Vaccine for use in prophylaxis and / or treatment of disease

Abstract

To provide virus-like particles (VLPs) for use in the prophylaxis and / or treatment of a disease derived from an endogenous retrovirus.SOLUTION: Provided is a virus-like particle (VLP), which is encoded by a nucleic acid molecule encoding an endogenous retrovirus (ERV) Gag protein and an ERV envelope (Env) protein, the Gag protein and the Env protein being linked via a self-cleavable peptide linker p2A, the nucleic acid molecule being an expression cassette.SELECTED DRAWING: Figure 3

Description

[Technical Field]

[0001] The present disclosure relates to vaccines for use in the prevention and / or treatment of diseases. In particular, the diseases may be caused by endogenous retroviruses, such as cancer. The vaccines of the present invention particularly relate to viruses that can form virus-like particles in eukaryotic cells. In certain embodiments of the present invention, virus-encoded virus-like particles (VE-VLPs) are produced in patients' bodies to develop immunogenic responses against endogenous retroviruses. [Background technology]

[0002] Over 100 years ago, it was recognized that cancer development is closely linked to the immune system, and today it is well established that the immune system protects against cancer on a regular basis, while malignant cells develop strategies to evade immune surveillance and develop lethal capabilities.

[0003] Although immune cells can detect and kill tumor cells, this system is not always functional, as evidenced by the nearly 9 million cancer deaths worldwide per year. Vaccination approaches to induce specific immune responses against tumor cells are a relatively old topic in cancer immunotherapy, but they are still under development and have only recently begun to yield relevant results. One vaccination strategy involves vaccination with attenuated tumor cells, such as irradiated autologous tumors or allogeneic tumor cell lines that often secrete granulocyte-macrophage colony-stimulating factor (GM-CSF). In both cases, the injected material contains cancer antigens likely to be present in the actual tumor. Other vaccination strategies involve the administration of peptides or proteins that induce specific immune responses. These antigens are either directly injected in combination with adjuvants or encoded by DNA plasmids or viral vectors.

[0004] Although immunotherapeutic approaches are constantly improving, a broadly acting and highly efficient vaccine is still lacking, and the detailed reason for this is the immunosuppression by tumor cells, which has already been described.

[0005] Endogenous retroviruses (ERVs) are evidence of ancient retroviral infections in our distant ancestors. During infection, viral RNA was reverse transcribed into proviral DNA, which was integrated into the host genome. Eventually, the provirus integrated into the cells of the genome, became heritable, and gave rise to endogenous retroviruses. Over millions of years, viral DNA was passed down and became fixed in the population. Today, every human genome consists of approximately 8% endogenous retroviral DNA, a mere relic of the former retrovirus. Due to mutations, deletions, and insertions, most retroviral genes have become inactive or completely lost from the genome. Today, functional full-length endogenous retroviruses no longer exist in humans. However, ERVs have undergone a replication process that resulted in the integration of multiple copies into the host genome using different functional proteins. Therefore, in some cases, a proportion of homologous ERVs still possess the ability to produce viral particles. Human ERV type K (HERV-K, HML2) is one of the most recently acquired ERVs in the human genome, and members of this family have retained full-length open reading frames for almost all viral proteins.

[0006] Various studies have highlighted the relationship between ERV expression and cancer development and progression. The detection of ERVs in human tumors has opened up new fields in anticancer therapy, with the prospect of new vaccination strategies. A prominent example of human ERVs (HERVs) is the K-type HERV (HERV-K), which is associated with prostate cancer, breast cancer, ovarian cancer, lymphoma, melanoma, leukemia, and sarcoma. Further examples are HERV-H, which is expressed in colon cancer, and syncytin 1, which is expressed in testicular cancer, ovarian cancer, breast cancer, lymphoma, and leukemia.

[0007] It is not always easy to determine whether expression of ERV proteins is a cause or a consequence of a developing tumor, but it is known that conditions within cancer cells allow ERV expression. The general state of hypomethylation in tumor cells promotes the activation of ERV genes that are normally inactivated by DNA methylation in healthy cells (Downey, RF, et al., Human endogenous retrovirus K and cancer: Innocent bystander or tumorigenic accomplice? Int J Cancer, 2015. 137(6): pp. 1249-1257. and Gimenez, J., et al., Custom human endogenous retroviruses dedicated microarray identifies self-induced HERV-W family elements reactivated in testicular cancer upon methylation control. Nucleic Acids Res, 2010. 38(7): pp. 2229-2246. Furthermore, exogenous factors can promote ERV expression. Activation of human ERVs has been observed, for example, by viral infection. HERV-W expression was detected after influenza and herpes simplex virus infection (Nellaker, C., et al., Transactivation of elements in the human endogenous retrovirus W family by viral infection. Retrovirology, 2006.3:p.44), whereas HERV-K was present after Epstein-Barr virus infection (Sutkowski, N., et al., Epstein-Barr virus transactivates the human endogenous retrovirus HERV-K18 that encodes a superantigen. Immunity, 2001.15(4):pp.579-589).Despite the mechanisms that lead to ERV expression, cancer cells maintain the activation of these proteins through selective pressure, demonstrating the beneficial effects of ERVs in tumors (Leong, SP, et al., Expression and modulation of a retrovirus-associated antigen by murine melanoma cells. Cancer Res, 1988. 48(17): pp. 4954-4958).

[0008] Not only are human tumors associated with ERV proteins, but mouse cancer cells also express ERVs. This provides a perfect model organism for testing the effects of ERVs on tumor progression and for testing ERV-targeting therapeutic approaches. One ERV model is the melanoma-associated retrovirus (MelARV), which arises from a murine leukemia virus (MuLV) provirus present in the mouse genome. Most inbred mouse strains contain one or two inactive MuLV copies (Li, M., et al., Sequence and insertion sites of murine melanoma-associated retrovirus. J Virol, 1999. 73(11):pp. 9178-9186). However, the AKR mouse strain, which has three insertions in its genome, is characterized by high production of MuLV early in life, resulting in a frequent incidence of spontaneous lymphomas. Other mouse strains, such as C57BL / 6, spontaneously produce MuLV particles only late in life. Several other mouse cancer models similarly express MuLV / MelARV, similar to human ERVs.

[0009] Because the host's immune system is a natural defense against infection, many viruses, especially retroviruses, have evolved strategies to evade this surveillance. One mechanism found in different virus families [Duch et al., WO 2013 / 050048] is the development of immunosuppressive domains in the envelope protein (Env), which result in different levels of immune system suppression. Immune cells, including natural killer (NK) cells, CD8 T cells, or regulatory T (Treg) cells, can be affected by viruses containing ISDs [Schlecht-Louf et al. (2010)].

[0010] Many ERVs contain proteins with immunosuppressive domains (ISDs), and such domains can also be found in the MelARV Env protein (Schlecht-Louf, G., et al., Retroviral infection in vivo requires an immune escape virulence factor encoded in the envelope protein of oncoretroviruses. Proc Natl Acad Sci USA, 2010. 107(8): pp. 3782-3787; and Mangeney, M. and T. Heidmann, Tumor cells expressing a retroviral envelope escape immune rejection in vivo. Proc Natl Acad Sci USA, 1998. 95(25): pp. 14920-14925). Env-transduced tumor cells grew more rapidly despite the addition of exogenous antigens. This observation was explained by the local immunosuppressive effect mediated by the Env protein. ISD affects both the innate and adaptive immune systems, as evidenced by the inhibition of macrophages, NK cells, and T cells (Lang, MS, et al., Immunotherapy with monoclonal antibodies directed against the immunosuppressive domain of p15E inhibits tumor growth. Clin Exp Immunol, 1995. 102(3): pp. 468-475). Furthermore, effects on regulatory T cell subsets have been suggested, which in turn suppress other immune cells (Mangeney, M., et al., Endogenous retrovirus expression is required for murine melanoma tumor growth in vivo. Cancer Res, 2005. 65(7): pp. 2588-2591).Although the detailed mechanisms of immunosuppression by ISDs are not yet fully understood, the effect appears to be largely mediated by the CKS-17 peptide within ISDs. CKS-17 has diverse effects on the immune system, mostly by altering cytokine expression (Haraguchi, S., RA Good, and NK Day-Good, A potent immunosuppressive retroviral peptide: cytokine patterns and signaling pathways. Immunol Res, 2008. 41(1): pp. 46-55.).

[0011] One of the first therapeutic approaches to target ERV-expressing tumor cells involved the administration of monoclonal antibodies. Thus, antibodies targeting HERV-K Env were able to reduce tumor growth in breast cancer cell lines. Wang-Johanning et al. showed that the observed effects of anti-HERV-K Env monoclonal antibodies were mediated by alterations in the cancer cell cycle and increased apoptosis. Another possible effect of such antibodies, not examined by Wang-Johanning et al. (Wang-Johanning, F., et al., Immunotherapeutic potential of anti-human endogenous retrovirus-K envelope protein antibodies in targeting breast tumors. J Natl Cancer Inst, 2012, 104(3):pp.189-210), could be the prevention of immunosuppression. Like MelARV Env, HERV-K Env proteins contain an ISD and have immunomodulatory functions (Morozov, V.A., Dao Thi, and J. Denner, The transmembrane protein of the human endogenous retrovirus--K (HERV-K) modulates cytokine release and gene expression. PLoS One, 2013. 8(8):p.e70399). The approach tested by Wang-Johanning et al. involved xenograft tumors in immunodeficient athymic mice. Therefore, the effects of HERV-K may only affect innate immune cells such as NK cells.

[0012] Another part of the adaptive immune response that can help eradicate tumors by targeting ERVs involves T cells. For example, adoptively transferred T cells against MuLV Env epitopes in combination with IL-2 were able to eradicate melanoma lung metastases (Yang, J.C. and D. Perry-Lalley, The envelope protein of an endogenous murine retrovirus is a tumor-associated T-cell antigen for multiple murine tumors. J. Immunother. 2000. 23(2): pp. 177-183). Similar experiments were performed in a humanized mouse model for HERV-K. T cells were genetically modified to express a chimeric antigen receptor (CAR) on the T cell surface that recognizes the HERV-K Env on cancer cells. Cytotoxic CAR + T cells were able to lyse tumor cells, preventing metastasis and tumor growth.

[0013] In addition to direct injection of antibodies or T cells, a more practical, inexpensive, and efficient strategy is to induce an immune response by vaccination. A simple approach is vaccination with virus-encoded antigens. However, this method is rather cumbersome because DCs must first be isolated and cultured, then pulsed with defined HLA-restricted peptides, and re-injected into mice or patients.

[0014] A simpler vaccine strategy is the presentation of antigens (e.g., viral envelope proteins) to the immune system on virus-like particles (VLPs) encoded by recombinant adenovirus (Figure 1). These particles do not contain viral nucleic acid and are therefore noninfectious. Nevertheless, VLPs are highly immunogenic, and the displayed proteins are presented in a natural context. For example, viral Env proteins incorporated into VLPs are displayed on a virus-like surface, which promotes correct folding and conformation. In addition to the advantages of potent immunogenicity, vaccination strategies using VLPs also offer practical advantages. Thus, because VLPs are constructed from only one or a few proteins, they are relatively easy to produce, and production can be carried out in cell culture.

[0015] To vaccinate against viruses or virus-related diseases (e.g., ERV-expressing cancers), the entire Env protein should ideally be presented to the immune system to ensure an immune response against the complete protein target. However, because the Env protein contains an ISD, the vaccine itself may have immunosuppressive potential, making it undesirable for an immunization approach. To circumvent this drawback, mutations were introduced into the ISD to prevent immunosuppression while maintaining the native conformation of the target protein.

[0016] Schlecht-Louf et al. (2010) were among the first to examine inactivating mutations in the ISD of viral proteins. Based on comparative studies between immunosuppressive syncytin and non-immunosuppressive syncytin 1 (Mangeney et al. (2007)), Schlecht-Louf et al. identified mutations that abolished the activity of the ISD without ablating the general structure and functionality of the Env protein. This mutational strategy was applied to proteins of other viral origin (e.g., HTLV and XMRV) and more broadly examined with Friend murine leukemia virus (F-MLV). This study not only demonstrated the suppression of both NK cells and T cells by the ISD, but also demonstrated that live attenuated F-MLV viruses containing mutated ISDs in the Env protein functioned as vaccines against the same virus with wild-type ISD sequences. Protection was due to elevated antibody levels and T cell responses to F-MLV epitopes. Their findings were ultimately revealed in patent application WO 2011 / 092199, which focuses on xenotropic murine leukemia virus-related virus (XMRV), which has been associated with human prostate cancer and chronic fatigue syndrome. WO 2011 / 092199 therefore specifically relates to ISD mutations in XMRV and the use of such ISD-mutated viruses for vaccination strategies.

[0017] Another application of ISD mutations was described in patent application WO 2014 / 195510. In this case, ISD mutations were introduced into feline immunodeficiency virus (FIV) to reduce viral immunosuppression while still maintaining its native conformation. WO 2014 / 195510 explains that specific mutations increased antibody responses to FIV Env proteins bound to MBP or transduced in implanted tumor cells when administered in a vaccination approach. Thus, WO 2014 / 195510 relates to mutations in the ISD of FIV Env and the use of such mutated proteins in vaccination approaches against infection with FIV or other lentiviruses.

[0018] Another approach, addressing a broader range of ISD mutations in viral Env proteins, is described in patent application WO 2013 / 050048. In particular, WO 2013 / 050048 relates to first identifying ISDs in enveloped RNA viruses and then mutating these domains to generate antigens that reduce immunosuppression during vaccination. The ISD identification strategy is based on four parameters: 1) the peptide must be located within the fusion protein of an enveloped RNA virus; 2) the peptide must be capable of interacting with membranes; 3) a high degree of homology in the primary structure (sequence) of the peptide must exist within either the viral order, family, subfamily, genus, or species; and 4) the surface location of the fusion protein in a given conformation must be characteristic of an immunosuppressive domain, as revealed by three-dimensional structure or antibody staining. After identifying potential ISDs in the Env of a virus of interest, their immunosuppressive function was confirmed, and then mutations were introduced into the ISD to confirm a reduction in immunosuppression by at least 25%. Overall, WO 2013 / 050048 describes the identification of ISDs in enveloped RNA viruses, the generation of mutated peptides of the ISDs, and their use as vaccines and the generation of antibodies.

[0019] The importance of simultaneous antigen presentation encoded in the adenoviral vector and on the surface of the viral capsid was demonstrated by Bayer et al. [Bayer et al. (2010)]. The advantage of presenting antigens in an ordered structure that helps cross-link B cell receptors is already known. However, by encoding different F-MLV proteins, such as Gag and Env subunits gp70 and p15E, and simultaneously presenting such antigens on the adenoviral capsid protein pIX, Bayer et al. showed that only the combination of antigens encoded and presented on the capsid can elevate the level of functional antibodies. This observation led to the conclusion that while presentation on the adenoviral capsid serves to cross-link B cell receptors, the encoded antigens do not necessarily bind to CD4 receptors, which promotes affinity maturation of B cells. + Using this vaccination strategy, Bayer et al. were able to reduce the viral load of F-MLV after challenge. However, CD8 T cell responses to the target antigen were not observed. + No signs of an increased T cell response could be observed.

[0020] Shoji et al. primarily focused on optimizing adenovirus-based HIV vaccines. Despite using a codon optimization strategy and diverse promoters, Shoji et al. simultaneously encoded Gag and Env proteins in adenoviruses linked via a cleavable furin site (F2A). This allowed for simultaneous expression of both proteins and thus the in situ formation of Gag-based VLPs. In their study, this setup demonstrated the highest immune response compared to other display strategies that did not promote in situ VLP formation [Shoji et al., 2012].

[0021] Duch et al. (2011) (US Patent Application Publication No. 2011 / 0305749) developed a VLP-based retroviral HIV vaccine and demonstrated enhanced immunogenicity of an HIV envelope protein with an ISD mutation. VLP immunogens were produced and purified in vitro.

[0022] US Patent Application Publication No. 2012 / 189647 relates to mutated envelope proteins resulting from mutations in the immunosuppressive domain of the transmembrane subunit of the wild-type envelope protein. US Patent Application Publication No. 2009 / 324553 relates to chimeric polytropic viral envelope polypeptides applicable to directed targeting and controlled fusion of viral particles with other cellular membranes.

[0023] Additionally, a publication by Hohn et al. [Hohn et al., 2014] described that when a codon-optimized version of HERV-K113 was expressed under a CMV promoter, the type and morphology of viral assembly was altered. In particular, VLPs were retained on the cell surface and lacked Env.

[0024] Despite previous strategies of mutating the ISD in viral Env proteins and using adenovirus to encode and present viral antigens, past vaccination strategies employing ISD mutations have been aimed solely at preventing viral infection (Schlecht-Louf et al. 2010, WO 2011 / 092199, WO 2014 / 195510, U.S. Patent Application Publication No. 2011 / 0305749, WO 2014 / 195510). Therefore, there remains a need to break tolerance to self-antigens. Moreover, systems for in situ synthesis of virus-like particles have been previously used for HIV Env and malaria antigens [Luo et al. (2003), Sohji et al. (2011), Andersson et al. (2016), Andersson & Holst (2016), Andersson et al. (2017)], but have not been used to display ERV Env with mutated ISDs on in situ synthesized VLPs. Furthermore, in light of the findings by Hohn et al., there is a need for an efficient system that allows for the production of VLPs, particularly HERV-K VLPs.

[0025] The present invention aims to produce a vaccine that is effective in preventing and / or treating diseases caused by endogenous retroviruses. The vaccine of the present invention exhibits improved immune responses derived from either CD4 T cell or CD8 T cell-initiated response pathways. Summary of the Invention The present invention relates to vaccines for use in the prevention and / or treatment of disease comprising an adenoviral vector capable of encoding a virus-like particle (VLP), the VLP presenting an inactive immunosuppressive domain (ISD).

[0026] Several viral vectors for producing VLPs, including HIV, baculovirus, lentivirus, and adenovirus, are used in vaccine development. The present inventors have surprisingly shown that adenovirus vectors encoding ERVs with inactive ISDs perform better than, for example, HIV vectors when combined with inactive ISDs. Thus, the present invention provides an unexpectedly high immune response, resulting in enhanced immunosuppression in tumors.

[0027] Although any adenovirus vector is expected to perform satisfactorily in the present invention, it is currently the case that the best results are obtained when the adenovirus vector is derived from a mammalian adenovirus, a human adenovirus, a chimpanzee adenovirus, or a gorilla adenovirus. Human adenovirus vectors exist in at least 52 different serotypes, such as types 1, 2, 5, 19, 28, 35, and 40. When a human adenovirus is selected, the human adenovirus vector is derived from a group D vector, human adenovirus serotype Ad5, human adenovirus serotype Ad19a, human adenovirus serotype Ad26, or chimpanzee adenovirus serotype. The inventors used adenovirus type 5 (Ad5) as the starting point for the vaccine vector of the present invention due to its favorable preclinical immunization results. Ad5 induces a sufficiently strong immune response against the target protein not only due to its efficient delivery into antigen-presenting cells (APCs) but also due to the adjuvant properties of the vector itself, which stimulate innate immunity. Additionally, it induces the transcription and release of immunostimulatory cytokines, such as IFN, IL-6, IL-12, IL-15, and TNF-α. These cytokines play important roles in the immune system and function as activators for cells of the adaptive immune response. A particular advantage of Ad5 is that it will prevent transgene expression, preventing an overly strong immune response against the vector. Ad5 balances innate immunity to a level that allows transgene expression while still activating the adaptive immune response. In light of the publication by Matthew J. Johnson et al. (J Immunol 2012;188:6109-6118) showing that recombinant adenovirus serotype 28 and recombinant adenovirus serotype 35 infect and more efficiently induce in vitro maturation and activation of both human and mouse dendritic cells compared to recombinant adenovirus serotype 5, Ad5 was not expected to demonstrate the desired response in the experiments reported herein.Additionally, another paper by Matthew J. Johnson et al. (Vaccine 32 (2014) 717-724) showed that recombinant adenovirus serotype 28 and recombinant adenovirus serotype 35 increased apoptosis of antigen-presenting cells such as monocytes compared to rAd5 and mock-infected controls.

[0028] Immunosuppressive domains (ISDs) can be seen as a mechanism for balancing antitumor immune responses against tumors while simultaneously maintaining a tumor-promoting inflammatory environment induced by ERV activation, similar to that of natural infection. ISDs affect both the innate and adaptive immune systems by inhibiting macrophages, NK cells, T cells, and other immune cells. However, the detailed mechanisms of immunosuppression by ISDs remain incompletely understood. As demonstrated by the present invention, inactivation of ISDs significantly enhances this response.

[0029] The ISD segment may be inactivated by mutation or deletion of one or more amino acids. When inactivation is performed by mutation, one or more of the amino acids are replaced with different amino acids, usually selected from the other 19 natural amino acids. In the case of deletion, any one or more of the amino acids in the ISD region may be deleted. Those skilled in the art will have the appropriate knowledge and experience to know which amino acids to replace to achieve a satisfactory immune response, possibly after initial trial assessment.

[0030] In certain embodiments of the present invention, the ISD has the peptide sequence LANQINDLRQTVIW (SEQ ID NO: 1), LASQINDLRQTVIW (SEQ ID NO: 2), LQNRRGLDLLTAEKGGL (SEQ ID NO: 3), LQNRRALDLLTAERGGT (SEQ ID NO: 4), LQNRRGLDMLTAAQGGI (SEQ ID NO: 5), or YQNRLALDYLLAAEGGV (SEQ ID NO: 6), in which at least one of the amino acids has been deleted or replaced with a different amino acid. It is preferred that the amino acid different from the original is selected from natural amino acids. The ISD segment of the ERV encoded in Ad5 used in the examples of the present application has the following amino acid sequence: LQNRRGLDLLFLKEGGL (SEQ ID NO: 7). The ISD can be inactivated by making one or more mutations in the amino acid sequence. It is currently suitable to replace a single amino acid; i.e., the ISD preferably used in the present invention has the following sequence: LQNRRGLDLLFLKRGGL (SEQ ID NO: 8).

[0031] It may be preferable to replace one or more amino acids in the region upstream or downstream of the ISD segment. This mutation is a compensatory mutation designed to preserve the structure of the domain so that the infecting virus can still function. In a specific embodiment shown in Figure 3, the third amino acid adjacent to the ISD region is replaced with an A→F mutation.

[0032] For an ISD to be inactivated by the present invention, the immunosuppressive capacity needs to be reduced by 70% or more compared to the immunosuppression provided by the original ISD. In a preferred embodiment of the present invention, the ISD is inactivated by 80% or more, such as 90% or more, such as 95% or more, such as 99% or more compared to the immunosuppression provided by the original ISD.

[0033] The present invention provides a general platform for presenting antigens to the body's immune system. Therefore, in principle, any type of protein against which an immune response is desired can be encoded within an adenoviral vector. In a preferred embodiment of the present invention, the antigen is an endogenous retroviral envelope protein (ERV Env) or an immunogenic protein derived from such a protein. It is generally believed that virus-encoded virus-like particle vaccines target ERV Env to dendritic cells (DCs), which present antigens to cells of the adaptive immune system. Presentation on MHC class I induces the activation and proliferation of CD8+ T cells. These cytotoxic T lymphocytes (CTLs), specific for antigens on ERV Env, infiltrate tumors and kill cells presenting the respective antigens. Presentation of antigens on MHC class II by professional antigen-presenting cells (APCs) activates CD4+ T cells, which then coactivate B cells. Activated B cells that encounter antigens presented on circulating ERV Env target proteins or VLPs release ERV Env-specific antibodies. These antibodies can bind their targets on cancer cells and induce the destruction and phagocytosis of malignant cells. Thus, ERV-specific antibodies act as a veil to prevent tumor growth and metastasis. The reacquired immunogenicity of tumor cells can prime a diverse set of tumor-specific T cells that recognize different tumor-associated and tumor-specific antigens. These newly primed and expanded CTLs infiltrate tumors and kill malignant cells.

[0034] Although the vaccine of the present invention can, in principle, be used to immunize several mammalian species, it has actually been developed in a mouse model. In a preferred embodiment of the present invention, the ERV protein is a human endogenous retroviral (HERV) protein or an immunogenic portion thereof. It has been estimated that each human genome consists of approximately 8% endogenous retroviral DNA. However, most endogenous retroviral DNA is merely a remnant of a previous retrovirus. ERVs are evidence of ancient retroviral infection in distant ancestors. During infection, viral RNA is reverse-transcribed into proviral DNA, which is integrated into the host genome. Eventually, the provirus is integrated into germline cells, becomes heritable, and gives rise to endogenous retroviruses. Over millions of years, viral DNA has been transmitted to later generations and established within the population. This suggests that a large portion of the human genome can be used as the antigen-encoding portion of an adenoviral vector. Currently, the HERV is preferably selected from the group consisting of HERV-K, HERV-H, HERV-W, HERV-FRD, and HERV-E. More specifically, HERV-K can be 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), HERV-K110 (=ERVK-18), HERV-H can be selected from the group consisting of HERV-H19 (=HERV-H_2q24.3), HERV-H_2q24.1, HERV-W can be selected as ERVW-1 (=syncytin 1), and HERV-FRD can be selected as ERVFRD-1 (=syncytin 2).

[0035] Adenoviral vectors are constructed to present the encoded ERV protein to the immune system so that an appropriate immunological response can be mounted. In a suitable embodiment of the invention, the ERV protein epitope, or an immunogenic portion thereof, is located between the transmembrane domain and the ISD.

[0036] The experiments reported herein demonstrate the application of adenovirus-encoded ISD-mutated HERV-K VLPs not only to Ad5 but also to another adenovirus serotype, i.e., Ad19 (see Examples 15-17). HERV-K has previously been implicated in the development of cancer and has been shown to contain a functional envelope ISD domain with in vitro activity similar to that of HIV (Morozov et al. 2013). In mice, HERV-K is a foreign antigen, and with similar ISD domain activity, it was a priori unlikely that ISD mutations would enhance immune responses. Surprisingly, however, ISD mutations were found to enhance antibody responses, T cell responses, and anticancer protection directed against HERV-K Env p15E and SU domain proteins. The mutations in HERV-K differ from those disclosed herein for MelARV because the virus families differ in ISD sequences. However, based on the information provided herein and general general knowledge, those skilled in the art can identify appropriate mutations that inactivate ISDs in other virus families. The HERV-K mutations used herein were derived from ISD mutations in HIV, which have been shown to preserve viral infectivity and site-specific conservation between HERV-K and HIV-1 (Morozov et al., 2012). Upon analysis of vector-transfected cells, increased intracellular and cell surface expression of HERV-K mutations was observed (see Example 15 and Figure 24), which can explain the increased immunogenicity and provide additional mechanistic rationale for making ISD mutations in HERV-K family Env proteins using any gene expression platform and construct that may or may not be capable of forming VLPs.

[0037] Thus, the present invention also relates to a nucleic acid molecule encoding an ERV envelope protein or an immunogenic portion thereof, wherein the ISD of the protein contains a mutation that renders the ISD inactive. Preferably, the ERV is a human endogenous retrovirus (HERV), more preferably the HERV is HERV-K. It is further preferred that the mutation in the ISD replaces Q525 with alanine, thereby resulting in the mutated ISD sequence NSQSSIDQKLANAINDLRQT (SEQ ID NO: 50) (instead of NSQSSIDQKLANQINDLRQT (SEQ ID NO: 49)). It is understood that corresponding mutations in the ISD using different sequences are also contemplated. In a further preferred embodiment, the nucleic acid molecule is contained within an adenoviral vector. More preferably, the adenoviral vector is adenoviral vector type 19 (Ad19). It is further preferred that the adenoviral vector containing this nucleic acid encodes a VLP. The present invention further relates to the nucleic acid molecule or the protein encoded by the vector. The nucleic acid molecule, vector, or encoded protein is to be used in the treatment or prevention of a disease, preferably cancer. The cancer to be treated is one that expresses the corresponding ERV. Preferably, the treatment involves a "prime-boost-administration regimen," in which a prime immunization using an adenovirus or nucleic acid molecule is administered first, followed by a later MVA virus, adenovirus, or DNA boost. Preferably, the boost is an MVA boost. Different timings for the prime and boost are contemplated. Longer intervals between the prime and boost are possible, particularly in cancer patients with minimal residual disease. In a preferred regimen, the boost is administered 4 to 8 weeks after the prime.

[0038] In a preferred embodiment of the vaccine according to the present invention, the protein products of the adenoviral vector include the gag protein, the 2A peptide, and the envelope protein (Env). The Env protein can further include a surface domain (gp70), a cleavage site, and a transmembrane domain (p15E). In addition, the transmembrane domain (p15E) can include a fusion peptide, an immunosuppressive domain (ISD), a transmembrane anchor, and a cytoplasmic tail.

[0039] To improve the immunosuppression of vaccines, it may be appropriate to link p15E or its immunogenic portion to the adenovirus capsid protein pIX. To achieve this, p15E was fused N-terminally to the C-terminus of pIX. The highly ordered structure of pIX and its binding antigen on the adenovirus surface aid in cross-linking B cell receptors. Another advantage is that pIX is normally presented as a trimer, which may also aid in presenting bound p15E antigen in its native trimeric form. This modification has been shown to increase the induction of specific antibodies in CD1 mice.

[0040] In certain embodiments of the present invention, the signal peptide encoded by the adenoviral vector is replaced with the signal peptide (LucSP) derived from Gaussia luciferase. This signal peptide enhances transport of proteins to the outer membrane of the cell without altering glycosylation status. Thus, including this signal peptide in place of the native sequence directs transport of synthesized proteins to the membrane, where they are incorporated into VLPs.

[0041] In another aspect of the present invention, the transmembrane anchor and cytoplasmic tail encoded by the adenoviral vector are replaced with the transmembrane domain and cytoplasmic tail derived from influenza A virus hemagglutinin. The insertion increases the expression of the recombinant protein on the cell surface and on VLPs, which results in a strong and broad antibody response. In a preferred embodiment, the transmembrane domain and cytoplasmic tail encoded by the adenoviral vector are replaced with the transmembrane domain and cytoplasmic tail derived from influenza A virus hemagglutinin H3N2 (HA-TMCT).

[0042] In another embodiment of the present invention, a trimerization sequence is provided adjacent to the signal peptide. Trimerization sequences can be added to proteins to facilitate natural presentation. In a preferred embodiment, the trimerization sequence is GCN4.

[0043] The protein products of adenoviral vectors typically include the gag protein, which may be an exogenous or endogenous retroviral gag protein.

[0044] Adenoviral vectors usually require cells to produce virus-like particles. Thus, adenoviral vectors infect cells and produce components for VLPs. In a specific embodiment of the present invention, VLPs are produced in isolated cell lines. Suitable examples include Sf9 cells, Vero cells, HeLa cells, etc. However, it is currently desirable for VLPs to be produced in the cells of the patient's body infected by adenoviral vectors. This production is also called virus-encoded virus-like particles (VE-VLPs), and has the advantage of avoiding the need for an intermediate host for VLP production.

[0045] The present invention also relates to a nucleic acid construct encoding a target protein capable of forming a virus-like particle (VLP), wherein the target protein comprises an immunosuppressive domain (ISD), and the ISD is inactive.

[0046] The present invention is particularly suitable for the prevention and / or treatment of cancer. The types of cancer that can be treated by the present invention are not particularly limited, and include prostate cancer, breast cancer, ovarian cancer, lymphoma, melanoma, leukemia, sarcoma, colon cancer, testicular cancer, ovarian cancer, breast cancer, lymphoma, lung cancer, and liver cancer.

[0047] Under certain conditions, it may be advantageous to treat patients with a prime-boost regimen. Thus, in one embodiment of the present invention, the use of the vaccine in the prevention and / or treatment of cancer comprises priming a patient with the above nucleic acid construct at least 5 days before boosting with the above-disclosed vaccine.

[0048] The present invention also relates to a vaccine for use in the prevention and / or treatment of cancer against a previously disclosed vaccine, comprising post-treating the patient 5 days or more after the patient's exposure to the previously disclosed vaccine with a VLP encoded by a virus other than the VLP derived from an adenoviral vector. In certain embodiments, the VLP encoded by a virus other than the VLP derived from an adenoviral vector is a VLP derived from Modified Vaccina Ankara (MVA).

[0049] Furthermore, in contrast to what was reported by Hohn et al. (2014) for codon-optimized HERV-K113 under a CMV promoter, the expression cassette used, i.e., Gag-p2A-Env, with Env expressed at a 1:1 ratio, again with Gag under a strong promoter, did not result in retention at the cell membrane. Instead, VLPs were expressed that also contained Env (again in contrast to the results reported by Hohn et al.). This indicates that the genetic platform using Gag-p2A-Env performs better than constructs without p2a (or the corresponding functional linker).

[0050] Thus, the present invention further relates to a nucleic acid molecule encoding a Gag protein and an ERV envelope protein (Env) or an immunogenic portion thereof, wherein the native genomic structure connecting Gag and Env is replaced by a functional linker. Preferably, the functional linker is p2A. In other words, the present invention also relates to a nucleic acid molecule comprising a Gag functional linker-Env expression cassette, preferably a Gag-p2A-Env cassette. Preferably, the ERV is HERV-K. More preferably, the ERV is HERV-K113. The HERV-K sequence is a HERV-K consensus sequence, more preferably a codon-optimized consensus sequence. Even more preferably, the HERV-K codon-optimized consensus sequence is the following amino acid sequence (SEQ ID NO: 55):

[0051] [ka]

[0052] [ka]

[0053] It is further preferred that HERV-K contains a mutation in its ISD (underlined and printed in bold in the sequence above). A particularly preferred sequence containing such a mutation is shown in SEQ ID NO:48.

[0054] It is further preferred that the nucleic acid molecule is an adenovirus vector.It is envisioned that the nucleic acid can be used as a genetic vaccine, particularly in the prevention and / or treatment of diseases, preferably cancer.Alternatively, the nucleic acid molecule can also be used to produce VLPs, particularly HERV-K VLPs, in vitro.The resulting VLPs can then be used in immunotherapy, particularly in the prevention and / or treatment of diseases, preferably cancer.In this context, it is understood that the cancer to be treated is an ERV-expressing cancer.

[0055] In addition, the present invention also relates to a VLP encoded by a nucleic acid molecule encoding a Gag protein and an ERV envelope protein (Env) or an immunogenic portion thereof, wherein the native genomic structure connecting Gag and Env is replaced by a functional linker. Preferably, the functional linker is p2A. It is further preferred that the ERV is HERV-K. More preferably, the ERV is HERV-K113. Preferably, the VLP contains a higher amount of Env compared to HERV-K113 VLPs produced by the method described by Hohn et al. As described above, the use of such VLPs in immunotherapy is envisioned. Furthermore, the present invention relates to a nucleic acid molecule or VLP for use in the prevention and / or treatment of a disease. Preferably, the disease is cancer. It is understood that the cancer is a cancer expressing the corresponding ERV.

[0056] In the following detailed section of the present disclosure, aspects, embodiments and implementations will be described in more detail with reference to exemplary embodiments shown in the drawings. [Brief explanation of the drawings]

[0057] [Figure 1] Figure 1 discloses the mechanism of viral vector-encoded virus-like particles. The vaccine contains a recombinant adenovirus (Ad5) encoding viral Gag and Env proteins. Upon injection, Ad5 infects cells and induces expression of the encoded proteins. Gag and Env are linked via a self-cleavable peptide (p2A), which ensures not only equimolar expression of both proteins but also their translational separation. The structural protein Gag alone is sufficient to induce cell membrane budding and virus-like particle (VLP) formation. During VLP formation, Env associates with Gag and is incorporated into the released VLP. Thus, vaccination with an Ad5 vector induces the production of VLPs that present the target protein Env on the surface of the VLP to the immune system. [Figure 2]Figure 2 shows the schematic structure of the MuLV / MelARV envelope protein. The envelope protein (Env) consists of two subunits. (Left) The transmembrane subunit p15E (TM) is anchored in the cell membrane and contains the immunosuppressive domain (ISD) and fusion peptide. p15E is covalently linked to the surface subunit gp70 (SU) via a disulfide bridge. p15E and especially the ISD are shielded by gp70. (Right) The protein subunits are expressed as precursor proteins that are cleaved during processing and transported to the membrane. The drawing has been modified from Mangeney et al. 2007. [Figure 3] Figure 3 shows the mutations in the ISD of the vaccine-encoded MelARV Env (p15E). Two amino acids in the ISD of p15E were mutated to inactivate the immunosuppressive mechanism. Therefore, the following amino acid changes were made: E14→R14 and A20→F20. [Figure 4] Figure 4 shows the vector maps of 768tet and capture pBGH. 768tet (A) and capture pBGH (B) are DNA vectors carrying relevant genes. Additional genes present in the plasmids are not shown in the vector maps. The target protein is first cloned into the expression vector 768tet (A). The expression cassette containing the target protein is then cloned into the human Ad5 (hAd5) genome vector, capture pBGH (B), by homologous recombination to generate recombinant virus in a producer cell line. [Figure 5] Figure 5 discloses the steps for recombinant Ad5 production. The scheme shows the process of virus production after cloning of the target protein into the capture pBGH. The production of recombinant Ad5 involves successive steps that result in "viral lysates," "3-day lysates," and "large-scale lysates" containing the recombinant virus. [Figure 6]Figure 6: Peptides used for ELISA analysis of p15E-specific antibody responses. The region of MelARV Env designated "TM(p15E)" by the arrow was synthesized as a peptide and used in ELISA analysis of serum samples from vaccinated mice. [Figure 7A] Figure 7: Antibody responses induced by Ad5-MelARV-ISD in CD1 mice. (A) p15E-specific antibodies in the serum of vaccinated CD1 mice (vaccination timeline IV). Mice were first vaccinated with DNA encoding MelARV, MelARV-ISD, or GFP (as indicated below the graph) followed by different Ad5 boosts (legend). The booster vaccines used were Ad5-MelARV (dark gray), Ad5-MelARV-ISD (light gray), or Ad5-GFP (white). Antibodies binding to p15E were analyzed by ELISA. Bars indicate the mean absorbance relative to LEV76 control serum using SE. Sample size was n = 5 per group. [Figure 7B] (B) B16F10-GP-specific antibodies. B16F10-GP cells were incubated with serum from the same mice as in (A). Bound antibodies were detected using flow cytometry with an APC-conjugated secondary antibody against mouse IgG. The mean fluorescence intensity for each vaccine group is presented as the mean with SEM. Asterisks indicate significant differences between groups: * (P ≤ 0.05), ** (P ≤ 0.01), *** (P ≤ 0.001). [Figure 8A] Figure 8: Antibody responses and metastasis counts in C57BL / 6 mice vaccinated with Ad-MelARV-ISD. Mice were vaccinated with DNA-MelARV and Ad5-MelARV (DNA + Ad5-MelARV) or DNA-MelARV-ISD and Ad5-MelARV-ISD (DNA + Ad5-MelARV-ISD) in a prime-boost regimen according to vaccination timeline III. (A) Binding of cancer-specific antibodies to B16F10-GP cells. Antibodies in the serum of vaccinated mice specific for tumor cells were analyzed by flow cytometry using an APC-conjugated secondary antibody against mouse IgG. Bars indicate the mean fluorescence intensity of bound antibodies in each group. [Figure 8B] (B) Antibody binding to p15E was analyzed by ELISA. Values ​​represent the mean with SEM for each group. [Figure 8C] (C) Vaccinated mice were challenged intravenously with B16F10-GP, and lung metastases were analyzed 14 days later. The horizontal lines indicate the mean number of metastases in each group. Groups contained n = 7 (DNA + Ad5-MelARV) or n = 8 (DNA + Ad5-MelARV-ISD) mice. [Figure 9] Figure 9: ELISPOT analysis of T cell responses induced by Ad5-MelARV-ISD in Balb / C mice. 21 days after a single vaccination with Ad5 (Ad5-MelARV or Ad5-MelARV-ISD), spleens were isolated from Balb / C mice. Splenocytes were stimulated with AH1, and activated immune cells were detected by IFNγ production in an ELISPOT assay. Results were calculated as the number of spots (IFNγ-producing cells) per 10 splenocytes. Bars indicate the mean number of spots (n = 59) in each group with SEM. Asterisks indicate significant differences from the PBS control: * (P ≤ 0.05), ** (P ≤ 0.01), *** (P ≤ 0.001). [Figure 10A] ICS analysis of T cell responses induced by Ad5-MelARV-ISD in Balb / C mice. The same splenocytes as in Figure 9 were analyzed for the production of the cytokines IFNγ and TNFα in T cells by intracellular staining (ICS) upon stimulation with AH1. The figure shows the total number of activated (CD44+), IFNγ- or TNFα-producing CD8+ T cells in the whole spleen. (A) IFNγ-positive CD8+ T cells. [Figure 10B] (B) TNFα-positive CD8+ T cells. [Figure 10C] (C) The integrated geometric mean of IFNγ-producing CD8+ T cells in each mouse was calculated from the number of IFNγ+ CD8+ T cells multiplied by the mean fluorescence intensity of IFNγ+ cells. [Figure 10D](D) Double-positive CD8+ T cells. Horizontal lines indicate the mean for each group. Asterisks indicate significant differences between groups: *(P≦0.05), **(P≦0.01), ***(P≦0.001). [Figure 11] Figure 11: Titers of Ad5-specific antibodies in CD1 mice vaccinated with Ad5-MelARV versus Ad5-MelARV-ISD. According to vaccination timeline IV, CD1 mice were vaccinated with Ad5-MelARV or Ad5-MelARV-ISD. Serum was analyzed by ELISA for Ad5-specific antibodies by coating ELISA plates with Ad5 particles. Serum from each mouse was tested in 1:2 serial dilutions to obtain antibody titers. The cutoff value for a positive result was 4 times the background OD450. Bars show the mean titers for each group with SEM. Groups contained n=5 mice. Asterisks indicate significant differences between groups: *(P≦0.05), **(P≦0.01), ***(P≦0.001). [Figure 12] Figure 12: Excerpt from the amino acid sequence of p15E displayed on the adenovirus pIX protein. The full sequence is presented in the sequence listing: pIX-p15E (SEQ ID NO: 51); pIX-p15E-ISD (SEQ ID NO: 52), pIX-p15E-with truncated-C (SEQ ID NO: 53), and pIX-p15E-without truncated-C (SEQ ID NO: 54). [Figure 13A] Characterization of adenoviral vectors displaying recombinant pIX. (A) The pcDNA3-pIX-taglinker-xxx plasmid encoding recombinant pIX was transfected into HEK293 cells to confirm correct expression. Cell lysates from transfected cells were analyzed by Western blotting using anti-pIX antibody. Line 1) pIX-p15E, line 2) pIX-p15E-ISD, line 3) with pIX-p15E_truncated-C, line 4) without pIX-p15E_truncated-C, line GFP pIX-GFP. [Figure 13B](B) The produced and purified viruses were analyzed for the integrity of recombinant pIX by Western blotting using anti-pIX antibody. The line numbers represent the same pIX modifications as in (A) presented on the Ad5 vector, whereas the line

number

number

number

number

[0058] Below is the raw sequence where the individual elements of the sequence are indicated as follows:

[0059] signal peptide Surface subunit Transmembrane subunit Immunosuppressive domain (ISD / ISD) * Transmembrane domain cytoplasmic tail

[0060] The present invention encompasses the sequences set forth below in which one, two or more of the amino acids in the immunosuppressive domain have been replaced with another naturally occurring amino acid.

[0061] 1. Amino acid sequence for Env protein (SEQ ID NO:9): [ka] and HERV-K108 (=ERVK-6) having the amino acid sequence (SEQ ID NO: 10): [ka] Gag protein with 2. Amino acid sequence for Env protein (SEQ ID NO:11): [ka] [ka] and ERVK-19 having the amino acid sequence (SEQ ID NO: 12): [ka] Gag protein with 3. Amino acid sequence for Env protein (SEQ ID NO: 13): [ka] HERV-K115 (ERVK-8) having the amino acid sequence (SEQ ID NO: 14): [ka] have 4. Amino acid sequence of Env protein (SEQ ID NO: 15): [ka] and the amino acid sequence of the Gag protein (SEQ ID NO: 16): [ka] and ERVK-9 5. Amino acid sequence of Env protein (SEQ ID NO: 17): [ka] and the amino acid sequence of the Gag protein (SEQ ID NO: 18): [ka] [ka] and HERV-K113 6. Amino acid sequence of Env protein (SEQ ID NO: 19): [ka] and having the amino acid sequence of the Env protein (SEQ ID NO: 20): [ka] ERVK-21 with 7. Amino acid sequence of Env protein (SEQ ID NO: 21): [ka] [ka] ERVK-25 with 8. Amino acid sequence of Env protein (SEQ ID NO: 22): [ka] and the amino acid sequence of the Gag protein (SEQ ID NO: 23): [ka] HERV-K102=ERVK-7 9. Amino acid sequence of Env protein (SEQ ID NO: 24): [ka] and the amino acid sequence of the Gag protein (SEQ ID NO: 25): [ka] HERV-K101=ERVK-24 10. Amino acid sequence of Env protein (SEQ ID NO: 26): [ka] HERV-K110=ERVK-18 11. Amino acid sequence of Env protein (SEQ ID NO: 27): [ka] HERV-H19 = HERV-H_2q24.3 12. Amino acid sequence of Env protein (SEQ ID NO: 28): [ka] [ka] and the amino acid sequence of the Gag protein (SEQ ID NO: 29): [ka] HERV-H_2q24.1 13. Amino acid sequence of Env protein (SEQ ID NO: 30): [ka] HERV-W = ERVM-1 = Syncytin 1 14. Amino acid sequence of Env protein (SEQ ID NO: 31): [ka] HERV-FRD = ERVFRD-1 = Syncytin 2 15. Amino acid sequence of Env protein (SEQ ID NOs: 32 and 33): [ka] and the amino acid sequence of the Gag protein (SEQ ID NOs: 34 to 38): [ka] HERV-E 16. Amino acid sequence of Env protein (SEQ ID NO: 339): [ka] and the amino acid sequence of the Gag protein (SEQ ID NO: 40): [ka] HERV-E

[0062] The target cancers for HERV-K are prostate cancer, breast cancer, ovarian cancer, lymphoma, melanoma, leukemia, and sarcoma. The target cancer for HERV-H is colon cancer. The target cancers for HERV-W are testicular cancer, ovarian cancer, breast cancer, lymphoma, and leukemia, and the target cancers for HERV-E are lung cancer and liver cancer. [Example]

[0063] The materials and methods shown below are common to the subsequent examples.

[0064] The prototype vaccines (DNA-MelARV and Ad5-MelARV) consisted of a DNA plasmid (768tet) or adenovirus type 5 (Ad5) encoding the gene MelARVgag_p2A_env under the strong human cytomegalovirus immediate-early promoter (CMV promoter). This gene simultaneously expressed the MelARV proteins Gag and Env linked via the self-cleavable peptide p2A. Gag induced the formation of virus-like particles (VLPs), whereas the target protein Env was incorporated into the resulting VLPs.

[0065] Furthermore, a vaccine was designed to target the envelope (Env) protein of human endogenous retrovirus type K (HERV-K or HML-2) expressed in tumor cells and tested for induction of cellular and humoral immune responses and anticancer efficacy.

[0066] The designed vaccines contain either a DNA plasmid (768tet), adenovirus type 5 (Ad5), or adenovirus type 19 (Ad19a), each of which encodes a group-specific antigen (Gag) and Env gene (HERV-KGag_p2A_Env) under the strong human cytomegalovirus immediate-early promoter (CMV promoter). These two proteins co-express the self-cleavable peptide p2A with a linker that remains associated with the Gag protein, which is involved in virus-like particle (VLP) formation. The Env protein is incorporated into the forming VLP and serves as a target, generating a specific immune response.

[0067] To improve the immune response of the vaccine, inactivating mutations were prepared in the ISD of the vaccine-encoded MelARV Env to prevent the immunosuppressive effect of the vaccine itself. Two point mutations were introduced in the sequence of the Env transmembrane subunit p15E: glutamic acid at position 14 of the ISD was replaced with arginine, and alanine at position 20 was replaced with phenylalanine (Figure 3).

[0068] In the HERV-K vaccine, the immune response induced by the vaccine was enhanced by introducing a point mutation in the immunosuppressive domain (ISD) of the transmembrane (TM) subunit of the HERV-K Env protein, i.e., p15E. This modification involved replacing glutamine with alanine at position 52 of the ISD (Schlecht-Louf et al. 2010) (see Figure 22). This change inactivated the domain to prevent the vaccine itself from producing an immunosuppressive effect.

[0069] cell culture Various cell lines were used in different experiments. All cell lines were maintained at 37°C in a humidified atmosphere with 5% CO2.

[0070] HEK293: HEK293 originates from human embryonic kidney cultures and was generated by transformation with shared adenovirus type 5 (Ad5) DNA [ATCC.293 [HEK-293]. [Cited June 8, 2017], available at https: / / www.lgcstandards-atcc.org / Products / All / CRL-1573.aspx?geo_country=de]. Advantages of this cell line include easy growth and efficient transfection. Another advantage is the expression of the Ad5 E1 gene [Kovesdi, I. and SJ Hedley, Adenoviral producer cells. Viruses, 2010.2(8):pp.1681-703]. Replication-deficient recombinant Ad5 vaccines are usually administered, meaning that they lack genes essential for viral replication, such as E1. In this case, the missing gene must be provided exogenously during virus production. HEK293 cells provide the proteins required for replication and can therefore be used as producer cells during virus production [Kovesdi, I. and SJ Hedley, Adenoviral producer cells. Viruses, 2010. 2(8): pp. 1681-703.] In this experiment, HEK293 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS), L-glutamine (2 mM), sodium pyruvate (1 mM), and penicillin and streptavidin (Pen / Strep).

[0071] HEK293_T-REx_Adenoviral Toxin-Producing (Avtoxic) (Adenoviral Toxin-Producing Cells): Adenoviral toxin-producing cells are modified HEK293 cells used to prevent the expression of recombinant proteins encoded by Ad5 during virus production. Inhibiting the expression of these recombinant proteins is necessary because some of the encoded target proteins are toxic to HEK293 cells and interfere with virus production [Cottingham, MG, et al., Preventing spontaneous genetic rearrangements in the transgene cassettes of adenovirus vectors. Biotechnol Bioeng, 2012. 109(3):719-28.] HEK cells were modified in two steps to include different protein suppression mechanisms. The first mechanism involved repression by the T-REx system [Fisher, T. Inducible Protein Expression - T-REx™ System. 2011 [cited June 8, 2017], available at https: / / www.thermofisher.com / dk / en / home / references / protocols / proteins-expression-isolation-and-analysis / protein-expression-protocol / inducible-protein-expression-using-the-trex-system.html]. T-REx-293 cells were genetically engineered to express the tetracycline repressor protein (Tet repressor), which binds to and represses the Tet operator. This results in expression of the recombinant target protein under the control of the strong CMV promoter.

[0072] Because the T-REx system is not completely effective in preventing expression of target proteins, the T-REx-293 cell line was further modified by Sirion Biotech GmbH (Martinsried, Germany). The new cell line, HEK293_T-REx_adenoviral toxin-producing (adenoviral toxin-producing cells), expresses a short hairpin RNA (shRNA) that targets a messenger RNA (mRNA) sequence called p2TS, which is co-transcribed with the target protein. The shRNA causes degradation of the p2TS-containing mRNA, thus further suppressing the recombinant protein. The adenoviral toxin-producing cells were maintained in DMEM supplemented with 10% heat-inactivated FBS, L-glutamine (2 mM), sodium pyruvate (1 mM), and Pen / Strep.

[0073] HEK293(CCS)-shmir-pIX_221-puro (pIX- cells): pIX- cells are modified HEK293 cells used to produce Ad5-pIX viruses that display antigens on the viral capsid protein pIX. The native pIX protein is encoded by the adenovirus E1 gene expressed in HEK293 cells. To prevent the integration of native pIX into viral particles and facilitate the integration of recombinant pIX, HEK293-encoded pIX was silenced in pIX- cells by shRNA expression. Transcription of the shRNA during virus production was induced by doxycycline. Additionally, the cells were transduced with the pac gene, which encodes puromycin N-acetyltransferase (PAC), allowing for the selection of shRNA-expressing cells with puromycin. Therefore, cells were maintained in DMEM supplemented with 10% heat-inactivated FBS, L-glutamine (2 mM), Na-pyruvate (1 mM), Pen / Strep, and 0.5 μg / mL puromycin.

[0074] B16F10-GP: The B16 cell line is a murine melanoma cell line originating from the C57BL / 6J mouse strain [ATCC.B16-F10. [cited June 8, 2017], available at https: / / www.lgcstandards-atcc.org / Products / All / CRL-6475.aspx?geo_country=de]. B16F10 is a more proliferative variant and is frequently used to analyze metastasis in C57BL / 6 mice. After intravenous injection of B16 cells into mice, the mutant was obtained by 10 consecutive rounds of selection for lung metastasis [Fidler, IJ, Selection of successive tumor lines for metastasis. Nat New Biol, 1973, 242(118): pp. 148-9; Fidler, IJ and GL Nicolson, Organ selectivity for implantation survival and growth of B16 melanoma variant tumor lines. J Natl Cancer Inst, 1976, 57(5): pp. 1199-202.] The cell line B16F10-GP used in this experiment additionally expresses the immunodominant epitope (GP33-41) of the glycoprotein of lymphocytic choriomeningitis virus (LCMV) [Prevost-Blondel, A., et al., "Tumor-Infiltrating Lymphocytes Exhibiting High Ex Vivo Cytolytic Activity Fail to Prevent Murine Melanoma Tumor Growth In Vivo." The Journal of Immunology, 1998, 161(5):2187-2194.] Cells were maintained in DMEM supplemented with 10% heat-inactivated FBS, L-glutamine (2 mM), sodium pyruvate (1 mM), and Pen / Strep.

[0075] CT26: CT26 is a murine colon carcinoma cell line derived from the Balb / C mouse strain and was obtained from Dr. Anders Elm Pedersen. This cell line was used to examine primary tumor growth in mice [ATCC.CT26.WT. [cited June 8, 2017], available at https: / / www.lgcstandards-atcc.org / products / all / CRL-2638.aspx?geo_country=de#generalinformation]. Cells were maintained in Roswell Park Memorial Institute medium (RPMI) supplemented with 10% heat-inactivated FBS, L-glutamine (2 mM), sodium pyruvate (1 mM), and Pen / Strep.

[0076] 4T1-Luc: 4T1 is a mouse breast cancer cell line originating from the Balb / C mouse strain. When injected into the mammary fat pad of mice, the cells form primary tumors that metastasize to the lungs, liver, lymph nodes, and brain [ATCC.4T1. [cited August 4, 2017], available at https: / / www.lgcstandards-atcc.org / Products / All / CRL-2539.aspx?geo_country=de#characteristics]. The cell line was stably transfected with a luciferase reporter protein (Luc). Cells were maintained in RPMI supplemented with 10% heat-inactivated FBS, L-glutamine (2 mM), sodium pyruvate (1 mM), and Pen / Strep.

[0077] Vero cells: Vero cells are a primate kidney cell line derived from African green monkeys (Cercopithecus aethiops) [ATCC. Vero. [cited June 8, 2017], available at https: / / www.lgcstandards-atcc.org / products / all / CCL-81.aspx?geo_country=de#characteristics]. This cell line is highly transducible by human Ad5 infection without the supportive production of new virions and was therefore used to analyze protein expression and VLP release by Ad5-vaccines. Cells were maintained in DMEM supplemented with 10% heat-inactivated FBS, L-glutamine (2 mM), sodium pyruvate (1 mM), and Pen / Strep.

[0078] A549 cells are human lung epithelial cells suitable for viral transfection. Therefore, A549 cells were used for adenovirus transfection containing the sequence of interest for VLP production. VLP secretion was analyzed via Western blot (WB) technique, and the presence of VLPs on the cell surface was detected using fluorescence-activated cell sorting (FACS) and visualized using electron microscopy (EM). These cells were maintained in Kaighn's modification of Ham's F-12 medium (Ham's F-12K medium) supplemented with 10% heat-inactivated FBS, Pen / Strep, and sodium pyruvate (1 mM).

[0079] Renca cells expressing Gag and Env proteins. Renca cells are mouse (Mus musculus) kidney epithelial cells. The cells were derived from a renal adenocarcinoma in balb / C mice. Tumor growth and progression closely resemble those observed in human renal cell carcinoma, mimicking spontaneous metastasis, particularly to the liver and lung. The cells used in the following examples were kindly provided by Professor Dr. Barbara Schnierle (Langen, Germany). In some of the following examples, cells were modified to express human endogenous retrovirus type K (HERV-K) Env or Gag proteins. This allowed for the induction of HERV-K protein-expressing tumors in mice, creating a suitable mouse model for testing our novel vaccination strategy aimed at human cancers expressing ERV proteins. The cells were maintained in Roswell Park Memorial Institute medium (RPMI-1640) supplemented with 10% heat-inactivated FBS at pH 7.2, 20 × 10 IU / L Pen and 5 g / L Strep, 2.9 g / L L-glutamine (2 mM), and 3.7 g / L sodium pyruvate (1 mm).

[0080] Primary cultures of chicken embryo fibroblasts (CEF) are widely used for virus cultivation. CEF cultures were prepared using 11-day-old chicken eggs from Jens Toft, Lohmann (Denmark) according to the protocol from (Staib et al. 2004). In this case, CEF cells were used for the production of modified Vaccina Virus Ankara (MVA), encoding the HERV-K Env and Gag foreign antigens. The reason for working with this specific cell type is that MVA replication is restricted to avian cells, meaning that MVA does not reproduce in the majority of mammalian cells, making it unsuitable for this purpose (Altenburg et al. 2014). CEF cells were cultured in CEF medium consisting of RPMI supplemented with 3.7 g / L sodium pyruvate, 10% heat-inactivated FBS, and 1% (v / v) antibiotic-antimycotic (Gibco™, 15240062).

[0081] Baby hamster kidney fibroblasts (BHK-21 cells) were originally derived from baby Syrian golden hamster kidney cells (Mesocricetus auratus). The specific cell line used in the following examples was provided by Professor Allan Randrup Thomsen (University of Copenhagen, Denmark). BHK-12 cells were used for titration of MVA Env and Gag because they are known to be one of the few cell lines capable of MVA replication. The cells were maintained in CEF medium, consisting of RPMI supplemented with 3.7 g / L sodium pyruvate, 10% heat-inactivated FBS, and 1% (v / v) antibiotic-antimycotic (Gibco™, 15240062).

[0082] Plasmid constructs To generate recombinant adenovirus, target proteins were cloned into the modified adenovirus vector capture pBGH. This vector contains an Ad5 genome with deletions in the E1 and E3 genes. Furthermore, the vector contains homology to the vector 768tet, which requires a CMV promoter and a 3' polyadenylation (polyA) tail and expresses recombinant proteins under the Tet operator (Figure 4) [Becker, TC, et al., Use of recombinant adenovirus for metabolic engineering of mammalian cells. Methods Cell Biol. 1994. 43 Pt A: pp. 161-89.] Therefore, target proteins were first inserted into 768tet by subcloning, PCR cloning, or Gibson assembly, and then cloned into capture pBGH (Figure 4) via homologous recombination (Figure 5). For pIX modification of adenovirus, the target protein was cloned into the common expression vector pcDNA3, which encodes pIX and a linker sequence (containing a FLAG tag) followed by an additional restriction site to insert the gene of interest (pcDNA3_pIX_taglinker_xxx, xxx = target antigen). The expression vector was transfected into producer cells to induce the expression of recombinant pIX in these cells. The different plasmid constructs used are listed in Table 1.

[0083] [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4]

[0084] Cloning Different cloning strategies were used to construct new DNA constructs for the production and testing of adenovirus vaccines.

[0085] Subcloning For subcloning, the target DNA sequence was transferred from one plasmid (donor vector) to another (target vector). The donor and target vectors were cleaved at the ligation site via restriction digestion. To prevent religation, the target vector was treated with calf intestinal alkaline phosphatase (CIP), which catalyzes dephosphorylation at the 5' and 3' ends of DNA. The digested DNA was separated on a 1% agarose gel containing GelGreen dye (#41004, Biotium). The desired DNA band was excised, and the DNA content was extracted using an EZNA Gel Extraction Kit (D2500; OMEGA bio-tek). Briefly, the gel was dissolved in 1 volume of binding buffer (XP2) and loaded onto a HiBind® DNA Mini Column. After two washes, the column was dried, and the DNA was eluted twice in elution buffer.

[0086] After purification, the vector and insert were mixed at a 1:3 stoichiometric ratio. Ligation of the two DNA fragments was catalyzed using Instant Sticky-end Ligase Master Mix (M0370; New England BioLabs). The ligated product was transformed into XL1-Blue Competent Cells (#200249, Agilent Technologies). For transformation, DNA was added to the bacterial suspension and incubated on ice for 10 minutes. The cells were then permeabilized by heat shock at 42°C for 45 seconds. After a 2-minute incubation on ice, Super Optimal Broth medium (SOC medium) was added, and the bacteria were incubated at 37°C for 1 hour with shaking. The bacterial suspension was streaked onto Lysogeny Broth medium (LB medium) agar plates containing the respective antibiotics and incubated overnight at 37°C.

[0087] To screen for the correct construct, several bacterial colonies were expanded for miniplasmid preparations (see "0 DNA preparations" below). Isolated plasmid DNA was excised by restriction digestion and analyzed by gel electrophoresis.

[0088] For the HERV-K constructs (and corresponding controls), subcloning was performed to insert DNA constructs containing the sequences of interest (DNA_ISD mutant_coHERV-K-P2TS and DNA_coHERV-K-P2TS) into the acceptor plasmid 768(TetO)-SO-alb-CIDR. To do this, the inserts and acceptors were first amplified using PIR1 and XL1-Blue cells and kanamycin and ampicillin selection markers, respectively. All constructs were digested with XbaI (New England Biolabs, R0145) and SwaI (New England Biolabs, R0604) with NEBuffer™ 3.1 (New England Biolabs, B7203) at 37°C for 1 hour and 30 minutes, because the activity of the XbaI enzyme is only 75% when used with NEBuffer 3.1. The DNA was separated by electrophoresis (100 V, 200 A, 1 h) using a 1% agarose gel with GelGreen dye. The bands containing the insert and 768 (TetO) were excised and purified using the EZNA® Gel Extraction Kit (Omega Bio-Tek, D2500) according to the manufacturer's instructions, and eluted in 20 μL of ultrapure water (UPW).

[0089] To ligate the constructs, 40 ng of acceptor vector and 120 ng of each insert were incubated with a 1:2 dilution of Instant Sticky End Ligase Master Mix (2x) at 37°C for 15–30 minutes. Transformations were performed using XL1-Blue cells, and DNA was obtained using a miniprep (as described below). Test cleavage was then performed to verify whether the sequence of interest had properly inserted into the acceptor vector. If so, a new transformation and miniprep (as described below) were performed to obtain a higher concentration of DNA.

[0090] PCR cloning In contrast to subcloning, PCR cloning is characterized by the generation of inserts in a polymerase chain reaction (PCR). Target sequences are amplified via PCR from donor vectors using specific extension primers to insert enzyme restriction sites. Primers were ordered from TAG Copenhagen and mixed with the template and PfuUltra II Hotstarter PCR Master Mix (#600850, Agilent Genomics). PCR was initiated by incubating at 95°C for 2 minutes to activate Taq polymerase and promote complete denaturation of the DNA template. The initial step was followed by 30 cycles of denaturation at 95°C, annealing at 60°C, and DNA extension at 72°C. PCR was completed with a final step at 72°C for 3 minutes to terminate DNA extension.

[0091] DNA was isolated from the reaction mixture using the EZNAGel Extraction Kit (D2500; OMEGA bio-tek) protocol "Purification from enzymatic reaction." To remove residual genomic DNA, the purified PCR product was treated with DpnI (R0176, New England BioLabs), an enzyme that cleaves methylated DNA. DNA was subjected to enzymatic digestion at specific restriction sites and purified using the EZNAGel Extraction Kit. Digestion and ligation of the target vector were performed using the subcloning protocol previously described.

[0092] In the context of the HERV-K construct (and corresponding control), to proceed using homologous recombination, the NotI site contained within the HERV-K wild-type / ISD mutant sequence had to be removed so that NotI could then be used to precisely linearize the plasmid and allow proper recombination. To do this, both sequences obtained from the previously described subcloning procedure (768(TetO)-SP-alb-CSP-HERV-K wild-type / ISD mutant) were cleaved with XbaI (New England Biolabs, R0145) and BspEI (New England Biolabs, R0540) together with NEBuffer™ 3.1 (New England Biolabs, B7203) for 1.5 hours at 37°C and separated by electrophoresis on a 1% agarose gel containing GelGreen dye. The DNA band containing the NotI site to be removed was digested and eluted using the EZNA® Gel Extraction Kit.

[0093] The forward primer used in the PCR reaction annealed at the 3' end of the HERV-K Env sequence, specifically at the BspEI restriction site (5'-CCCGTGTCCGGACCTGAG-3', SEQ ID NO: 45), whereas the reverse primer annealed at the 5' end of the HERV-K Env sequence at the XbaI restriction site (5'-GTTCTAGACTTGTCCTGAATTTTCTGGTTA-3', SEQ ID NO: 46). The reverse primer contained a modification at the NotI site to remove it. Primers were obtained from TAG Copenhagen A / S (Copenhagen, Denmark).

[0094] A reaction mixture was prepared for each DNA construct using 10 ng of template DNA (1 ng / μL), 10 μM of each primer, and a 1:2 dilution of PfuUltra II Hotstart PCR Master Mix (Agilent Technologies, 600850). The PCR reaction consisted of an initial denaturation step (95°C, 5 min), followed by a 35-cycle loop including a denaturation step (95°C, 30 s), an annealing step (58°C, 25 s), and a final extension step (72°C, 45 s). Finally, a final extension step was performed (72°C, 10 min), and the samples were stored at 4°C.

[0095] The PCR products, together with the acceptor plasmid, were separated by gel electrophoresis, and the desired bands were collected and processed as previously described in the "Subcloning" section of this specification, thus obtaining the 768(TetO)-HERV-K-Gag-p2A-Env wild-type and ISD mutant constructs, which no longer contained a restriction site for the NotI enzyme in their sequences.

[0096] Gibson Assembly Gibson assembly was used to combine several DNA fragments into one construct. The fragments were amplified by extension PCR to add overhangs homologous to the target vector. The PCR product was treated and purified as described for PCR cloning. The target vector was opened via restriction digestion at the insertion site. To assemble the fragments, the opened target vector and purified insert were mixed in a 1:3 stoichiometric ratio and incubated with Gibson Assembly Master Mix (E2611, New England BioLabs) for 1 hour at 50°C. Three key enzymes in the Master Mix facilitated assembly: an exonuclease removes DNA from the 5' end of the fragment, creating a single-stranded 3' overhang that anneals to other fragments at the homologous region. Nucleotides are inserted into the remaining gap by DNA polymerase. Finally, DNA ligase joins the nicks in the assembled DNA. Similar to the cloning techniques described above, the assembled DNA was transformed into bacteria and then screened for the correct construct.

[0097] Homologous recombination to generate recombinant adenovirus genomes Insertion of the target gene into the adenovirus genome (Ad5) was carried out by homologous recombination in E. coli. The insert (target gene) from 768tet containing the homologous region to the target vector was excised via restriction digestion and purified by gel electrophoresis. The acceptor vector, capture pBGH (Ad5 genome), was similarly linearized by restriction digestion. To prevent religation, the excised vector was subjected to CIP treatment (see subcloning). The vector DNA was then purified by ethanol precipitation. Briefly, the DNA was precipitated in 0.3 M sodium acetate and 70% ethanol, frozen at -80°C for 20 minutes, and centrifuged at 16,000 g for 15 minutes (4°C). The pellet was washed in 70% ethanol and centrifuged for another 5 minutes. After drying at room temperature (RT), the DNA was resuspended in water. To prevent further religation, adenosine overhangs were generated using Tempase Hot Start DNA Polymerase (#230306, Ampliqon). The DNA was then purified via phenol-chloroform extraction. For this purpose, phenol-chloroform was added to the reaction mixture, followed by centrifugation at 16,000 g for 10 minutes. The upper aqueous phase was transferred to a new reaction tube, and the DNA was extracted by ethanol precipitation as previously described.

[0098] To integrate the vector and insert by homologous recombination, both components were mixed at a 1:3 stoichiometric ratio and added to electroporation-competent BJ5183 cells. Bacteria were transferred to an electroporation cuvette (#1652086, Bio-Rad) and permeabilized by electroporation at 25 μFD, 2.5 kV, and 200 Ω in a Gene Pulser (Bio-Rad). After electroporation, cells were transferred into SOC medium and further processed as described in the heat shock protocol (see "Subcloning").

[0099] The following plasmids were provided by Sirion biotech:

[0100] cDNA_HERV-K(Gag_p2A_Env) cDNA_HERV-K(Gag_p2A_Env-(Q6A)ISD-mutant).

[0101] The same construct but encoded by the Ad19a vector was also provided by Sirion.

[0102] The cDNA construct is amplified and used as a DNA vaccine and insert vector, for the ultimate purpose of cloning strategy to obtain the Ad5 vector that codes for the above-mentioned sequence that can be used as a vaccine.Specifically, for the HERV-K construct coded in hAd5 (and corresponding control), the gene of interest is cloned into the pBGH plasmid that codes the human Ad5 genome with deletions in E1 and E3 genes.The transgene is inserted into the 768tet plasmid that codes the gene of interest instead of E1 by homologous recombination.This strategy is chosen because conventional cloning using restriction digestion and ligation is not very effective, and the pBGH vector is a very large plasmid, exceeding 38 kbp.

[0103] Homologous recombination between 768tet and the pBGH capture plasmid was carried out in E. coli. The capture vector contained green fluorescent protein (GFP) as an insert to be replaced by the gene of interest.

[0104] Because the pBGH plasmid encoding the human Ad5 genome is too large (<38 kbp) to employ the usual cloning strategy of inserting the desired construct using restriction enzyme digestion, the pBGH plasmid was inserted in place of E1 using homologous recombination.

[0105] First, the pBGH acceptor vector was linearized using SwaI enzyme (New England BioLabs, R0604) at 37°C for 2 hours. Meanwhile, the 768(TetO)-HERV-K-Gag-p2A-Env wild-type and ISD mutants were digested with NotI enzyme (New England BioLabs, R3189) for 1 hour. The reaction products were separated by electrophoresis in a 1% agarose gel containing GelGreen. The HERV-K sequences flanked by the homologous regions required for recombination were recovered from the gel, and the DNA was isolated using an EZNA® Gel Extraction Kit (Omega Bio-Tek, D2500) according to the manufacturer's instructions and eluted in UPW.

[0106] After digestion of pBGH, both the 3' and 5' ends were phosphorylated using Calf Intestinal Alkaline Phosphatase (30 min, 37°C, M0290) to prevent religation. The vector then underwent ethanol precipitation in 0.3 M sodium acetate and 70% (v / v) ethanol at -80°C for 20 min. Immediately after, the sample was centrifuged (15 min, 4°C, 16,000g), and the pellet was washed with 70% (v / v) ethanol. The vector underwent a further centrifugation (5 min, 4°C, 16,000g), and the resulting pellet was allowed to dry at RT and finally resuspended in UPW.

[0107] To prevent further religation of the pBGH vector, it was treated with Tempase Hot Start DNA polymerase (Ampliqon, A230306) at 72°C for 30 minutes, which added adenosine overhangs. The DNA was purified by adding phenol / chloroform and centrifugation (10 minutes, 4°C, 16,000g), and then the upper aqueous phase containing the DNA was transferred to a microcentrifuge tube. The DNA underwent ethanol precipitation as before to further purify the DNA, and the DNA was diluted into UPW.

[0108] All plasmids were stored in water rather than in elution buffer because salt content interferes with the efficiency of electroporation. The pBGH vector and HERV-K wild-type / ISD mutant insert were combined with electroporation-competent BJ5183 cells (Agilent, 200154) at a molar ratio of 1:3. The mixture was then transferred to an electroporation cuvette (Bio-Rad, 1652086), which was used to permeabilize the cells using a gene pulser (Bio-Rad) at 25 μFD, 2.5 kV, and 200 Ω. SOC medium was then added to recover the transformed E. coli competent cells. The cells were then incubated at 37°C for 1 hour in a shaking incubator. Finally, the mixture was plated onto LB agar plates containing kanamycin and incubated overnight at 37°C.

[0109] To confirm that homologous recombination had occurred properly, DNA was isolated using a miniprep as described below. The DNA was then digested with restriction enzymes and separated in a 1% agarose gel containing GelGreen dye. Bands corresponding to the correct size for pBGH and the insert were excised and transformed into E. coli, and the DNA was finally reisolated via a miniprep as described below.

[0110] DNA preparation Escherichia coli (E. coli) transformation For transformation, chemically competent E. coli XL1-Blue Supercompetent Cells (Agilent, 200236) and One Shot™ PIR1 Chemically Competent Cells (Thermofisher Scientific, C101010) were used. 20 μL of the latter was mixed together with 10 ng of plasmid DNA and kept on ice for 3 minutes. The mixture was then heat-shocked in a Waterbath TW80 (Julabo) at 42°C for 45 seconds and placed again on ice for 3 minutes. Immediately after, 200 μL of Super Optimal Broth containing 200 μL of Suppression of Catabolite (SOC) medium (20 g tryptone, 5 g yeast extract, 0.58 g NaCl, 0.19 g KCl, 3.96 g glucose, and 5.04 g MgSO 7H O) was added to the sample, which was then placed in a shaking incubator at 37°C for 1 hour. The final step consisted of plating the samples onto LB agar plates containing the corresponding antibiotics to which our plasmids were resistant (ampicillin (Amp): 100 μg / mL, kanamycin (Kan): 50 μg / mL), and placing the E. coli agar plates (Binder) in an incubator at 37°C overnight.

[0111] Agarose gel electrophoresis To check whether the transformation was accurate, the DNA-purified constructs were run on a 1% (w / v) agarose gel containing ethidium bromide or GelGreen™ dye (Biotium, 41004) to visualize the DNA under ultraviolet (UV) light. 1x loading buffer (6x) was added to the sample, and the sample was loaded onto the gel along with the size marker GeneRuler 1kb Plus DNA Ladder (Thermo Fisher Scientific, SM1331). The buffer used was Tris-acetate-ethylenediaminetetraacetic acid (EDTA) (TAE) buffer (4.86 g / L Trizma® base, 0.37 g / L Na2EDTA·2H2O, and 0.11% (v / v) acetic acid, pH = 8.3). Electrophoresis was performed at 120 V for 1 hour using an EPS3501XL electrophoresis power supply (GE Healthcare).

[0112] Mini Preparation To screen for the correct construct after cloning, small-scale DNA amplification was performed. Bacterial colonies were transferred into 3 mL or 5 mL of LB medium (containing 100 μg / mL of Amp or 50 μg / mL of Kan, depending on the resistance gene in the plasmid of interest) and grown overnight at 37°C. Plasmid DNA isolation was performed using the EZNA® Plasmid DNA Mini Kit I (D6943, Omega Bio-Tek). Briefly, bacteria were pelleted by centrifugation and resuspended in RNase-containing solution I (resuspension buffer). Solution II (lysis buffer) was added to release DNA from the cells. Solution III (neutralization buffer) was added to terminate the reaction and precipitate genomic DNA along with cell debris. The precipitate was pelleted by centrifugation, and the supernatant was transferred to a HiBind® DNA Mini Column. DNA was bound to the column membrane by centrifugation, and after adding HB buffer, the column was washed twice with DNA wash buffer and then dried. Finally, the plasmid DNA was eluted in elution buffer.

[0113] Midi Preparation To obtain larger and more purified DNA yields, midi preparations were performed using the NucleoBond® Xtra Midi Kit (#740410, AH Diagnostics) from E. coli grown overnight in 100 mL of LB medium (again containing the appropriate antibiotics). The principle was similar to minipreparation, starting with resuspending and lysing the bacteria. After neutralization, the lysate was loaded onto an equilibrated NucleoBond® Xtra Column and washed with equilibration buffer. The inserted column filter containing residual cell debris was removed, and the column was washed with wash buffer. DNA was eluted in elution buffer and then precipitated in isopropanol. The precipitated DNA was pelleted by centrifugation and washed with 70% ethanol. After an additional centrifugation step, the supernatant was removed, and the DNA pellet was dried at RT. DNA was reconstituted in 100 μL of 10 mM Tris-HCl buffer (pH 8.0) or with 100 μL of elution buffer from the EZNA® Plasmid DNA Mini Kit and the concentration was determined with a NanoDrop™ 2000.

[0114] 2.6 Virus production Different viruses were produced and tested in this experiment (Table 2). In addition to the usual recombinant adenovirus, an Ad5 vector displaying recombinant pIX on its surface was tested and had to be produced by a different procedure.

[0115] [Table 3-1] [Table 3-2] [Table 3-3]

[0116] Sequence of MelARV Env protein with modified ISD The Env protein has the following sequence (SEQ ID NO: 41):

[0117] [ka]

[0118] The sequence has been modified by exchanging the original E to R in the grey background letters in the ISD sequence and A to F in the third amino acid outside the ISD, also marked by grey.

[0119] Recombinant Ad5 generation The starting point for Ad5 production is the adenoviral genome plasmid pBGH. The plasmid contains all the genes required for the formation of infectious Ad5 particles, but is missing the E1 and E3 genes. E1 is required for viral replication and is instead provided by the producer cell line HEK293 / adenoviral toxin production (Kovesdi, I. and SJ Hedley, Adenoviral producer cells. Viruses, 2010. 2(8): pp. 1681-703). E3, a gene nonessential for viral production, is deleted from the genome, creating space for recombinant target genes. During the process of capture cloning (see "0. Homologous recombination to generate recombinant adenoviral genomes"), these target genes are inserted into the vector via homologous recombination. The process of cloning target proteins into capture pBGH and subsequent viral production is summarized in Figure 5.

[0120] Adenoviral toxin-producing cells were infected with the recombinant capture pBGH vector. To this end, cells were seeded into T75 culture flasks and grown to 50-70% confluency. The vector DNA was linearized by restriction digestion with PI-SceI (#R0696S, New England BioLabs) in PI-SceI buffer for 1 h at 37°C. Phenol-chloroform purification was then performed as described in "Capture Cloning," and the DNA was dissolved in OptiMEM (#11058-021, Invitrogen). A portion of the DNA solution was loaded on a 1% agarose gel to confirm accurate cleavage of the plasmid. The remaining DNA was mixed with polyethyleneimine (PEI) at a DNA:PEI ratio of 1:3. After 15 min of incubation at RT, the mixture was added dropwise to the adenoviral toxin-producing cells. Transfected cells were incubated under normal cell culture conditions (see "Error! Reference source not found. Cell Culture"), but the medium was changed after 16 hours and every 2-3 days thereafter. When cell lysate was visible due to detached cells (after 2-3 weeks), the cell medium containing lysed cells (referred to as "viral lysate") was collected and stored at -80°C.

[0121] In the next step, cells were reinfected with the "viral lysate" to obtain a "3-day lysate." For this purpose, adenosine toxin-producing cells were grown in 6-well plates to 70% confluency and infected with 1:10 serial dilutions of the "viral lysate" from well to well. Three days after infection, the supernatant from the most diluted, completely lysed well was harvested and frozen at -80°C. This virus sample was called the "3-day lysate."

[0122] To produce this virus on a large scale ("large scale lysate"), adenosine toxin-producing cells were cultured in four Nunc™ Cell Culture Treated TripleFlasks™ (500 cm 2) (#132913, Thermo Fisher). When the cells reached 70% confluency, the flask was infected with 150 μL of "3-day lysate." After complete lysis of the cells (approximately 3 days), the supernatant was harvested and frozen at -80°C.

[0123] Recombinant Ad5 purification In the first step of virus purification, 0.5% Igepal CA-630 (#56741, Sigma-Aldrich) was added to the harvested large-scale lysate. During a 10-minute incubation at room temperature, the detergent disrupted the remaining cells and released the virus content into the medium. To remove cell debris, the lysate was centrifuged at 12,186 g for 20 minutes at 4°C. The supernatant was collected, and half of the volume was added as a 20% polyethylene glycol (PEG) + 2.5 M NaCl solution, followed by gentle shaking overnight at 4°C. During this step, the virus in the supernatant precipitated, allowing for virus concentration in the next step. The precipitated virus was pelleted by centrifugation at 12,186 g for 20 minutes. The virus pellet was resuspended in 5 mL of cold phosphate buffered saline (PBS) and transferred to a 15 mL Falcon tube. The sample was centrifuged at 784 g for 5 minutes to remove remaining cell debris. The supernatant was transferred to a new 15 mL Falcon tube, and the previous centrifugation step was repeated several times until only a small pellet of cellular debris remained in the tube, which could not be completely removed. Most of the saturated CsCl solution was added to the virus-containing supernatant to reach a final density of 1.34 g / mL. The resulting solution was transferred to an ultracentrifuge tube (#342413, Beckman Coulter), which was then sealed and centrifuged overnight at 257,300 g in a Beckman Coulter Ti 70.1 rotor. A clearly visible virus band was extracted with a needle and syringe and loaded onto an equilibrated PD-19 desalting column (#17-0851-01, GE Healthcare). The flow-through fraction was collected in 70% glycerol, with a final glycerol concentration of 10%. The fraction with the highest virus concentration (maximum turbidity) was pooled, aliquoted, and stored at -80°C. Virus aliquots were thawed and frozen no more than twice.

[0124] Generation and purification of recombinant Ad5 vectors displaying antigens on pIX The Ad5-pIX virus was produced using a strategy different from that used for conventional recombinant Ad5 viruses. The producer cell line was the HEK293(CCS)-shmir-pIX_221-puro cell line (pIX cells) described previously. pIX cells were cultured at 175 cm 2 The cells were seeded into flasks (four flasks per virus) and grown to 70% confluency. To produce recombinant pIX protein, the cells were transfected with the pcDNA3_pIX plasmid, in which pIX was coupled to the recombinant protein via gene fusion. Doxycycline was added to the medium (0.5 μg / mL) prior to transfection, which induced transcription of a pIX-specific shRNA that inhibited the translation of native pIX. The cell culture medium was replaced 18 hours after transfection, and doxycycline was added again. The cells were then infected with a 5 MOI (multiplicity of infection) of each base adenovirus (encoding the recombinant protein of interest). Viral replication was allowed to proceed for 48 hours under normal culture conditions until the viral cytopathic effect was visible. The cells were harvested and pelleted by centrifugation at 750 g for 10 minutes. The pellet was resuspended in PBS containing 0.5% sodium deoxycholate and incubated at RT for 30 minutes to lyse the cells and release the virus. To digest genomic DNA from the producer cell line, 0.2 M MgCl2 and 0.05 mg / mL DNAse I (A3778, AppliChem) were added and incubated at 37°C for 1 hour. Cell debris was removed by centrifugation at 3000 g for 15 minutes, and CsCl was added to the virus-containing supernatant to a final concentration of 1.34 g / mL. The virus was ultracentrifuged in a CsCl gradient as previously described for Ad5 purification. The extracted virus band was transferred to a dialysis membrane (Spectra / Por® Dialysis Membrane, 300 kDa, #131450, Biotech CE Tubing) and dialyzed in PBS overnight at 4°C. Finally, the virus was aliquoted in 10% glycerol and stored at -80°C.

[0125] Virus titration For experimental reproducibility, purified virus was titrated to obtain infectious units per mL (IFU / mL). Flat-bottom 96-well plates with a Δ-treated surface were coated with polylysine for 15 minutes and washed three times with PBS. HEK293 cells were placed in the wells at 5 × 10 in 100 μL of medium. 4 The cells were seeded at a concentration of 10 x 10 cells. The virus was diluted in 10-fold serial dilutions in medium, starting at a dilution of 1:50. 50 μL of dilution factor 5 x 10 4 ~5×10 7 The antibody was added in duplicate to cell suspensions in 96-well plates. Infected cells were incubated for 48 hours under standard cell culture conditions. After removing the medium, the wells were dried at room temperature, and the cells were fixed with cold methanol at -20°C for 10 minutes. The wells were then washed three times with PBS containing 1% bovine serum albumin (BSA). To detect virus-infected cells, anti-Ad5 hexon antibody (1E11, #sc-51746, Santa Cruz Biotechnologies) was added at a dilution of 1:1000 in PBS+BSA and incubated for 1 hour at 37°C. After washing three times with PBS+BSA, a secondary antibody against mouse immunoglobulin conjugated to horseradish peroxidase (HRP) (#P0447, Dako) diluted 1:500 in PBS+BSA was added to the wells for 1 hour at 37°C. Residual antibody was washed out, and viral plaques were visualized using 3,3'-diaminobenzidine (DAB) substrate for 10 min at RT.

[0126] To determine the virus titer, plaques at the appropriate dilution were counted under a microscope at 20x magnification. Several fields were counted in each well until approximately 100 plaques were detected. The final number of IFU per mL was calculated using the following formula:

[0127]

number

[0128]

number

[0129] As an additional quality control, the measured concentration of infectious units per mL (IFU / mL) was compared with viral particle (VP) counts. VP / mL was determined by measuring absorbance at 260 nm using a NanoDrop™ 2000. One absorbance unit is 10 12 This corresponds to a concentration of VP / mL. The ratio of IFU / mL to VP / mL indicates the viability of the virus, with the ideal / typical ratio being 1:30 to 1:100.

[0130] Genomic DNA purification from recombinant Ad5 DNA isolation from the recombinant adenovirus was performed to confirm the correct insertion of the recombinant gene into the adenovirus genome. DNA was extracted using a modified GenElute™ Mammalian Genomic DNA Miniprep Kit (G1N70, Sigma-Aldrich). For this purpose, 100 μL of purified virus sample was mixed with 100 μL of resuspension solution. Proteinase K and lysis solution C were added, followed by incubation at 70°C for 10 minutes. After adding 96% ethanol, the solution was loaded onto the prepared column. Subsequent steps followed the original protocol, except for two washing steps and subsequent drying of the column. Viral DNA was eluted in the elution solution. To ensure the quality of the virus, DNA was sent for sequencing (GENEWIZ UK Ltd.) to rule out mutations in the homologous recombination region. Additionally, viral DNA was digested with restriction enzymes and the correct band size was confirmed by gel electrophoresis.

[0131] VLP generation and purification Virus-like particle (VLP) production and purification was performed primarily to test the functionality of the VLP-encoded vaccine. 7 at a cell density of 175 cm 2 VLP production was examined in Vero cells seeded into culture flasks and allowed to adhere for 2 hours. The cells were then infected with 50 MOI of Ad5 (5 × 10 8 Cells were infected with 100 IFU / flask (100 IFU / flask) for 5 hours. After removing the medium, the cells were washed twice with PBS and incubated in serum-free medium for 48 hours. The supernatant (SN) was centrifuged at 282g for 10 minutes and filtered through a 0.45 μM membrane to remove cellular contaminants. VLPs were purified by pelleting through a 20% sucrose cushion at 82.700g in a Beckman Coulter Ti 70 rotor using an open 32 mL thick-walled tube (#355631, Beckman Coulter). The SN was removed and the pellet resuspended in 100 μL of PBS (160x the original concentration).

[0132] Adv vaccines encoding HERV-K-Gag-p2A-Env wild-type (WT) / ISD mutants were translated into functional proteins capable of generating VLPs, cell lysates were produced from infected cells, and VLPs were purified from cell culture supernatants (SN).

[0133] VLPs were produced and purified using Vero, A549, and HEK293 cell lines. 6 Vero cells, 10 x 10 6 A549 cells or 10 x 10 5 HEK293 cells were cultured in a T175 (175cm) 2 ) flask or T25 (25cm 2) flasks containing the corresponding medium in the case of HEK cells. After 2 hours, cells were infected with different viral vectors encoding our sequences of interest (see Table 2b) using a multiplicity of infection (MOI) of 50 or 20 (HEK293), indicating the number of virions / cell for a given infection. After 5 hours, cells were washed twice with phosphate-buffered saline (PBS) containing 8 g / L NaCl, 0.2 g / L KCl, 1.15 g / L NaHPO 2H O, and 0.2 g / L KHPO at pH 7.4. The medium was then replaced with the corresponding cell culture medium, but without FBS. Cells were incubated for 48 hours within optimal maintenance conditions, or 16 hours when using HEK293 cells.

[0134] Two different procedures were then followed to obtain VLPs from the cell culture: on the one hand, SNs were maintained to generate and analyze VLPs secreted by the cells, and on the other hand, cells were lysed to analyze VLPs contained within the cells.

[0135] For the first step, cells were centrifuged at 12,000 rpm for 10 minutes at 4°C, and the supernatant was filtered through a 0.45 μm membrane (Sartorius, 16555) to remove cellular impurities. 13.5 mL of SN was added dropwise to 3 mL of 20% (w / v) sucrose dissolved in PBS in a 32 mL thick-walled ultracentrifuge tube (Beckman Coulter, 355631). The tube was weighed to an equal volume and placed in a Ti70 rotor (Beckman Coulter, 337922), which was then introduced into an ultracentrifuge set at 82,700 g, 4°C, and 2.5 hours. Upon completion, the SN was carefully removed, and the remaining pellet was resuspended in 100 μL PBS and stored at -20°C.

[0136] The second procedure consisted of a first step of washing with cold PBS. Next, 10 mL of cold PBS was added to the flask, and the cells were mechanically scraped. 4 mL was transferred to a 15 mL conical tube and centrifuged at 12,000 rpm at 4°C for 5 minutes. The SN was discarded, and a mixture containing 1,300 μL of NP40 cell lysis buffer (Invitrogen, FNN0021) with 7 μL / mL protease inhibitor cocktail (Sigma-Aldrich, P8340) was added to each tube. The tubes were then placed on ice for 30 minutes, vortexing every 10 minutes using a Shaker Vortex 3 (IKA). Finally, the tubes were centrifuged at 13,000 rpm at 4°C for 10 minutes to remove cell debris, and the SN was transferred to a new tube and stored at -20°C.

[0137] [Table 4]

[0138] MVA generation and titration The procedures for the production, purification, and titration of MVA were carried out using the guidelines described by Staib et al. 2004. The starting MVA expressing HERV-K Gag or Env protein seed lysates used to perform this experiment were provided by Prof. Dr. Barbara Schnierle (Langen, Germany). 2 Before producing MVA on a large scale using flasks, the amount of virus was increased to 175 cm 2 The cells were expanded on a smaller scale using flasks, in each case seeded with CEF cells.

[0139] In this case, MVA titration was performed in BHK-21 cells. Infected cells were detected using a primary polyclonal rabbit anti-vaccinia virus antibody (BioRad, 9503-2057) diluted 1:1000 and a secondary HRP-conjugated polyclonal goat anti-rabbit Ig antibody (Dako, P0448) diluted 1:500. To determine the titer (IFU / mL), the number of stained foci was counted for diluted samples of approximately 20 to 100 viral foci / well to maximize accuracy.

[0140] Animal experiments Six- to eight-week-old female C57BL / 6, Balb / C, and CD1 mice were obtained from Taconic (C57BL / 6) or Envigo (Balb / C and CD1). Mice were allowed to acclimate for one week before starting experiments. All experiments were performed in accordance with national guidelines and experimental protocols approved by the national animal testing inspectorate (Dyreforsogstilsynet).

[0141] Separation of serum samples To obtain serum samples, approximately 10% of the total blood volume was collected from mice by puncturing the facial vein with a Goldenrod lancet.

[0142] Alternatively, for terminal bleeding (complete bleeding), animals were anesthetized with an intraperitoneal (ip) injection of 1 mg / mL xylazine and 10 mg / mL ketamine in PBS at a dose of 100 μL per 10 g mouse. The maximum amount of blood was collected by puncturing the facial vein, and the mice were then euthanized by cervical dislocation.

[0143] For the HERV-K experiments, mice were anesthetized with isoflurane for whole-blood cardiac puncture. Immediately afterward, mice were placed face-up under a face mask continuously delivering isoflurane, and cardiac puncture was performed using a G27 needle attached to a 1 mL syringe. Approximately 800–1000 μL was collected, and the mice were then euthanized by cervical dislocation.

[0144] Alternatively, mice were anesthetized with isoflurane. They were then examined for involuntary reflexes. Only after confirming that none were present was a maximum blood volume collected from one eye via the orbital sinus. The mice were then immediately euthanized by gentle cervical dislocation.

[0145] Blood samples were stored overnight at 4°C to allow clotting, and blood cells were removed from the serum by centrifugation twice at 800g for 10 minutes. The serum was then stored at -20°C.

[0146] Injection: intravenous, subcutaneous, intramuscular, intraperitoneal Different injection procedures were performed. For intravenous (iv) injection, mice were warmed in a heating chamber to increase superficial venous blood flow. A maximum of 200 μL was injected into the tail vein. In HERV-K-related experiments, 10 6 A volume of 100 μL containing RLZ Gag and Env cells (derived from B. Schnierle) was injected intravenously into mice to induce metastasis to the lungs.

[0147] Subcutaneous (sc) injections into the plantar (fp) were performed under isoflurane anesthesia by injecting 30 μL under the skin of the plantar surface. For HERV-K experiments, this type of injection was used to administer 10 6 RLZ Gag and Env cells (derived from B. Schnierle) were injected (in 100 μL) to grow subcutaneous tumors in mice, establishing a mouse tumor model expressing HERV-K Env.

[0148] For intramuscular (im) injections, a maximum volume of 60 μL was injected into the thigh muscle.

[0149] In the context of HERV-K experiments, this type of injection was primarily used to immunize (prime) and boost mice with the vaccine of interest (see Table 2c below). 50 μL per mouse was used for each vaccination / boost of adenovirus or MVA. Injections were performed in the thigh muscle under isoflurane anesthesia, which provided both analgesia and muscle relaxation.

[0150] [Table 5]

[0151] Intraperitoneal (ip) injections were performed by administering up to 500 μL into the peritoneal cavity.

[0152] vaccination Five different vaccination trials were performed in mice.

[0153] Vaccination timeline I. Balb / C mice were vaccinated with a prime-boost regimen consisting of two DNA vaccinations, followed by one Ad5 vaccination, or either DNA or Ad5 alone. As a control, mice were injected with PBS. Four weeks after Ad5 vaccination, blood samples were collected and spleens were isolated from some mice. Mice were then challenged subcutaneously with CT26 tumor cells in the right flank, and tumor growth was measured.

[0154] Vaccination timeline II. Balb / C mice were subcutaneously challenged with CT26 tumor cells. Mice were vaccinated with Ad5-MelARV either 2 days postchallenge (d.2 pc) or 5 days postchallenge (pre-primed with DNA). In addition, one group was vaccinated at d.2 pc and then received four injections of anti-PD1 antibody as soon as tumors became palpable (d.8 pc). As control groups, mice were injected with PBS or anti-PD1 alone.

[0155] Vaccination timeline III. C57BL / 6 mice were vaccinated with two DNA-MelARV injections in a prime-boost regimen, followed by Ad5 vaccination. Blood samples were collected 3 weeks after the last vaccination, and mice were challenged intravenously with 2 x 10 B16F10-GP cells. The number of metastases in the lungs was determined 2 weeks after challenge.

[0156] Vaccination timeline IV. CD1 mice were first vaccinated with DNA plasmids encoding MelARVgag_p2A_env (DNA-MelARV) or the ISD-mutated version MelARVgag_p2A_env_ISD (DNA-MelARV-ISD). DNA priming was followed by adenovirus vaccination with either Ad5-MelARV or Ad5-MelARV-ISD. Blood samples were taken 4 weeks after vaccination and analyzed for serum antibodies.

[0157] Vaccination timeline V: C57BL / 6 mice were vaccinated twice with either adenovirus Ad5-MelARV_pIX-p15E or Ad5-MelARV. Ad5-GFP was used as a control. Blood samples were then collected, and the mice were challenged intravenously with 2 x 10 B16F10-GP cells. Lungs were isolated 2 weeks postchallenge and analyzed for metastases.

[0158] For DNA vaccination, 50 μg of DNA in 50 μL Tris / PBS (142 mM) was injected intramuscularly. Adenovirus was injected at 2 × 10 in 30 μL PBS. 8 In experiments involving pIX-modified viruses (vaccine timelines IV and V), 10 IFU was injected into the footpad. 10 Virus particles were injected intramuscularly. The lower concentration of pIX virus did not allow injection of small amounts into the sole of the foot.

[0159] Another experiment involved administering anti-PD1 antibody (RMP1-14, #BE0146, BioXCell) to tumor-burdened mice (see "0 Tumor Burden"). Anti-PD1 was administered intraperitoneally with 200 μg antibody in 200 μL PBS. Treatment began 8 days after tumor challenge, when subcutaneously growing tumors became palpable. Mice were injected four times, every four days (8, 12, 16, and 20 days after tumor challenge) (Kim, K., et al., Eradication of metastatic mouse cancers resistant to immune checkpoint blockade by suppression of myeloid-derived cells. Proc Natl Acad Sci USA, 2014. 111(32): pp. 11774-9 and Shindo, Y., et al., Combination immunotherapy with 4-1BB activation and PD-1 blockade enhances antitumor efficacy in a mouse model of subcutaneous tumor. Anticancer Res, 2015. 35(1): pp. 129-36).

[0160] In the HERV-K experiments, Adv and / or MVA booster immunizations were administered approximately 4 or 8 weeks after the primary immunization (day 0) with Adv or DNA vaccine. Blood samples were collected both before and after the primary vaccination (day 14). Mice were also bled 14 and 28 days after the MVA / Adv / DNA booster immunization. Blood samples were used to analyze the humoral response (antibody production against HERV-K Env) of vaccinated mice. Additionally, mice were euthanized 10 days after the MVA booster immunization to examine the cellular immune response (generation of CD8+ T HERV-K Env-specific T cells).

[0161] To test the therapeutic efficacy of the novel vaccination strategy, only a single dose of vaccine was given 10 days after tumor challenge.

[0162] Tumor burden To assess in vivo metastasis of B16F10-GP cells, cultured cells were washed three times with PBS and detached by incubation in Versene at 37°C for 15 minutes. Cells were then centrifuged at 282g, washed with PBS, and diluted to a concentration of 2 x 10^6 cells / mL in PBS. 2 x 10^5 cells in 100 μL PBS were injected intravenously into the tail vein of mice, resulting in tumor metastasis in the lungs. The challenged mice were euthanized 14 days later. Lungs were isolated and fixed overnight in a solution of 2% paraformaldehyde (PFA) in PBS and then stored in PBS at 4°C. Metastases were counted under a dissecting microscope as black nodules on the lung surface. Samples were examined blinded, and metastases were counted by at least two individuals. To analyze the primary growth of CT26 tumors, CT26 cells were prepared as described for B16F10-GP cells, and subcutaneous injection of 5 x 10^5 cells in PBS into the right thigh resulted in the formation of tumors at the injection site. Tumor size was measured three times a week in length and width. Tumor burden was calculated as length x width. 2 The IR was determined as × 0.5236 (Janik, P., et al., The Effect of Estrone-Progesterone Treatment on Cell Proliferation Kinetics of Hormone-dependent GR Mouse Mammary Tumors. Cancer Research, 1975. 35(12): pp. 3698-3704). Mice were euthanized when tumors exceeded 16 mm on any side, necrotic wounds developed, or mouse mobility was significantly reduced. During tumor measurement, the different vaccination groups were blinded to prevent biased evaluation.

[0163] In addition to CT26 challenge, Balb / C mice were injected with 2.5 x 10^4 4T1-Luc cells in 100 μL PBS into the thoracic mammary fat pad. To visualize tumor formation after 6 weeks, mice were injected intraperitoneally with luciferin (1.5 mg per 10 g mouse) and imaged 12 minutes after injection using an IVIS Spectrum in vivo imaging system. IVIS imaging was performed by Andreea-Cornelia Udrea and Melanie Schwerdtfeger.

[0164] To analyze tumor growth and metastasis of RLZ Gag and Env cells in vivo, cells were grown to 60–80% confluency. Once the desired confluency was reached, RLZ cells were washed three times with PBS, followed by the addition of Versene at 37°C for 15 minutes to detach the cells. Cells were then spun down at 282 g, washed with PBS, and finally diluted to 107 cells / mL in PBS. Each mouse received 106 cells / 100 μL intravenously for lung metastasis and subcutaneously for subcutaneous tumors. To assess lung metastasis, mice were weighed at 0, 7, and 14 days and every two days thereafter. Mice were euthanized if they lost approximately 15–20% of their body weight within several days. The termination endpoint was set at 40 days after tumor challenge. Mice bearing subcutaneous tumors were examined at the same time points as intravenously challenged mice and were euthanized when tumors exceeded 16 mm in diameter.

[0165] Both subcutaneous tumors and lungs were isolated and immersed in 4% paraformaldehyde (PFA) and 0.01 mol / L phosphate buffer, pH 7.2 (Rigshospitalet, Copenhagen, Denmark) and stored at 4° C. Samples were processed, and tissues were analyzed for HERV-K Env-specific staining using high-titer serum from vaccinated mice.

[0166] Western blotting For detection of pIX-protein, cell lysates (approximately 10 μg) or purified viruses (10 10The 1000 virions (1000 virions) were mixed with 6x SDS loading buffer containing DDT and heated to 95°C for 5 minutes. To demonstrate MelARV protein expression, cell lysate (5 μg), cell supernatant (15 μg), and purified VLPs (approximately 2 μg) were similarly mixed with DDT-containing loading buffer, but the samples were not heated. This mixture was loaded onto a NuPAGE™ 4-12% Bis-Tris Protein Gel (#NP0322, Thermo Fisher Scientific) and run at 150 V in MOPS buffer for 1 hour. The protein content in the gel was transferred to a nitrocellulose membrane in a wet transfer system at 30 V for 1 hour.

[0167] The membrane was then blocked with 5% nonfat milk in Tris-buffered saline + Tween 20 (TBS-T) for 1 hour. The membrane was then washed three times with TBS-T for 10 minutes on a shaker and incubated overnight at 4°C with diluted primary antibodies (Table 3) (in TBS-T + 3% nonfat milk). After the following three washing steps, HRP-conjugated secondary antibodies in TBS-T were added, and the membrane was incubated for 1 hour at RT. Unbound secondary antibodies were washed out, and target proteins were visualized on an ImageQuant LAS 4000 using LumiGLO Reserve Chemiluminescent Substrate (54-61-00 or 54-71-02).

[0168] [Table 6-1] [Table 6-2]

[0169] In HERV-K-related experiments, VLP expression at the protein level was analyzed via WB technique. To ensure equal loading of samples, the protein concentrations of both VLPs (SN) and cell lysates were measured using the Pierce™ Bicinchoninic Acid (BCA) Protein Assay Kit (Thermo Fisher Scientific, 23225) according to the manufacturer's instructions. A 6x sodium dodecyl sulfate (SDS) loading buffer containing dithiothreitol (DTT) was added to different samples, which were then placed in a 95°C block heater SBH130DC (Stuart) for 5 minutes. Five micrograms of protein and 7 μL of RunBlue™ Prestained Marker (Expedeon, NXA05160) were then loaded onto a NuPAGE™ 4-12% Bis-Tris Protein Gel (Thermo Fisher Scientific, NP0322) along with NuPAGE™ MOPS SDS Running Buffer (Thermo Fisher Scientific, NP0001). Samples were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) at 180 V for 45 minutes.

[0170] The samples were then transferred to a 0.45 μm nitrocellulose blotting membrane (Bio-Rad, 1620115) for 45 min at 30 V. For this step, a transfer buffer containing 20% ​​ethanol (3.75 g / L Trizma® base, 18.1 g / L glycine, pH 8.5) was used.

[0171] To prevent nonspecific binding, the membranes were blocked with 5% (w / v) nonfat dry milk in Tris-buffered saline with Tween (TBS-T) (6.06 g / L Trizma® base, 8.76 g / L NaCl, 0.25% (v / v) Tween-20, pH 7.6) for 1 h at room temperature (RT). Afterwards, the membranes were washed with TBS-T for 10 min and incubated overnight (o / n) with the corresponding primary antibodies (see Table 3a) in 3% (w / v) nonfat dry milk in TBS-T on a CERTOMAT® MO II shaker (Sartorius) at 4°C.

[0172] [Table 7]

[0173] The membrane was then washed three times for 10 min with TBS-T and then incubated with the corresponding secondary antibody (see Table 3b) diluted in TBS-T for 1 h at RT.

[0174] [Table 8]

[0175] The membrane was then washed three times with TBS-T (10 min each time). Peroxidase chemiluminescent substrate (KPL, 54-16-00) was used for protein detection on an ImageQuant Las 4000 camera (GE Healthcare Life Sciences).

[0176] Enzyme-linked immunosorbent assay (ELISA) For the detection of MelARV-specific antibodies in vaccinated mice, a peptide of MelARV Env subunit p15E conjugated to BSA was purchased from Schafer-N (Copenhagen, Denmark).

[0177] [Table 9]

[0178] MaxiSorp flat-bottom plates (Thermo Fisher) were coated overnight at 4°C with 100 μL of peptide solution (2 μg / mL in PBS) per well and then washed twice with wash buffer (PBS + 2.07% NaCl + 0.1% Tween-20). The wells were blocked with dilution buffer (PBS + 2.07% NaCl + 0.1% Tween-20) for 2 hours at 37°C, washed once with wash buffer, and incubated with diluted mouse serum (1:50 in dilution buffer) for 3 hours at 37°C. After two washes, the peptide-bound serum antibodies were incubated with HRP-conjugated goat anti-mouse immunoglobulin antibody (Dako, P0447) at a 1:2000 dilution for 2 hours at 37°C. After two additional wash steps, 100 μL of TMB PLUS2 (Kem-En-Tec Diagnostics, 4395A) was added and incubated for 8 minutes at room temperature. The reaction was terminated with 100 μL of 0.2 M H 2 SO 4 and quantified by measuring the optical density at 450 nm.

[0179] Detection of Ad5-specific antibodies in mouse serum was performed by incubating ELISA plates with 5 x 10 cells of heat-inactivated Ad5. 9 The assay was performed by coating with 1000 viral particles / mL (30 min, 56°C). The assay was performed as described above, but with 1 h blocking at RT and a shorter incubation time for antibody binding. Primary antibodies were mouse serum diluted in a 1:2 serial dilution starting at 1:200.

[0180] Detection of MelARV proteins in cell lysates, supernatants, and purified VLPs from infected Vero cells was achieved by coating ELISA plates with individual samples. Cell lysates were diluted 1:2 in PBS (100 μL), supernatants were applied undiluted (100 μL), and purified VLPs were diluted 1:25 in PBS (50 μL). Detection was achieved using the same procedure as described above, with anti-p2A (1:500), MM2-9B6 (1:100), 4F5 (1:100), and 19F8 (1:100) as primary antibodies and the secondary antibodies listed in Table 3.

[0181] Flow cytometry In the HERV-K-related experiments, FACS was used to detect both extracellular and intracellular markers of activated immune cells from vaccinated mice and the presence of HERV-K Env protein on the surface of infected A549 cells. The machine used for cell sorting was a BD LSR II flow cytometer (BD Biosciences).

[0182] The following buffers were used for FACS:

[0183] [Table 10]

[0184] Extracellular staining with serum antibodies In non-HERV-K experiments, flow cytometry was performed to detect binding of serum antibodies to cancer cells. B16F10-GP or CT26 cells were resuspended and plated at 4 × 10 cells per well in a round-bottom 96-well plate. 5Cells were seeded at 1000 x g. The plate was centrifuged at 784 x g for 3 min (4 °C) to fix the cells to the bottom of the wells. The medium was removed by flicking the plate upside down, and the cells were resuspended in 50 µL of fluorescence-activated cell sorting (FACS) medium (PBS + 1% BSA + 0.1% NaN3) containing mouse serum at a 1:50 dilution. After 20 min of incubation at 4 °C, the plate was centrifuged at 784 x g for 3 min (4 °C), and the medium was removed. The cells were washed twice with 200 µL of wash medium (PBS + 0.1% NaN3) and resuspended in 50 µL of FACS medium containing a 1:100 dilution of fluorescently labeled secondary antibody against mouse immunoglobulin G (IgG) (goat anti-mouse IgG_APC, #405308, Biolegend). Cells were incubated for 20 minutes at 4° C., washed twice with wash buffer, and fixed in 200 μL of PFA solution (1% in PBS) for 15 minutes at 4° C. Cells were resuspended twice in FACS medium and analyzed for fluorescence on a BD LSR II Flow Cytometer.

[0185] Detection of MelARV Env on the surface of infected Vero cells was performed using the same protocol with monoclonal antibodies against different epitopes (Table 5). The secondary antibodies were anti-mouse IgG_APC (1:100) or goat anti-mouse IgM heavy chain_RPE (1:100, A10689, Invitrogen).

[0186] Furthermore, this technique was implemented to characterize a new vaccine strategy based on Ad19 vectors encoding HERV-K wild-type and HERV-K ISD mutant transgenes (Sirion) and to compare the use of different adenoviral vectors (Ad19 vs. Ad5). Surface staining was used to detect the presence of HERV-K Env protein on the surface of infected A549 cells by flow cytometry.

[0187] 3×10 6A549 cells were seeded into 75 cm flasks in 15 mL of Ham's F-12K medium and incubated for 2 hours at 37° C. Each flask was infected with the following viruses at an MOI of 50 (1.5×10 IFU / flask):

[0188] Ad5-(TetO)-CMV-SIVgag_p2A_HERV-K108env_P2TS Ad19a(II)-(TetO)-CMV-ISD mutant_MelARV-P2TS Ad19a(II)-(TetO)-CMV-coHERV-K-P2TS from Sirion Sirion's Ad19a(II)-(TetO)-CMV-ISD mutant_coHERV-K-P2TS

[0189] The virus was then incubated for 5 hours at 37° C., after which the medium was replaced with Ham's F-12K FBS-free medium. The cells were then incubated at 37° C. for 48 hours.

[0190] Cells were maintained on ice inside the LAF bench. The medium was aspirated, and the cells were carefully washed with cold PBS. After scraping with cold PBS, the cells were separated by centrifugation (4°C, 784g, 3 min). The cells were resuspended in PBS and distributed into a round-bottom 96-well plate (Thermo Fisher Scientific, 163320). The plate was centrifuged (4°C, 784g, 3 min), and the SN was removed by flicking the plate. The cells were resuspended in 50 μL of FACS buffer containing 2 μg / mL of mouse monoclonal (IgG) primary antibody (Austral Biologicals, HERM-1811-5) directed against the p15E™ domain of the HERV-K Env protein for 20 min at 4°C. The cells were then washed with FACS wash buffer (150 μL first volume followed by 200 μL) and centrifuged three times (4°C, 784g, 3 min). The plate was incubated with 100 μL of FACS buffer, which had previously contained a 1:100 dilution of goat anti-mouse IgG APC secondary antibody (BioLegend, 405308). The plate was incubated for 20 minutes at 4°C in the dark. The cells were centrifuged (4°C, 784 g, 3 minutes) and washed three times with 200 μL of FACS wash buffer. The plate was then incubated in 200 μL of 1% (w / v) paraformaldehyde (PFA) (Rigshospitalet, Copenhagen, Denmark) for 15 minutes at 4°C in the dark. The plate was then centrifuged (4°C, 784 g, 3 minutes), resuspended in 100 μL of FACS buffer, and centrifuged again (4°C, 784 g, 3 minutes). The plate was finally resuspended in 200 μL and stored overnight in the dark at 4°C. The next day, cell fluorescence was analyzed using a flow cytometer BD LSR II, and data were processed and analyzed using FlowJo 10 (FlowJo LLC).

[0191] Intracellular staining (ICS) of stimulated non-cells Mice were euthanized 3-4 weeks after vaccination, and spleens were isolated. The excised spleens were transferred to HANKS BSS and mashed through a sterile net to obtain a single-cell suspension. After centrifugation and resuspension in complete RPMI, the splenocyte concentration was determined, and the cells were diluted to the required concentration.

[0192] Splenocytes were cultured in a round-bottom 96-well plate at 2.5 x 10 6 Cells were plated at 100 cells / well. Cells were centrifuged at 784 g for 3 minutes and resuspended in complete RPMI (+50 μM 2-mercaptoethanol) containing 3 μM monensin (pathway inhibitor) and 1 μg / mL peptide (AH1), whereas negative controls received no peptide. Cells were then incubated at 37°C for 5 hours. After washing the cells in FACS medium (PBS + 1% BSA + 0.1% NaN3 + 3 μM monensin), the cells were incubated with fluorescently labeled surface antibodies (anti-CD4, anti-CD8, anti-CD44, anti-B220) diluted 1:100 in FACS medium for 20 minutes at 4°C. Cells were washed twice with PBS + 3 μM monensin and fixed in 1% PFA for 15 minutes at 4°C. After washing in FACS medium, the cells were permeabilized with 0.5% saponin in PBS for 10 minutes at room temperature. Intracellular antibodies (anti-IFNγ, anti-TNFα) were added at a 1:10 dilution in PBS + 0.5% saponin and incubated for 20 minutes at 4°C. Cells were washed twice and finally resuspended in PBS + 1% BSA + 0.1% NaN3. Cell fluorescence was analyzed on a BD LSR II Flow cytometer. Analysis of the flow cytometry data is shown in accompanying Figure 5.

[0193] [Table 11-1] [Table 11-2] [Table 11-3]

[0194] In a HERV-K-related experiment, splenocyte ICS was performed to evaluate specific cellular responses from vaccinated mice. To enable this experiment, different strong binding (SB) HERV-K peptides consisting of 8 to 10 amino acids from both C57BL / 6 and BALB / c mouse strains were previously tested for their ability to stimulate CD8+ T cells from HERV-K-vaccinated mice. Only a BALB / c 10-mer peptide (TYHMVSGMSL, SEQ ID NO: 47) at position 192 of the HERV-K Env sequence conferred a response. Therefore, this peptide, designated P-HKE, was used together with an improved Ad19 vaccine containing a mutation in the Env ISD to stimulate splenocytes from BALB / c mice immunized with Ad5 and Ad19 vectors encoding the HERV-K Env.

[0195] [Table 12-1] [Table 12-2]

[0196] To compare the efficacy of different vaccines containing different vectors and insert improvement strategies, Ad5 and Ad19 HERV-K / ISD mutant vaccinated (primed) mice were used in this experiment. The mice were euthanized 10 days after the MVA vector booster immunization, and the spleens were collected in 5 mL of Hank's BSS medium. To obtain a single cell suspension, the spleens were mashed with a sterile Corning® 70 μm cell strainer (Sigma-Aldrich, CLS431751). The cells were then counted and seeded at the desired number of cells per well, providing the total number of cells per spleen for subsequent calculation of the absolute number of IFNγ CD8+ and IFNγ CD4+ T cells per spleen.

[0197] Approximately 3 x 10 6Cells were seeded at 1 / well into a round-bottom 96-well plate, centrifuged (4°C, 784 g, 3 min), and suspended in RPMI medium. The previously described HERV-K Env 10-mer peptide, designated P-HKE, TYHMVSGMSL (SEQ ID NO: 47), was dissolved in dimethyl sulfoxide (DMSO) to a concentration of 400 ng / μL. It was then redissolved in PBS to a concentration of 100 ng / μL, and finally, RPMI was added to the previous dilution to obtain a concentration of 6.67 ng / μL. Prior to adding the P-HKE peptide, 50 μL of the protein transport inhibitor monensin (3 μM) was added to the wells to prevent cytokines from exiting the cells. Additionally, 30 μL / well of the aforementioned P-HKE peptide was added to stimulated wells to induce T cell cytokine production. The rest of the wells did not receive any peptide but only DMSO at the same concentration as the stimulated samples and served as negative controls. Cells were incubated at 37°C for 5 hours.

[0198] After the incubation period, the cells were centrifuged (4°C, 784 g, 3 min) and washed twice with 100 μL of FACS buffer containing monensin (3 μM). Surface antibodies (PerCP / Cy5.5-CD8, FITC-CD4, Pacific Blue™-B220, APC / Cy7-CD44) were diluted 1:100 in FACS buffer containing monensin (3 μM). Splenocytes were resuspended in 50 μL of the previous solution, and 50 μL of FACS / monensin (3 μM) containing 1:10 diluted antibodies, i.e., PerCP / Cy5.5-CD8, FITC-CD4, Pacific Blue™-CD8, APC / Cy7-CD8, APC-CD8, and PE / Cy7-CD8, was used for compensation. The plate was incubated at 4°C in the dark for 20 min. Wells were washed twice with 100 μL of 3 μM monnexin in PBS. Next, 100 μL of 3 μM PBS / monnexin was added along with 100 μL of 2% PFA, and cells were fixed in the dark at 4°C. Cells were again washed twice with FACS / monnexin (3 μM) and resuspended in 150 μL of 0.5% saponin in OBS for 10 min at 20°C (in the dark). Once cells were permeabilized, intracellular antibodies (APC-IFNγ, PE / Cy7-TNFα) were diluted 1:100 in 0.5% saponin / PBS, and 50 μL was added to the wells. The plates were incubated at 4°C for 10 min in the dark. Cells were washed with PBS containing 1% BSA and 0.1% NaN3, and finally resuspended in 200 μL of the same buffer. The plates were kept at 4°C overnight.

[0199] Additionally, intracellular staining of A549 transfected cells was performed to confirm the presence of HERV-K Env protein inside the cells. In this case, production (and not secretion to the cell membrane) was assessed. The latter protocol was followed by an additional 10-minute incubation step with 150 μL of 0.5% (w / v) saponin (Sigma-Aldrich, 47036) diluted in PBS at 4°C in the dark. This extra step is required to permeabilize the cell membrane. Antibodies were also diluted in 0.5% saponin.

[0200] Gating Strategy FlowJo10 (FlowJo LLC) was used to analyze data from extracellular and IC FACS staining (see Figure 27). Cells were first plotted and gated in forward scatter (FSC)-H and FSC-A. This gate was used to separate the lymphocyte population in a plot of side scatter (SSC)-A and FSC-A. The latter population was gated on CD8+ CD4- cells and then on CD8+ B220 cells to obtain the CD8+ T cell population, removing both CD4+ T-cells and B cells (B220 marker) from the analysis (Coffman & Wei, 2001). Next, cells were gated for CD8+ CD44+ T cells to obtain only activated CD8+ T cells. These were then further gated for IFNγ+ CD44+ cells, both of which are markers expressed as a consequence of T cell activation. Furthermore, IFNγ is known to be a more sensitive marker for activated CD8+ T cells compared with TNFα cytokines (Badovinac & Harty 2000), (Kristensen et al. 2004). In addition, CD8+ CD44+ T cells were gated for IFNγ+ TNFα+ cells because CD4+ T cells producing multiple cytokines are known to have higher levels of activation and memory activity (Kannanganat et al. 2007).

[0201] To estimate the absolute number of IFNγ+ CD44+ B220- CD8+ T cells, the percentage of IFNγ+ CD44+ B220- CD8+ T cells among lymphocytes was multiplied by the number of lymphocytes per spleen. Additionally, the percentage of IFNγ+ CD8+ double-positive (IFNγ+ TNFα+) cells was calculated by dividing IFNγ+ TNFα+ cells by the sum of IFNγ+ TNFα+ and IFNγ+ TNFα- cells.

[0202] Enzyme-linked ImmunoSpot (ELISPOT) ELISPOT assays were performed to detect antigen-specific T cells. The peptide used in this experiment was AH1(SPSYVYHQF), a known H2-Ld-restricted T cell epitope in Balb / C mice located within the MelARV Env subunit gp70 (Huang, AY, et al., The immunodominant major histocompatibility complex class I-restricted antigen of a murine colon tumor derived from an endogenous retroviral gene product. Proc Natl Acad Sci USA, 1996. 93(18):9730-5).

[0203] Splenocytes from vaccinated mice were prepared as described for ICS.

[0204] The assay was performed using a mouse IFN-γ T cell ELISPOT kit (CT317-PR5, U-CyTech). Briefly, the membrane of a polyvinylidene difluoride (PVDF) 96-well plate (MSIP S4510, Millipore) was activated with 70% ethanol and then coated overnight with anti-mouse IFN-γ antibody. After removing the coating antibody and blocking the membrane, splenocytes were seeded at 2 × 10^5 cells / well in complete RPMI medium containing 1 μg / mL AH1. As a control, splenocytes were either left unstimulated or stimulated with the potent T cell activator concanavalin A (ConA) (2 μg / mL). After 48 h of incubation under normal cell culture conditions, cells were removed, the wells were washed, and then incubated with a biotinylated detection antibody targeting IFN-γ. Streptavidin-HRP complex was added, and IFN-γ spots were visualized using AEC substrate solution. The spots were counted using a CTL ImmunoSpot analyzer.

[0205] Positive control (control serum LEV76) The positive control serum LEV76 was used as a standard for flow cytometry and ELISA analysis of mouse serum samples. The LEV76 serum originated from an early pilot study in which C57BL / 6 mice were vaccinated against MelARV Env and showed protection from B16F10-GP lung metastasis. Therefore, the antibody response in this serum corresponded to a level potentially capable of protecting against tumor burden and therefore served as a reference for successful antibody responses. In addition, the use of the LEV76 control serum allowed for comparison between different experiments.

[0206] statistical analysis All statistical analyses were performed using GraphPad Prism software (version 5.03). Groups were compared using an unpaired two-tailed Mann-Whitney test. Significance is indicated by an asterisk. * (P ≤ 0.05), ** (P ≤ 0.01), *** (P≦0.001). When different groups of vaccinated mice were compared, the results were presented as the mean of each group together with the standard error of the mean (SEM).

[0207] Mouse survival curves were compared using the Kaplan-Meier approximation. This test measures the fraction of subjects surviving over time after a given treatment. Significant results are marked with an asterisk ( * ) and * (P ≤ 0.05), ** (P ≤ 0.01), *** (P≦0.001).

[0208] To assess correlation between responses, Spearman correction was used, followed by adjustment of p values ​​by the Holm-Sidak method.

[0209] Example 1 Mutations in vaccine-encoded immunosuppressive domains (ISDs) As a first improvement strategy, two point mutations were introduced into the sequence of MelARV Env to inactivate the immunosuppressive domain (ISD) (Figure 3). These specific mutations were previously examined and analyzed in murine leukemia viruses by Schlecht-Louf et al. (Schlecht-Louf, G., et al., Retroviral infection in vivo requires an immune escape virulence factor encoded in the envelope protein of oncoretroviruses. Proc Natl Acad Sci USA, 2010. 107(8):3782-3787). The virus encoding this modified version of MelARV Env is called Ad5-MelARV-ISD.

[0210] Effect of Ad5-MelARV-ISD on antibody responses in CD1 mice Outbred CD1 mice were primed with DNA-MelARV or DNA-MelARV-ISD and then boosted with either AD5-MelARV or Ad5-MelARV-ISD according to vaccination timeline IV. Four weeks after adenovirus vaccination, blood samples were collected and analyzed by ELISA.

[0211] As shown in Figure 7A, p15E-specific antibodies increased in mice vaccinated with Ad5-MelARV-ISD. In particular, the combination of DNA-MelARV-ISD and Ad5-MelARV-ISD (bar D) produced a high antibody response comparable to that of the LEV76 control serum.

[0212] In addition, vaccination with Ad5-MelARV (bars A and C) and Ad5-MelARV-ISD (bars B and D) increased the levels of tumor cell-specific antibodies compared with the GFP control (bar E) (Fig. 7B). However, Ad5-MelARV-ISD induced significantly lower levels of tumor-associated antibodies than Ad5-MelARV (bars A vs. B, and bar C vs. D, but not significantly).

[0213] Both p15E- and B16F10-GP-binding antibody levels suggested that priming with DNA-MelARV-ISD generally elevated antibody responses compared with DNA-MelARV-primed mice, although these results were not significant.

[0214] Example 2 Effect of Ad5-MelARV-ISD on antibody responses and metastasis in C57BL / 6 mice C57BL / 6 mice were vaccinated and challenged according to vaccination timeline III. Mice received either DNA-MelARV or DNA-MelARV-ISD followed by individual adenoviruses. Analysis of antibody responses revealed that MelARV-ISD slightly elevated the levels of B16F10-GP cell-specific antibodies (Figure 8A). However, the elevation was not significant and was slightly above background in PBS-inoculated mice. As shown in Figure 8B, no effect on p15E-specific antibodies was observed. Corresponding to tumor cell-bound antibodies, metastasis was slightly reduced in MelARV-ISD-inoculated mice, but the difference was not significant (Figure 8C).

[0215] Example 3 Effect of Ad5-MelARV-ISD on T cell responses in Balb / C mice In addition to antibody responses, the effect of Ad5-MelARV-ISD on T cell priming and activation was analyzed. Both ELISPOT (Figure 9) and ICS (Figure 10) showed elevated levels of AH1-specific T cells in mice vaccinated with Ad5-MelARV-ISD compared to Ad5-MelARV. As observed by ICS, double-positive IFNγ + TNF-alpha + CD8 + T cells were significantly elevated in mice inoculated with Ad5-MelARV-ISD compared to untreated mice. +The integrated geometric mean (IGM) of cells shows a significant difference compared to untreated Ad5-MelARV. The IGM combines the number of positive cells with the mean fluorescence intensity and therefore also takes into account the amount of activated immune cells. The IGM of TNFα remained insignificant (data not shown).

[0216] Example 4 Effect of Ad5-MelARV / Ad5-MelARV-ISD on immunosuppression To analyze the mechanism underlying the increased immune response of Ad5-MelARV-ISD, vaccine-induced immunosuppression was analyzed. The same mouse sera as in Figure 7 from mice vaccinated with Ad5-MelARV or Ad5-MelARV-ISD were analyzed for immune responses to the viral vector by ELISA. The ISD-inactivated MelARV Env vaccine (Ad5-MelARV-ISD) showed significantly increased titers of Ad5-binding antibodies compared to the naive version of MelARV Env (Ad5-MelARV with a functional ISD).

[0217] Example 5 Antigen presentation on the capsid protein pIX of adenovirus vectors In an attempt to boost protective antibody responses, p15E was linked to the adenovirus capsid protein pIX in a previously tested adenovirus vaccine. The different constructs tested are shown in Figure 12. Native p15E (minus the transmembrane subunit and cytoplasmic tail) was added to either pIX (1) or, alternatively, an ISD-mutated version (2). In addition, truncated p15E variants with (3) or without (4) an additional cysteine ​​were tested relative to the ISD. The core of the viral vectors was consistent with the p15E presented: Ad5-MelARV for pIX-p15E, Ad5-MelARV with and without pIX-p15E-truncated-C for pIX-p15E-ISD, and Ad5-MelARV-ISD.

[0218] Characterization of Ad5 vectors displaying p15E on the capsid protein pIX. The new pIX plasmid constructs (pcDNA3-pIX-tag linker-xxx, xxx = p15E antigen) were tested for correct expression of recombinant pIX by transfecting HEK293 cells. Lysates of transfected cells were analyzed by Western blotting using the anti-pIX antibody in Figure 13A. All four constructs showed expression of recombinant pIX with a lower band than expected for the truncated p15E version (lines 3 and 4). GFP linked to pIX was used as a positive control with a higher band of approximately 50 kDa. To confirm the integration of recombinant pIX into the viral vector, purified virus was analyzed by Western blotting using the anti-pIX antibody in Figure 13B. All constructs showed expression of recombinant pIX next to the intact pIX band (approximately 10 kDa). Unmodified Ad5

number

[0219] [Table 13]

[0220] Example 6 Analysis of antibody responses induced by pIX-modified viruses in CD1 mice CD1 mice were vaccinated with a DNA prime (DNA-MelARV or DNA-MelARV-ISD) followed by an adenovirus boost (regular virus vs. pIX-modified) according to vaccination timeline IX. Sera were analyzed for p15E-specific antibodies by ELISA (Figure 14A). Because the p15E peptide sequence used in ELISA was not included in the truncated pIX-modified plates, only Ad5-MelARV_pIX-p15E and Ad5-MelARV-ISD_pIX-p15E-ISD could be assessed in this setting. In most cases, presentation of p15E on pIX increased the levels of p15E-specific antibodies (A vs. B, C vs. D, E vs. F). However, in these comparisons, the only significant difference was observed for DNA-MelARV + Ad5-MelARV (A vs. B). In the case of DNA-MelARV-ISD + Ad5-MelARV-ISD (G vs. H), presentation of pIX-p15E-ISD had a worse effect and significantly reduced antibody responses compared to the unmodified vaccine. In addition, serum antibody binding to B16F10-GP cells was analyzed (Figure 14B). Presentation of intact p15E on pIX did not affect antibody responses to tumor cells. Meanwhile, Ad5-MelARV-ISD_pIX-p15E-ISD was able to reduce the loss of B16F10-GP-specific antibodies reduced by ISD-mutated MelARV Env (compare Figure 7).

[0221] Example 7 Effect of Ad5-MelARV_pIX-p15E on antibody responses and metastasis in C57BL / 6 mice The pIX-modified virus Ad5-MelARV_pIX-p15E was tested in a pilot study for antibody responses and protection from metastases in C57BL / 6 mice. Mice were vaccinated and challenged twice according to vaccination timeline V. As shown in Figures 15A and 15B, the vaccine did not significantly elevate antibody responses to either B16F10-GP cells (15A) or p15E (Figure 15B). Furthermore, the number of metastases was not significantly reduced by vaccination (Figure 15C). However, no correlation was detected between tumor cell-specific antibodies and metastasis counts (Figure 15D), whereas a significant negative correlation was observed between the level of p15E-specific antibodies and the number of metastases (Figure 15E).

[0222] Example 8 In an attempt to improve the display of MelARV Env on VLPs not only in quantity but also in quality (in a more native conformation), functional domains were applied to the native sequence. These modifications were applied not only to the full-length MelARV Env but also to p15E alone (Figure 16). The modifications included a signal peptide from Gaussia luciferase (LucSP), a transmembrane domain and cytoplasmic tail from influenza A virus hemagglutinin H3N2 (HA-TMCT), and a trimerization sequence (GCN4) (Figure 16). The chimeric Env or p15E protein was co-encoded with the Gag protein of SIV.

[0223] Characterization of vaccines encoding chimeric MelARV Env or p15E Although the modified vaccines were not tested in mice, expression from the adenovirus was examined by flow cytometry in infected Vero cells (Figure 17). This experiment not only demonstrates protein expression but also characterizes some of the anti-MelARV Env antibodies for the protein's target epitopes. Both 19F8 (Figure 17A) and 4F5 (Figure 17B) showed significantly higher binding to the modified versions of MelARV Env and p15E compared to the unmodified vaccines (Ad5-MelARV and Ad5-MelARV-ISD). Since binding to Ad5-LucSP_GCN4_p15E_HA-TMCT was similarly observed, this experiment indicates that both antibodies bind the transmembrane subunit p15E. Furthermore, no binding of 19F8 to Ad5-MelARV-ISD-infected cells was observed, whereas a clear signal was detected for 4F5, confirming ISD as the target epitope for 19F8. Although none of the MM2 antibodies showed binding to the p15E construct, all three antibodies demonstrated directivity to the surface subunit gp70. MM2-9B6 (Fig. 17C) and MM2-3C6 (Fig. 17D) showed similar characteristics, with equally strong antibody binding to Ad5-MelARV- and Ad5-LucSP_MelARV_HA-TMCT-infected cells. Meanwhile, Ad5-MelARV-ISD-infected cells showed very low antibody binding. MM2-9A3 (Fig. 17E) showed similar characteristics, with the exception that Ad5-MelARV-infected cells showed lower antibody binding than Ad5-LucSP_MelARV_HA-TMCT-infected cells.

[0224] The new constructs were also tested for their ability to produce and present target proteins upon infection. Lysates of infected Vero cells and purified VLPs were analyzed by Western blot (Figure 18). Anti-p2A antibody binding (Figure 18A) revealed bands indicative of MelARV Gag (lines 1 and 2) and SIV Gag (lines 3 and 4) expression in both the lysate and VLP. As shown in Figure 18B, p15E (bound by 4F5) was detected only in the lysate of Ad5-MelARV-infected cells (line 1), with a lower band of approximately 20 kDa corresponding to p15E and a higher band at 70 kD, indicating full-length Env (gp70 + p15E). Full-length Env was also detectable for Ad5-MelARV-ISD (line 2), whereas no single p15E band was visible. Only in Ad5-MelARV-derived VLPs were p15E and full-length Env detectable. Other weak bands were present for the different constructs, but it is unclear to which proteins they correspond.

[0225] Similar results to those for 4F5 are shown in Figure 18C, in which gp70 was visualized by MM2-9B6. Only VLPs derived from Ad5-MelARV (line 1) displayed gp70 that was detected by MM2-9B6.

[0226] In addition to cell lysates, the supernatants of infected cells were analyzed to determine whether the protein was secreted (Fig. 18D,E). p15E (bound by 4F5) in the supernatant (Fig. 18D) was detectable only for Ad5-MelARV (line 1), which may be due to VLPs in the supernatant. MM2-9B6 (Fig. E), on the other hand, revealed that Ad5-MelARV-ISD-infected cells (line 2) released large amounts of gp70, detected as complexes of different sizes (Fig. 18E). In contrast, Ad5-MelARV induced the release of small amounts of gp70 (line 1).

[0227] None of the new modified MelARV Env proteins (lines 3 and 4) showed protein expression in lysates, supernatants, or purified VLPs, despite detectable expression on the cell surface under non-denaturing conditions.

[0228] Due to the absence of bands for the new constructs in Western blot analysis, the assumption was that the synthesized proteins were unable to bind to nitrocellulose membranes. Therefore, ELISA analysis was performed using ELISA plates coated with cell lysates, supernatants, or VLPs (Figure 19). As expected, Gag protein detected by anti-p2A was present in all samples (Figure 19A). In contrast, MelARV Env gp70 (bound by MM2-9B6) was detected only in the VLPs of AD5-MelARV-infected cells (line 1), but not in cells infected with the modified MelARV virus (Figure 19B). Similar results were observed for p15E expression (bound by 4F5 and 19F8) (Figures 19C, D). Ad5-MelARV (line 1) induced high expression of the transmembrane subunit, which was successfully incorporated into VLPs. In contrast, for Ad5-MelARV-ISD (line 2), almost no protein was detected in any of the samples. The modified vaccines (lines 3 and 4) induced p15E expression and VLP assembly to some extent, but at much lower levels than the native MelARV Env vaccine (Fig. 19C, D).

[0229] Example 9 (Comparative) Balb / c mice were immunized with the following constructs: HIV B gag P2A ConB gp140 G / CD (wild-type), HIV B gag P2A ConB gp140 G / CD ISD#4(Y75G), HIV B gag P2A ConB gp140 G / CD ISD#19(L70Q), and HIV B gag P2A ConB gp140 G / CD G19Rdb(G83K,S88F). Antibody responses were analyzed 4 weeks (day 28 - Figure 20A) and 7 weeks (day 49 - Figure 20B) after immunization against the HIV ConB gp140 wild-type protein. IiGP-P2A-IFNalpha4 and HIV B gag P2A ConB gp140 G / CD (wild-type) (420B) served as controls for type I interferon-induced responses.

[0230] c57 / bl6 mice or c57 / bl6IFN-g knockout mice were immunized with the following constructs: adenoviruses encoding HIV B clade gag p2A followed by the gp140 sequence of the B clade consensus sequence (HIV B gag P2A ConB gp140 G / CD) (wild-type) and HIV B gag P2A ConB gp140 G / CD ISD#19(L70Q). Antibody responses against the HIV ConB gp140 CF protein were determined 4 weeks after immunization (day 26—Figure 20C).

[0231] Example 10 The following constructs were used to immunize balb / c mice: HIV B gag P2A ConB gp140 G / CD (wild-type), HIV B gag P2A ConB gp140 G / CD ISD#4(Y75G), HIV B gag P2A ConB gp140 G / CD ISD#19(L70Q), and HIV B gag P2A ConB gp140 G / CD G19Rdb(G83K,S88F). Four months after immunization (d. 114), mice were analyzed for T cell responses to a single pool covering the Gag gene (MA (p17, matrix) (peptides 1–31), CA (p24, capsid) (peptides 32–89), p2, NC (nucleocapsid), p1, and p6 (peptides 90–124), as well as pools of peptides covering gp120(1) (peptides 1–62), gp120(2) (peptides 63–124), and gp41 (peptides 125–211).

[0232] Example 11 BALB / c mice were vaccinated with MVA-expressing gag, env, gag+env, VE-VLPs or adenovirus-expressing gag-env or gag+env ISD mutant VE-VLPs, and combinations of these, and immune responses directed against putative MHC binding 9-amino acid-long peptides were measured using ELISPOT or intracellular cytokine staining.

[0233] In particular, gag+env or gag+envISD mutant VEVLP in adenoviral vectors is highly expected to outperform the previously described MVA vectors in inducing T cell responses.

[0234] Example 12: BALB / c mice are vaccinated with either MVA expressing gag, env, gag+env, or gag+envISD mutant as VE-VLPs, or adenovirus expressing gag-env or gag+envISD mutant VEVLPs, and combinations of these, and peptide responses derived from sequences of the extracellular portion of the transmembrane domain p15E of HERV-Kcon are measured.

[0235] The gag-env or gag+env ISD mutant VEVLP vectors are expected to outperform the previously described MVA vectors in inducing T cell responses.

[0236] Example 13 Animals are subcutaneously challenged with RENCA renal carcinoma cells expressing HERVcon-gag and HERVcon-env, respectively, and then vaccinated with MVA expressing gag, env, gag+env, or gag+env ISD mutants as VE-VLPs, or adenoviruses expressing gag-env or gag+env ISD mutant VEVLPs, or combinations thereof, and tumor growth is monitored.

[0237] Tumor control is expected to be improved with VE-VLP and gag-envVEVLP vaccines, which are uniquely able to control tumor growth in both cell lines.

[0238] Example 14 Animals are intravenously challenged with RENCA renal carcinoma cells expressing HERVcon-gag and HERVcon-env, respectively. Then, the animals are vaccinated with either MVA expressing gag, env, gag+env, or gag+env ISD mutants as VE-VLPs, or adenovirus expressing gag-env or gag+env ISD mutant VEVLPs, or combinations thereof. Tumor growth is monitored by vivisection, and metastases are counted 30 days after tumor challenge.

[0239] Tumor control is expected to be improved with VE-VLP and gag-envVEVLP vaccines, which are uniquely capable of controlling tumor growth of both cell lines.

[0240] Example 15 In relation to translational studies of the immunotherapeutic strategies described in the previous examples, a human-relevant version of the vaccine was designed using an adenoviral vector (Ad5 / Ad19a) (Dewannieux et al. 2006) encoding the consensus sequence human endogenous retrovirus type K (HERV-K) envelope (Env) and group-specific antigen (Gag) proteins, designed to induce VLP formation in transduced cells (Muster et al. 2003). To improve the vaccination strategy, the ISD contained in the p15E subunit of the HERV-K Env protein was inactivated by a single point mutation (see Figure 22), the selection of which was based on conservation between HERV-K and HIV (Morozov et al. 2012) and between HERV-K and HIV (van der Kuyl 2012) (Dewannieux et al. 2005).

[0241] The HERV-K Gag-p2A-EnvISD mutant had the amino acid sequence (SEQ ID NO: 48).

[0242] [ka] [ka]

[0243] These vaccines were tested for immunogenicity in BALB / c, C57BL / 6, and CD1 mice, and their efficacy, as measured by mouse survival curves, was tested in BALB / c mice challenged with murine renal carcinoma (Renca or RLZ) cells expressing the HERV-K Env target protein. Immune responses were assessed for their ability to induce cellular and humoral responses, and for the presence of INFγ+ CD8+ T cells (by FACS analysis) and specific antibodies against the HERV-K Env target protein (detected by ELISA) in mice immunized with the DNA / Adv-HERV-K wild-type / ISD vaccine and boosted with MVA Env.

[0244] The Ad19-HERV-K wild-type vaccine and its improved version containing the ISD mutation were tested and compared for their ability to induce the expression of VLPs formed by the Gag_p2A_Env HERV-K Adv-encoded protein. Pre-existing immunity in humans, which leads to neutralizing antibodies (NAbs) that block the immune response, may be a drawback to using the Ad5 vector. Furthermore, Ad19 vectors are known to be more successful at transducing different cell types (Kiener et al. 2018). Therefore, the use of different adenoviral vectors (Ad19 vs. Ad5) was also analyzed and compared.

[0245] To analyze the functionality of the novel strategy, the vaccines were analyzed for induction of HERV-K Gag and Env target proteins. Therefore, VLP production and secretion were examined in Vero and A549 cell lines transfected with different virus-based vaccines containing different sequences of interest (see Figure 23). Supernatants (SN) and cell lysates from the above-mentioned transfected cell lines were examined for the presence of HERV-K Gag and Env proteins by Western blot (WB). HERM-1811-5 and HERM-1821-5, monoclonal antibodies against p15E(TM) and gp70(SU), were specifically used to detect the HERV-K Env domain, while a polyclonal rabbit anti-p2A antibody was used to detect Gag protein linked to p2A. An HRP-conjugated secondary antibody was employed for detection.

[0246] The WB results showed the presence of HERV-K Gag_p2A and HERV-K Env proteins in both the SN and cell lysates of Vero and A549 cells transfected with Ad19_HERV-K wild-type / ISD mutants. The higher expression of both Gag and Env proteins from cells transfected with Ad19_HERV-K ISD mutants (shown in rows 2 and 8 of Figure 23) suggests the enhanced functionality and superior potency of the modified prototype vaccine compared with the Ad19_HERV-K_wild-type and Ad5_HERV-K_Env vaccines. Furthermore, the absence of Gag and Env proteins in the SN of Vero cells transfected with Ad19_HERV-K could be explained by the low concentrations of proteins obtained after VLP purification of the corresponding samples.

[0247] To further confirm the expression of the HERV-K Env target protein, A549 cells were transfected with the adenovirus vaccine encoded by the VLP (see Figure 24). Forty-eight hours after infection, cells were incubated with a primary anti-HERV-K Env antibody (HERM-1811) and then labeled with a goat anti-mouse IgG APC secondary antibody, with or without prior fixation and permeabilization. The intracellular and extracellular fluorescence of the bound antibody, and therefore the expression of HERV-K Env inside and outside the infected cells, were analyzed by FACS. This result suggests a better transfection efficiency using the Ad19 vector compared to Ad5, because although both vectors encode the same target protein, a higher signal was detected with Ad19. Comparing the Ad19_HERVK wild-type and ISD mutant vaccines, a larger cell surface signal and a similar intracellular signal were detected in Ad19_HERV-K_ISD mutant-transfected cells, indicating improved cell surface selection of the mutated sequence.

[0248] To visually confirm the production of the structural protein Gag and the subsequent release of Env HERV-K, A549 cells were infected with the Ad19_HERV-K_ISD mutant at an MOI of 50 and fixed at 24 and 48 hours post-infection. Budding and secreted VLPs were then detected by electron microscopy (see Figure 29), demonstrating that the vaccine was fully capable of expressing the HERV-K Gag and Env target proteins, which were incorporated into secreted VLPs.

[0249] Example 16 To examine the T cell responses induced by the Ad19_HERV-K wild-type / ISD mutant vaccine, we analyzed T lymphocyte responses to P-HKE (a 10-mer peptide of HERV-K Env with the sequence TYHMVSGMSL) in BALB / c mice. Because P-HKE is an MHC class I-restricted epitope, CD8+ T cell activation and, therefore, secretion of interferon gamma (IFNγ) and tumor necrosis factor alpha (TNFα) cytokines after peptide stimulation in BALB / c mice were measured by intracellular cytokine staining (ICS) using FACS.

[0250] BALB / c mice were primed with various vaccines consisting of different vectors (Ad5 / Ad19 / MVA) encoding the HERV-K protein. Subsequently, half of the mice received an MVA Env boost to examine whether the cellular response elicited by the prime immunization regimen could be augmented. Ten days after the MVA boost, mice were euthanized and splenocytes were analyzed by FACS upon P-HKE stimulation (see Figure 25). The groups receiving the Ad19_HERV-K_wildtype / ISD mutant vaccine were compared between boosted (MVA-Env) and non-boosted immunizations.

number

[0251] Example 17 To test and compare vaccine efficacy, mice were challenged and subsequently vaccinated, and survival was assessed in correlation with tumor progression (see Figure 26). For this experiment, BALB / c mice were intravenously challenged with RENCA cells expressing HERV-K Env. Ten days after tumor challenge, mice were inoculated with MVA Env, Ad19_HERV-K wild-type / ISD mutant, and an irrelevant vaccine as a control. This experiment, based on (Kraus et al., 2013 PLoS One. Aug 30;8(8):e72756), aimed to score metastatic tumor burden 40 days after injection. Animals were weighed longitudinally, and mice were euthanized if any physical, behavioral, or physical changes were observed in the animals or if weight loss exceeded 10%. Once mice were sacrificed, lungs were harvested and preserved in 4% PFA for further analysis of the presence of metastases. Notably, all animals sacrificed due to weight loss had substantial tumor burden. Unexpectedly, significant mortality was recorded during this experiment, and survival curves were established and compared between different groups. This indicated a more rapid progression of RENCA-HERV-K tumors compared with previously reported data. Under this rather strict tumor burden model, mice receiving the Ad19_HERV-K_ISD mutant vaccine showed a significantly increased life expectancy compared with controls. Three different statistical tests (log-rank, Wilcoxon, and Tarone-Ware) showed significant p values ​​(0.037, 0.046, and 0.040). This suggests that the Ad19_HERV-K_ISD mutant vaccine delayed lung tumor progression and metastasis in BALB / c mice, consistent with the above results showing increased antibody and CD8+ T cell responses. None of the other vaccines extended survival time.

[0252] Example 18 To further confirm this finding in the human system, tissue samples were obtained from human breast tumors. Sections were made at 4 μm and stained with a 1:1000 dilution of primary antibody obtained from non-immunized mice (prebleed serum). Mice primed with Ad5_HERV-K_Env were boosted with the vaccination regimen of Ad19_HERV-K_ISD (8 weeks later) and MVA_Env (2 months later). As shown in Figure 28, the HERV-K antibody from vaccinated mice can stain cancer tissues expressing the HERV-K target protein.

[0253] Various aspects and implementations have been described in conjunction with various embodiments herein. However, other variations to the disclosed embodiments can be understood and effected by those skilled in the art in carrying out the claimed subject matter, from a study of the drawings, the disclosure, and the appended claims. In the claims, the word "comprising" does not exclude other elements or steps, and the indefinite articles "a" or "an" do not exclude a plurality. A single processor or other unit may fulfill the functions of several items recited in a claim. The mere fact that certain means are recited in mutually different independent claims does not indicate that a combination of these measures cannot be used to advantage. A computer program can be stored / distributed on an appropriate medium, such as an optical storage medium or a solid-state medium, together with or as part of other hardware, but can also be distributed in other forms, such as via the Internet or other wired or wireless communication systems.

[0254] Any reference signs used in the claims should not be construed as limiting the scope.

[0255] Sequences are disclosed in the main body of the specification and in a separate sequence listing in accordance with WIPO Standard ST.25. SEQ ID NOs designated by specific numerical values ​​must be the same in the main body of the specification and in the separate sequence listing. For example, SEQ ID NO: 1 must define the same sequence in both the main body of the specification and the separate sequence listing. In the event of a conflict between a sequence definition in the main body of the specification and a separate sequence listing (e.g., SEQ ID NO: 1 in the main body of the specification incorrectly corresponds to SEQ ID NO: 2 in the separate sequence listing), the reference to a specific sequence in the application, particularly in a specific embodiment, shall be understood as a reference to the sequence in the main body of the application, not as a reference to the separate sequence listing. In other words, the sequence definition / designation in the main body of the specification shall be resolved by amending the separate sequence listing to the sequence and its designation disclosed in the main body of the application, including the specification, examples, figures, and claims.

[0256] Patent items 1. A vaccine for use in the prevention and / or treatment of disease, comprising an adenoviral vector capable of encoding a virus-like particle (VLP), wherein the VLP displays an inactive immunosuppressive domain (ISD).

[0257] 2. The vaccine according to item 1, which is for the prevention and / or treatment of cancer.

[0258] 3. The vaccine of item 1 or 2, wherein the ISD has LANQINDLRQTVIW (SEQ ID NO: 1), LASQINDLRQTVIW (SEQ ID NO: 2), LQNRRGLDLLTAEKGGL (SEQ ID NO: 3), LQNRRALDLLTAERGGT (SEQ ID NO: 4), LQNRRGLDMLTAAQGGI (SEQ ID NO: 5), or YQNRLALDYLLAAEGGV (SEQ ID NO: 6), with at least one of the amino acids deleted or replaced with a different amino acid.

[0259] 4. The vaccine according to item 3, wherein the non-original amino acid is selected from among the naturally occurring amino acids.

[0260] 5. The vaccine of any one of items 1 to 4, wherein at least one of the amino acids in a 10 amino acid region upstream or downstream of the ISD has been replaced with a different amino acid.

[0261] 6. The vaccine of any one of items 1 to 5, wherein the VLP further displays an endogenous retrovirus (ERV) envelope protein or an immunogenic portion thereof.

[0262] 7. The vaccine of any one of paragraphs 1 to 6, wherein the ERV envelope protein is a human endogenous retrovirus (HERV) protein or an immunogenic portion thereof.

[0263] 8. The vaccine of any one of paragraphs 1 to 7, wherein the HERV is selected from the group consisting of HERV-K, HERV-H, HERV-W, HERV-FRD, and HERV-E.

[0264] 9. The vaccine of any one of items 1 to 8, 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), and HERV-K110 (=ERVK-18), the HERV-H is selected from the group consisting of HERV-H19 (=HERV-H_2q24.3), and HERV-H_2q24.1, the HERV-W is selected as ERVW-1 (=Syncytin 1), and the HERV-FRD is selected as ERVFRD-1 (=Syncytin 2).

[0265] 10. The vaccine of any one of paragraphs 1 to 9, wherein the adenoviral vector is derived from a mammalian adenovirus type, a human adenovirus type, a chimpanzee adenovirus type, or a gorilla adenovirus type.

[0266] 11. The vaccine of any one of paragraphs 1 to 10, wherein the human adenovirus vector is derived from a group D vector, human adenovirus serotype Ad5, human adenovirus serotype Ad19a, human adenovirus serotype Ad26, or a chimpanzee adenovirus serotype.

[0267] 12. The vaccine of any one of paragraphs 1 to 11, wherein the adenoviral vector is adenovirus, serotype 5 (Ad5).

[0268] 13. The vaccine of any one of paragraphs 1 to 12, wherein the protein products of the adenoviral vector include gag protein, 2A peptide, and envelope protein (Env).

[0269] 14. The vaccine of any one of paragraphs 1 to 13, wherein the gag protein is an exogenous retroviral gag protein or an endogenous retroviral gag protein.

[0270] 15. The vaccine of any one of items 1 to 14, wherein the Env protein comprises a surface unit (gp70), a cleavage site, and a transmembrane unit (p15E).

[0271] 16. The vaccine of any one of items 1 to 15, wherein the transmembrane unit (p15E) comprises a fusion peptide, an immunosuppressive domain (ISD), a transmembrane anchor, and / or a cytoplasmic tail.

[0272] 17. The vaccine according to any one of items 1 to 16, wherein p15E or an immunogenic portion thereof is linked to the adenovirus capsid protein pIX.

[0273] 18. The vaccine of any one of items 1 to 17, wherein the signal peptide encoded by the adenoviral vector has been exchanged for the signal peptide from Gaussia luciferase (LucSP).

[0274] 19. The vaccine of any one of items 1 to 18, wherein the transmembrane anchor and the cytoplasmic tail encoded by the adenoviral vector are replaced with the transmembrane domain and cytoplasmic tail from influenza A virus hemagglutinin H3N2.

[0275] 20. The vaccine of any one of items 1 to 19, wherein the transmembrane anchor and the cytoplasmic tail encoded by the adenoviral vector are replaced with the transmembrane domain and cytoplasmic tail from influenza A virus hemagglutinin H3N2 (HA-TMCT).

[0276] 21. The vaccine of any one of items 1 to 20, wherein a trimerization sequence is provided adjacent to the signal peptide.

[0277] 22. The vaccine of any one of items 1 to 21, wherein the trimerization sequence is GCN4.

[0278] 23. The vaccine of any one of items 1 to 22, wherein the VLP comprises a gag protein.

[0279] 24. The vaccine according to any one of items 1 to 23, wherein the gag protein is an exogenous retroviral gag protein or an endogenous retroviral gag protein.

[0280] 25. The vaccine of any one of items 1 to 24, wherein the VLPs are produced in cells of the body of a patient infected with the adenoviral vector.

[0281] 26. The vaccine of any one of items 1 to 25, wherein the VLPs are produced in isolated mammalian cells.

[0282] 27. A nucleic acid construct encoding a target protein capable of forming a virus-like particle (VLP), wherein the target protein comprises an immunosuppressive domain (ISD), and the ISD is inactive.

[0283] 28. The nucleic acid construct of item 27, wherein the ISD has LANQINDLRQTVIW (SEQ ID NO: 1), LASQINDLRQTVIW (SEQ ID NO: 2), LQNRRGLDLLTAEKGGL (SEQ ID NO: 3), LQNRRALDLLTAERGGT (SEQ ID NO: 4), LQNRRGLDMLTAAQGGI (SEQ ID NO: 5), or YQNRLALDYLLAAEGGV (SEQ ID NO: 6), with at least one of the amino acids deleted or replaced with a different amino acid.

[0284] 29. The nucleic acid construct according to item 27 or 28, wherein the amino acid different from the original is selected from among the naturally occurring amino acids.

[0285] 30. The nucleic acid construct of item 27 or 28, wherein at least one of the amino acids in a 10 amino acid region upstream or downstream of the ISD has been replaced with a different amino acid.

[0286] 31. The nucleic acid construct of any one of items 27 to 30, wherein the VLP further displays an endogenous retroviral (ERV) envelope protein or an immunogenic portion thereof.

[0287] 32. The nucleic acid construct of any one of items 27 to 31, wherein the ERV envelope protein is a human endogenous retrovirus (HERV) protein or an immunogenic portion thereof.

[0288] 33. The nucleic acid construct of any one of items 27 to 32, wherein the HERV is selected from the group consisting of HERV-K, HERV-H, HERV-W, HERV-FRD, and HERV-E.

[0289] 34. The nucleic acid construct according to any one of items 27 to 33, 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), and HERV-K110 (=ERVK-18), the HERV-H is selected from the group consisting of HERV-H19 (=HERV-H_2q24.3), and HERV-H_2q24.1, the HERV-W is selected as ERVW-1 (=syncytin 1), and the HERV-FRD is selected as ERVFRD-1 (=syncytin 2).

[0290] 35. The nucleic acid construct of any one of items 27 to 34, wherein the adenoviral vector is derived from a mammalian adenovirus type, a human adenovirus type, a chimpanzee adenovirus type, or a gorilla adenovirus type.

[0291] 36. The nucleic acid construct of any one of items 27 to 35, wherein the human adenovirus vector is derived from a group D vector, human adenovirus serotype Ad5, human adenovirus serotype Ad19a, human adenovirus serotype Ad26, or a chimpanzee adenovirus serotype.

[0292] 37. The nucleic acid construct of any one of items 27 to 36, wherein the adenoviral vector is adenovirus, serotype 5 (Ad5).

[0293] 38. The nucleic acid construct of any one of items 27 to 37, wherein the protein products of the adenoviral vector include gag protein, 2A peptide, and envelope protein (Env).

[0294] 39. The nucleic acid construct of any one of items 27 to 38, wherein the gag protein is an exogenous retroviral gag protein or an endogenous retroviral gag protein.

[0295] 40. The nucleic acid construct of any one of items 27 to 39, wherein the Env protein comprises a surface unit (gp70), a cleavage site, and a transmembrane unit (p15E).

[0296] 41. The nucleic acid construct of any one of items 27 to 40, wherein the transmembrane unit (p15E) comprises a fusion peptide, an immunosuppressive domain (ISD), a transmembrane anchor, and / or a cytoplasmic tail.

[0297] 42. The nucleic acid construct according to any one of items 27 to 41, wherein p15E or an immunogenic part thereof is linked to the adenovirus capsid protein pIX.

[0298] 43. The nucleic acid construct of any one of items 27 to 42, wherein the signal peptide encoded by the adenoviral vector has been exchanged for the signal peptide from Gaussia luciferase (LucSP).

[0299] 44. The nucleic acid construct of any one of items 27 to 43, wherein the transmembrane anchor and the cytoplasmic tail encoded by the adenoviral vector are replaced with the transmembrane domain and cytoplasmic tail from influenza A virus hemagglutinin H3N2.

[0300] 45. The nucleic acid construct of any one of items 27 to 44, wherein the transmembrane anchor and the cytoplasmic tail encoded by the adenoviral vector are replaced with the transmembrane domain and cytoplasmic tail from influenza A virus hemagglutinin H3N2 (HA-TMCT).

[0301] 46. ​​The nucleic acid construct of any one of items 27 to 45, wherein a trimerization sequence is provided adjacent to the signal peptide.

[0302] 47. The vaccine of any one of items 27 to 46, wherein the trimerization sequence is GCN4.

[0303] 48. A protein comprising the expression product of the nucleic acid construct according to any one of items 27 to 47.

[0304] 49. A virus-like particle (VLP) comprising the nucleic acid construct of any one of items 27 to 47.

[0305] 50. The vaccine according to any one of items 1 to 27 for use in the prevention and / or treatment of cancer.

[0306] 51. The vaccine of any one of items 1 to 27 for use in the prevention and / or treatment of cancer, comprising a step of priming the patient with the nucleic acid construct of any one of claims 27 to 47 and then boosting the patient with the vaccine of any one of items 1 to 26 at least 5 days later.

[0307] 52. The vaccine of any one of items 1 to 26 for use in the prevention and / or treatment of cancer, comprising a step of post-treating the patient 5 days or more after exposure of the patient to the vaccine of any one of items 1 to 26 with a VLP encoded by a virus that is different from the VLP derived from an adenoviral vector.

[0308] 53. The vaccine of item 52, wherein the VLP encoded by the virus different from the VLP derived from an adenoviral vector is a VLP derived from Modified Vaccina Ankara (MVA).

[0309] 54. A vaccine for use in the prevention and / or treatment of disease, comprising a viral vector capable of encoding a virus-like particle (VLP), wherein the VLP displays an inactive immunosuppressive domain (ISD).

[0310] 55. The vaccine of claim 54, wherein the viral vector is derived from Modified Vaccina Ankara (MVA), an adeno-associated virus (AAV), or a lentivirus.

[0311] 56. A method for the prevention and / or treatment of cancer, comprising administering a vaccine according to any one of items 1 to 26.

[0312] 57. A method for the prevention and / or treatment of cancer, comprising priming said patient with a nucleic acid according to any one of items 27 to 47, followed at least 5 days later by boosting said patient with the vaccine according to any one of items 1 to 26.

[0313] 58. A method for the prevention and / or treatment of cancer, comprising the step of post-treating the patient 5 days or more after exposure of the patient to the vaccine of any one of items 1 to 20 with a virally encoded VLP that is different from the VLP derived from an adenoviral vector.

[0314] 59. The method of item 58, wherein the virally encoded VLP, which is different from the VLP derived from an adenoviral vector, is a VLP derived from Modified Vaccina Ankara (MVA).

Claims

1. A nucleic acid molecule encoding an endogenous retrovirus (ERV) envelope protein or an immunogenic portion thereof for use in the prevention and / or treatment of disease, wherein the ISD of the protein contains a mutation that renders the ISD inactive.

2. 2. A vector comprising the nucleic acid molecule of claim 1 for use according to claim 1, preferably an adenovirus vector, more preferably an adenovirus vector derived from a mammalian adenovirus type, a human adenovirus type, a chimpanzee adenovirus serotype, or a gorilla adenovirus serotype, wherein the human adenovirus vector is derived from a group D vector, human adenovirus serotype Ad5, human adenovirus serotype Ad19a, human adenovirus serotype Ad26, or a chimpanzee adenovirus serotype, and the adenovirus vector is adenovirus, serotype 5 (Ad5) or adenovirus, serotype 19 (Ad19).

3. The vector of claim 2 for use as described in claim 2, wherein the vector encodes a virus-like particle (VLP), and the VLP displays an endogenous retrovirus (ERV) envelope protein or an immunogenic portion thereof having an inactive immunosuppressive domain (ISD).

4. A protein comprising the expression product of the vector according to claim 3 for use in the prevention and / or treatment of a disease.

5. A vaccine comprising a nucleic acid molecule or vector according to any one of claims 1 to 3 or comprising a protein according to claim 4 for use in the prevention and / or treatment of a disease.

6. 6. The nucleic acid molecule, vector, protein, or vaccine of any one of claims 1 to 5 for use as claimed in claim 5, wherein the disease is selected from cancer, more preferably ERV-expressing cancer, even more preferably prostate cancer, breast cancer, ovarian cancer, lymphoma, melanoma, leukemia, sarcoma, colon cancer, testicular cancer, lung cancer, and liver cancer.

7. The nucleic acid molecule, vector, or vaccine of any one of claims 1 to 6, for use as described in claim 6, wherein the ISD has LANQINDLRQTVIW (SEQ ID NO: 1), LASQINDLRQTVIW (SEQ ID NO: 2), LQNRRGLDLLTAEKGGL (SEQ ID NO: 3), LQNRRALDLLTAERGGT (SEQ ID NO: 4), LQNRRGLDMLTAAQGGI (SEQ ID NO: 5), YQNRLALDYLLAAEGGV (SEQ ID NO: 6) or NSQSSIDQKLANQINDLRQT (SEQ ID NO: 49), in which at least one of the amino acids has been replaced with a different amino acid.

8. A nucleic acid molecule, vector, or vaccine described in any one of claims 1 to 7 for use as described in claim 7, wherein at least one of the amino acids in a 10 amino acid region upstream or downstream of the ISD has been replaced with a different amino acid.

9. The ERV protein is a human endogenous retrovirus (HERV) protein or an immunogenic portion thereof, the HERV is selected from the group consisting of HERV-K, HERV-H, HERV-W, HERV-FRD, and HERV-E, and 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 10. The nucleic acid molecule, vector or vaccine of any one of claims 1 to 8 for use according to claim 8, wherein HERV-H is selected from the group consisting of HERV-H19 (=HERV-H_2q24.3) and HERV-H_2q24.1, HERV-W is selected as ERVW-1 (=Syncytin 1), and HERV-FRD is selected as ERVFRD-1 (=Syncytin 2).

10. The protein products of the adenoviral vector include gag protein, 2A peptide, and envelope protein (Env), the Env protein includes a surface unit (gp70), a cleavage site, and a transmembrane unit (p15E), the transmembrane unit (p15E) includes a fusion peptide, an immunosuppressive domain (ISD), a transmembrane anchor, and a cytoplasmic tail, and p15E or an immunogenic portion thereof is linked to adenoviral capsid protein pIX and / or encoded by the adenoviral vector.

10. The vector of any one of claims 3 to 8 or the vaccine of any one of claims 1 to 9 for use according to claim 9, wherein the signal peptide encoded by the adenoviral vector is exchanged for the signal peptide derived from Gaussia luciferase (LucSP) and / or the transmembrane anchor and the cytoplasmic tail encoded by the adenoviral vector are exchanged for the transmembrane domain and cytoplasmic tail derived from influenza A virus hemagglutinin H3N2 (HA-TMCT).

11. 11. The vaccine of any one of claims 1 to 10 for use in the prevention and / or treatment of cancer, comprising a step of boosting the patient with the vaccine of any one of claims 1 to 10 at least 5 days after priming the patient with the adenoviral vector.

12. 12. The vaccine of claim 1 for use in the prevention and / or treatment of cancer, comprising a step of post-treating the patient 5 days or more after exposure of the patient to the vaccine with a VLP encoded by a virus other than a VLP derived from an adenoviral vector, wherein the VLP encoded by the virus other than a VLP derived from an adenoviral vector is a VLP derived from Modified Vaccina Ankara (MVA).

13. A nucleic acid molecule encoding Gag protein and ERV envelope protein (Env) or immunogenic portions thereof, wherein the intact genomic structure connecting Gag and Env is replaced by a functional linker, said linker preferably being p2A.

14. A VLP encoded by the nucleic acid molecule of claim 13.

15. A nucleic acid molecule according to claim 13 or a VLP according to claim 14 for use in the prevention and / or treatment of a disease.

16. The nucleic acid molecule according to claim 1, the VLP according to claim 14, or the VLP according to claim 15, for use according to claim 15, wherein the ERV is HERV-K, preferably HERV-K 113.

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