (TMD), andpolypeptide D is a cytoplasmic tail (CT) of a viral envelope protein or fragment thereof, wherein said first polypeptide and second polypeptide is encoded by the same nucleic acid molecule or by two different nucleic acid molecules.

4. At least one nucleic acid molecule encoding(1) a first polypeptide A, wherein the polypeptide A is a group-specific antigen (Gag) protein of a beta-retrovirus; and(2) a second polypeptide, wherein the second polypeptide comprises the polypeptides B, C and D in the order B-C-D or D-C-B; whereinpolypeptide B is an antigenic polypeptide of a negative sense RNA virus, preferably an antigen of Orthornavirae, more preferably an antigen of Negarnaviricota, even more preferably an antigen of Orthomyxoviridae, Paramyxoviridae or Pneumoviridae, and preferably wherein the Orthomyxoviridae are selected from the group consisting of Alphainfluenzavirus, Betainfluenzavirus, Gammainfluenzavirus and Deltainfluenzavirus, and preferably wherein the Paramyxoviridae are selected from the group consisting of Parainfluenza Virus 1, Parainfluenza Virus 2, Parainfluenza Virus 3, Parainfluenza Virus 4, and preferably wherein the Pneumoviridae are selected from the group consisting of human metapneumovirus (HMPV), human respiratory syncytial virus A2 (HRSV-A2) and human respiratory syncytial virus B1 (HRSV-B1), polypeptide C is a transmembrane domain (TMD), andpolypeptide D is a cytoplasmic tail (CT) of a viral envelope protein or fragment thereof, wherein said first polypeptide and second polypeptide is encoded by the same nucleic acid molecule or by two different nucleic acid molecules.

5. A composition comprising at least one nucleic acid molecule or a pharmaceutically acceptable salt thereof, encoding(1) a first polypeptide A, wherein the polypeptide A is a group-specific antigen (Gag) protein of a beta-retrovirus; and(2) a second polypeptide, wherein the second polypeptide comprises the polypeptides B, C and D in the order B-C-D or D-C-B; whereinpolypeptide B is an antigenic polypeptide,polypeptide C is a transmembrane domain (TMD), andpolypeptide D is a cytoplasmic tail (CT) of a viral envelope protein or fragment thereof, wherein said first polypeptide and second polypeptide is encoded by the same nucleic acid molecule or by two different nucleic acid molecules,wherein the composition comprises a buffering agent, buffering the composition at a pH in the range of 7 to 8, preferably in a range of pH 7,3 to 7,7.

6. At least one nucleic acid molecule encoding at least three polypeptides(1) a first polypeptide A, wherein the polypeptide A is a group-specific antigen (Gag) protein of a beta-retrovirus;(2) a second polypeptide, wherein the second polypeptide comprises the polypeptides Bl, Cl and DI in the order Bl -Cl -DI or DI -Cl -Bl; whereinpolypeptide Bl is an antigenic polypeptide,polypeptide Cl is a transmembrane domain (TMD), andpolypeptide DI is a cytoplasmic tail (CT) of a viral envelope protein or fragment thereof; and(3) a third polypeptide, wherein the third polypeptide comprises the polypeptides B2, C2 and D2 in the order B2-C2-D2 or D2-C2-B2; whereinpolypeptide B2 is an antigenic polypeptide,polypeptide C2 is a transmembrane domain (TMD), andpolypeptide D2 is a cytoplasmic tail (CT) of a viral envelope protein or fragment thereof,wherein said first polypeptide, said second polypeptide and said third polypeptide are encoded by the same nucleic acid molecule,wherein the polypeptides Bl and B2 are different antigenic polypeptides, preferably wherein the different antigenic polypeptides are from different pathogens.

7. VLP comprising the polypeptides A, B, C and D as defined in any one of claims 1 to 6.

8. The at least one nucleic acid according to any one of claims 1 to 4 and 6, the composition of claim 5, or the VLP according to claim 2, wherein polypeptide D is a cytoplasmic tail (CT) of a viral envelope protein or fragment thereof capable of binding to the first polypeptide A so that when the first and second polypeptides are expressed in the same cell, virus like particles (VLPs) are formed that comprise said first and second polypeptide.

9. The at least one nucleic acid, the composition or the VLP according to any of the preceding claims, wherein(i)(a) polypeptides C, D and A are from the same beta-retrovirus and preferably of HERV-K or IAPE; and(b) polypeptide B is an antigenic part of a protein of a virus, preferably wherein the protein is Influenza A Hemagglutinin or human metapneumovirus glycoprotein F; or wherein(ii)(c) polypeptides B, C, D are from the same virus and preferably of Influenza A; and (d) polypeptide A is of a beta-retrovirus, preferably of HERV-K.

10. The at least one nucleic acid molecule, the composition or the VLP according to claim 9, wherein polypeptide D (CT) is of a cytoplasmic tail of an envelope (Env) protein of said betaretrovirus or a fragment thereof; and / orwherein the c-terminus of said first polypeptide comprises a GSG linker andwherein said nucleic acid molecule encodes downstream of said GSG linker a p2A peptide capable of inducing ribosomal skipping during translation, wherein said GSG-linker and p2A peptide preferably have the amino sequences of SEQ ID NO: 55 and SEQ ID NO: 56, respectively.

11. The at least one nucleic acid, the composition or the VLP according to any of the preceding claims, wherein the polypeptide D comprises at least 6 continuous amino acid residues of the C-terminus of a beta-retroviral envelope protein and wherein the C-terminal amino acid of said continuous amino acid residues is the C-terminal amino acid of said envelope protein;and preferably wherein polypeptide D comprises the amino acid residues IVTVSV (SEQ ID NO: 9), or comprises the amino acid residues the amino acid residues QIVTVSV (SEQ ID NO: 10), or comprises the ammo acid residues KRKGGNVGKSKRDQIVTVSV (SEQ ID NO: 11), or comprises the ammo acid residues AVLSKRKGGNVGKSKRDQIVTVSV (SEQ ID NO: 12), or comprises the amino acid residues RAMMTMAVLSI< RI< GGNVGI< SI< RDQIVTVSV (SEQ ID NO: 27), or wherein polypeptide D comprises a polypeptide according to any of SEQ ID NOs: 9, 10, 11, 12 or 27 in which the polypeptide comprises at least one conservative amino acid substitution compared with the respective reference sequence SEQ ID NO: 9, 10, 11, 12 or 27.

12. The at least one nucleic acid, the composition or the VLP according to any one of claims 1 to 11, wherein(a) in polypeptide D at least the 6 continuous terminal amino acid residues of the C-terminus of polypeptide D are deleted, preferably wherein at least the 20 continuous terminal amino acid residues of the C-terminus of polypeptide D are deleted, more preferably wherein at least the 30 continuous terminal amino acid residues of the C-terminus of polypeptide D are deleted, or wherein polypeptide D is deleted; or(b) wherein polypeptides C and D together comprise the polypeptide sequence TIGSTTIINLILILVCLFCLLLVCRCTQQLRRDSDHRERAMMTMAVLSKRKGGNVGKS KRDQ (SEQ ID NO: 29), or comprises the polypeptide sequence TIGSTTIINLILILVCLFCLLLVCRCTQQLRRDSDHRERAMMTMAVLSKRKGGNVGKS KRDQ (SEQ ID NO: 29) in which the polypeptide comprises at least one conservative amino acid substitution compared with the reference sequence SEQ ID NO: 29; or(c) wherein polypeptides C and D together comprise the polypeptide sequence TIGSTTIINLILILVCLFCLLLVCRCTQQLRRDSDHRERAMMTMAVLS (SEQ ID NO: 30), or comprises the polypeptide sequence TIGSTTIINLILILVCLFCLLLVCRCTQQLRRDSDHRERAMMTMAVLS (SEQ ID NO: 30) in which the polypeptide comprises at least one conservative amino acid substitution compared with the reference sequence SEQ ID NO: 30; or(d) wherein polypeptides C and D together comprise the polypeptide sequence HGSTTIINLILILVCLFCLLLVCRCTQQLRRDSDHRE (SEQ ID NO: 31), or comprises the polypeptide sequence TIGSTTIINLILILVCLFCLLLVCRCTQQLRRDSDHRE (SEQ IDNO: 31) in which the polypeptide comprises at least one conservative amino acid substitution compared with the reference sequence SEQ ID NO: 31; or(e) polypeptide A is HERV-K Gag and polypeptide C and D are as defined in (a), (b), (c) or (d), or(f) polypeptide A is HERV-K Gag, polypeptide B is an antigenic part of a protein of a virus, preferably wherein the protein is Influenza A Hemagglutinin or human metapneumovirus glycoprotein F, and polypeptide C and D are as defined in (a), (b), (c) or (d).

13. The at least one nucleic acid, the composition or the VLP according to any of the preceding claims, wherein at the C-terminus of polypeptide D, 6 continuous amino acid residues of the C-terminus of a beta-retroviral envelope protein are attached, and preferably wherein the 6 continuous amino acid residues of the C-terminus of a beta-retroviral envelope protein comprise the amino acid sequence IVTVSV (SEQ ID NO: 9) or an amino acid sequence according to SEQ ID NO: 9 in which the amino acid sequence comprises at least one conservative amino acid substitution compared with the respective reference sequence SEQ ID NO: 9.

14. The at least one nucleic acid, the composition or the VLP according to any of the preceding claims, wherein polypeptide C comprises at least on alpha helical polypeptide structure, more preferably wherein polypeptide C comprises from about 15 to 55 amino acid residues, even more preferably wherein polypeptide C is a transmembrane domain (TMD) of a bitopic type I integral transmembrane protein, even more preferably wherein polypeptide C is a TMD of a virus, preferably of a beta-retrovirus, even more preferably wherein polypeptide C is of the same beta-retrovirus as the first polypeptide A (Gag), most preferably wherein polypeptide C is of an envelope (Env) protein of said same beta-retrovirus; orwherein polypeptide C and D together comprise the polypeptide TIGSTTIINLILILVCLFCLLLVCRCTQQLRRDSDHRERAMMTMAVLSKRKGGNVGKS KRDQIVTVSV (SEQ ID NO: 8) or comprise the polypeptide TIGSTTIINLILILVCLFCLLLVCRCTQQLRRDSDHRERAMMTMAVLSKRKGGNVGKS KRDQIVTVSV (SEQ ID NO: 8) in which the polypeptide comprises at least one conservative amino acid substitution compared with the reference sequence SEQ ID NO: 8.

15. The at least one nucleic acid molecule or the VLP according to any of the preceding claims, wherein polypeptide B is an antigenic polypeptide that is a type I membrane protein, preferably a type I membrane protein associated with a diseases selected from cancer and senescence, more preferable wherein polypeptide B is an antigenic polypeptide selected from the group consisting of CEACAM1, CEACAM1.2, CEACAM3, CEACAM5, CEACAM6, EGFR, EphA2, F0LR1, HER2, Mucinl iso2, Mucinl iso3, Mucinl6, PAP, PSCA, PSMA (isol), PSMA (iso3), uPAR, SAGP, CD153, a peptide of amyloid-beta (A0), a peptide of Microtubule-associated protein tau (Tau), DPP4, and alpha-synuclein.

16. The at least one nucleic acid molecule or the VLP according to any of claims 1 to 14, wherein polypeptide B is an antigenic polypeptide of a virus or an antigenic part thereof.

17. The at least one nucleic acid, the composition or the VLP according to any of the preceding claims, wherein the first polypeptide A is a Gag protein of an endogenous beta-retrovirus (beta- ERV) or of a foreign beta-retrovirus,preferably wherein the beta-ERV is selected from the group consisting of human beta-ERV and non-human beta-ERV, more preferably wherein the human beta-ERV is HERV-K and wherein the non-human beta-ERV is selected from IAPE (Intracisternal A-type Particles elements with an Envelope) murine endogenous retrovirus and simian retrovirus 2 (SRV2), even more preferably wherein the HERV-K is selected from the group consisting of HERV- K108 (=ERVK-6), ERVK-19, HERV-K115 (=ERVK-8), ERVK-9, HERV-K113, ERVK-21, ERVK-25, HERV-K102 (=ERVK-7), HERV-K101 (=ERVK-24), andHERV-Kl 10 (=ERVK- 18).

18. The at least one nucleic acid, the composition or the VLP according to any of the preceding claims, wherein the at least one nucleic acid molecule encoding polypeptides A, B, C and D comprises the following amino acid sequences:(a) Polypeptide A comprises a sequence comprising at least 90%, more preferably at least 95%, and most preferably 100%, sequence identity with SEQ ID NO: 5 or 13, Polypeptide B comprises a sequence comprising at least 90%, more preferably at least 95%, and most preferably 100%, sequence identity with SEQ ID NO:s 64, 67, 71, 73 or 75, and preferably with SEQ ID NO: 64 or 67, andPolypeptides C and D together comprise a sequence comprising at least 90%, more preferably at least 95%, and most preferably 100%, sequence identity with SEQ ID NO: 8 or 47; or(b) Polypeptide A comprises a sequence comprising at least 90%, more preferably at least 95%, and most preferably 100%, sequence identity with SEQ ID NO: 5 or 13, and Polypeptide B comprises a sequence comprising at least 90%, more preferably at least 95%, and most preferably 100%, sequence identity with SEQ ID NO:s 64, 67, 71, 73 or 75, and preferably with SEQ ID NO: 64 or 67, andPolypeptides C and D together comprise a sequence comprising at least 90%, more preferably at least 95%, and most preferably 100%, sequence identity with SEQ ID NO: 77, 78, 80, 82 or 84,and preferably wherein polypeptide B comprises a sequence comprising at least 95% with SEQ ID NO: 64 and polypeptides C and D together comprise a sequence comprising at least 95% sequence identity with SEQ ID NO: 77.

19. The at least one nucleic acid molecule according to any one of claims 1 to 4, 6 or 8 to 18, wherein the at least one nucleic acid is an RNA, preferably an RNA selected from the group consisting of mRNA, circular RNA and self-amplifying RNA.

20. The at least one nucleic acid molecule according to any one of claims 1 to 4, 6 or 8 to 19, wherein the first polypeptide A in (1) and the second polypeptide in (2) are encoded on the same nucleic acid molecule, wherein the first polypeptide A in (1) is encoded by a first open reading frame (ORF) and wherein the second polypeptide c in (2) is encoded by a separate second ORF.

21. The at least one nucleic acid molecule according to claim 20, wherein the sequence of the first ORF encoding the first polypeptide in (1) and the sequence of the separate second ORF encoding the second polypeptide in (2) are connected by a sequence enabling separate translation of the first and second polypeptide,preferably wherein said sequence enabling separate translation encodes a p2A self-cleaving peptide or an internal ribosomal entry site (IRES).

22. The at least one nucleic acid molecule according to any one of claims 1 to 4, 6 or 8 to 21, wherein the first polypeptide A in (1) is encoded on a first nucleic acid molecule and wherein the second polypeptide comprising polypeptides B, C and D in (2) is encoded on a separate second nucleic acid molecule.

23. The at least one nucleic acid molecule according to any one of claims 1 to 4, 6 or 8 to 22, wherein the at least one nucleic acid molecule comprises at least 60 adenosine nucleotides at the 3’-UTR.

24. The at least one nucleic acid molecule according to any one of claims 1 to 4, 6 or 8 to 21, wherein the at least one nucleic acid molecule is codon optimized for expression in a human.

25. The at least one nucleic acid molecule according to any one of claims 1 to 4, 6 or 8 to 24, wherein the at least one nucleic acid molecule comprises at least 300 nucleotides.

26. The VLP according to any one of claims 7 to 18, wherein the polypeptide B is displayed on the surface of the VLP.

27. The VLP according to any one of claims 7 to 16 or 26, wherein the VLP when administered into a subject or expressed from the at least one nucleic acid molecule in a subject as a vaccine generates an immune response against polypeptide B.

28. A viral vector comprising the at least one nucleic acid molecule according to any one of claims 1 to 4, 6 or 8 to 27, preferably wherein the vector is an adenoviral vector, more preferably a human adenoviral vector, even more preferably human adenoviral vector is selected from subtype C and subtype D human adenoviral vectors, even more preferably wherein the subtype C human adenoviral vector is Ad5F35 and wherein the subtype D human adenoviral vector is Adi 9a, most preferably Adl9a / 64.

29. A method of producing the VLP according to any one of claims 7 to 16, 24 or 27 comprising the step of transfecting a nucleic acid molecule according to any one of the preceding claims into a cell and preferably a cell of a A549 cell line.

30. A pharmaceutical composition comprising the at least one nucleic acid molecule according to any one of claims 1 to 4, 6 or 8 to 25, the VLP according to any one of claims 7 to 18, 26 or 27, or the viral vector according to claim 26, wherein the pharmaceutical composition comprises a pharmaceutically acceptable excipient.

31. The pharmaceutical composition according to claim 30 comprising an adjuvant.

32. The at least one nucleic acid molecule according to any one of claims 1 to 4, 6 or 8 to 25, the VLP according to any one of claims 7 to 18, 26 or 27, or the viral vector according to claim 26, or the pharmaceutical composition according to any one of claims 28 or 29 for use as a medicament.

33. The at least one nucleic acid molecule according to any one of claims 1 to 4, 6 or 8 to 23, the VLP according to any one of claims 7 to 18, 26 or 27, or the viral vector according to claim 28, or the pharmaceutical composition according to any one of claims 30 or 31 for the manufacture of a medicament.

34. The at least one nucleic acid molecule according to any one of claims 1 to 4, 6 or 8 to 23, the VLP according to any one of claims 7 to 18, 26 or 27, or the viral vector according to claim 28, or the pharmaceutical composition according to any one of claims 30 or 31 for use in the prophylaxis and / or treatment of a disease, preferably for immunizing a subject against a disease.

35. The pharmaceutical composition according to any one of claims 30 or 31 for use in the manufacture of a medicament for the prophylaxis and / or therapeutic treatment of a disease, preferably for immunizing a subject against a disease.

36. A method of treatment or prophylaxis of a disease comprising administering the at least one nucleic acid molecule according to any one of claims 1 to 4, 6 or 8 to 25, the VLP according to any one of claims 7 to 18, 26 or 27, or the viral vector according to claim 28, or the pharmaceutical composition according to any one of claims 30 or 31 to a subject.

37. The nucleic acid molecule, the VLP, the viral vector or the pharmaceutical composition for use according to claims 34 or 35, or the method according to claim 36, wherein the disease is an infectious disease,preferably wherein the infectious disease is a respiratory disease, more preferably a disease caused by a viral pathogen selected from the group consisting of influenza virus, coronavirus, respiratory syncytial virus (RSV), human metapneumovirus, adenovirus, rhinovirus, enterovirus, parainfluenza virus and parvovirus.

38. The nucleic acid molecule, the VLP, the viral vector or the pharmaceutical composition for use according to claims 34 or 35, or the method according to claim 36, wherein the disease is a disease related to the process of senescence.

39. The pharmaceutical composition of claims 34, 35 or 37-38 or the method according to claims 36- 38, wherein(i) the pharmaceutical composition is administered in a first dose; and(ii) the pharmaceutical composition is administered in a second dose, wherein the second dose is administered about 4 weeks after administration of the first dose.

40. The pharmaceutical composition of claims 34, 35 or 37-38 or the method according to claims 36-38, wherein(i) the pharmaceutical composition is administered in a first dose; and(ii) the pharmaceutical composition is administered in a second dose;whereinthe second dose is administered about 25 to 35 days after administration of the first dose, and preferably about 28 days after administration of the first dose.

41. The pharmaceutical composition or method of claims 39 or 40, wherein the amount of viral particles administered to the subject is in the range of 106to 109infectious units.

42. The pharmaceutical composition or method according to any one of claims 39 to 41, wherein the pharmaceutical composition is administered via an administration route selected from thegroup consisting of intramuscular (i.m.), subcutaneous (s.c.), intravenous, and administration by inhalation, preferably via an administration route selected from intramuscular and subcutaneous administration.

43. The pharmaceutical composition or method according to claim 42, wherein the pharmaceutical composition elicits local immunity.

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