Vaccine for use in the prevention and / or treatment of a disease

Abstract

This invention relates to adenovirus vectors capable of encoding virus-like particles (VLPs) exhibiting an inactive immunosuppressive domain (ISD). The vaccines of this invention demonstrate improved immune responses originating from either CD4 T cell or CD8 T cell-initiated pathways.

Description

Technical Field

[0001] This disclosure relates to vaccines for use in the prevention and / or treatment of diseases. It is noteworthy that the disease can be derived from endogenous retroviruses, such as cancer. The vaccines of this invention particularly relate to viruses capable of forming virus-like particles in eukaryotic cells. In some embodiments of the invention, virus-encoded virus-like particles (VE-VLPs) are generated in a patient to develop an immunogenic response to an endogenous retrovirus. Background of the Invention

[0003] It was observed more than a century ago that cancer development is closely linked to the immune system, and it is now well-established that the immune system provides long-term protection against new cancers. On the other hand, malignant cells have developed strategies to evade immune surveillance and exert their lethal potential.

[0004] While immune cells can detect and kill tumor cells, this system doesn't always work, as evidenced by the nearly 9 million cancer deaths worldwide each year. Vaccination strategies 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 results. One vaccination strategy involves inoculating with attenuated tumor cells that frequently secrete granulocyte-macrophage colony-stimulating factor (GM-CSF), such as irradiated autologous tumors or allogeneic tumor cell lines. In both cases, the injected material covers cancer antigens that may be present in the actual tumor. Other vaccination strategies involve administering peptides or proteins to induce specific immune responses. These antigens are either injected directly in combination with adjuvants or encoded by DNA plasmids or viral vectors.

[0005] Despite continuous improvements in immunotherapy, a widely effective and highly efficient vaccine remains lacking. A specific reason for this is the previously described immunosuppression through tumor cells.

[0006] Endogenous retroviruses (ERVs) are evidence of ancient retrovirus infection in our ancestors. After infection, viral RNA is reverse transcribed into proviral DNA, which integrates into the host genome. Eventually, the provirus integrates into the cells of the germline and becomes heritable, producing the endogenous retrovirus. Over millions of years, the viral DNA has been passed down through generations and become fixed in the population. Today, each human genome consists of approximately 8% endogenous retroviral DNA, but these are merely remnants of previous retroviruses. Due to mutations, deletions, and insertions, most retroviral genes have become inactive or completely lost from the genome. Currently, no functional full-length endogenous retroviruses exist in humans. However, ERVs undergo replication, resulting in several copies integrating into the host genome with unique functional proteins. Therefore, in some cases, many homologous ERVs still have the potential to produce viral particles. Human ERV type K (HERV-K, HML2) is one of the most recently discovered ERVs in the human genome, and members of this family retain full-length open reading frames for almost all viral proteins.

[0007] Various studies have highlighted the link between ERV expression and cancer development and progression. ERV detection in human tumors opens new avenues in cancer treatment and holds promise for novel vaccination strategies. A prominent example of human ERV (HERV) is HERV K (HERV-K), associated with prostate cancer, breast cancer, ovarian cancer, lymphoma, melanoma, leukemia, and sarcoma. Further examples include HERV-H expressed in colorectal cancer, and cytosine-1 in testicular cancer, ovarian cancer, breast cancer, lymphoma, and leukemia.

[0008] Determining whether ERV protein expression is a cause or consequence of developing tumors is not always straightforward. However, conditions within cancer cells are known to allow for ERV expression. The general state of hypomethylation in tumor cells promotes the activation of ERV genes, which are normally silenced 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): 1249-57; 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): 2229-46. Endogenous factors can also promote ERV expression. For example, activation of ERV has been observed in people due to viral infection. HERV-W expression has been 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), while HERV-K exists after EBV infection (Sutkowski, N. et al., Epstein-Barr virus transactivates the human endogenous retrovirus HERV-K18 that encodes a superantigen. Immunity, 2001. 15(4): 579-89). Regardless of the mechanism leading to ERV expression, cancer cells maintain the activation of these proteins through selective pressure, suggesting a beneficial effect of ERV in tumors (Leong, SP et al., Expression and modulation of a retrovirus-associated antigen by murine melanoma cells. Cancer Res, 1988. 48(17): 4954-8.

[0009] Not only are human tumors associated with ERV proteins, but mouse cancer cells also express ERV. This provides a perfect model organism for studying the role of ERV in tumor progression and testing ERV-targeting therapies. One ERV model is melanoma-associated retrovirus (MelARV), which is derived from a provirus of murine leukemia virus (MuLV) present in the mouse genome. Most inbred mouse strains contain one or two inactivated copies of MuLV (Li, M. et al., Sequence and insertion sites of murine melanoma-associated retrovirus. J Virol, 1999. 73(11): 9178-86. However, the AKR mouse strain has three insertions in its genome and is characterized by a large amount of MuLV production early in life, leading to frequent spontaneous lymphomas. Other mouse strains, such as C57BL / 6, only spontaneously produce MuLV granules late in life. Similar to human ERV, several other mouse cancer models also express MuLV / MelARV.

[0010] Because the host's immune system is a natural defense mechanism against infection, many viruses, especially retroviruses, have developed strategies to evade this surveillance. One mechanism that can be seen throughout different viral families [Duch et al., WO2013 / 050048] is the development of an immunosuppressive domain in the envelope protein (Env), leading to suppression of the immune system at different levels. Immune cells, including natural killer (NK), CD8 T, or regulatory T (Treg) cells, can be affected by viruses containing ISD [Schlecht-Louf et al. (2010)].

[0011] Many ERVs contain proteins with immunosuppressive domains (ISD), and such domains can also be found in MelARV Env proteins (Schlecht-Louf, G. et al.). Retroviral infection in vivo requires an immune escape virulence factor encrypted in the envelope protein of oncoretroviruses. Proc Natl Acad Sci USA, 2010. 107(8): 3782-7, 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): 14920-5. ). The importance of ISD in MuLV or MelARV has been demonstrated by introducing the murine leukemia virus Env protein into tumor cells that are normally rejected by immune cells (Mangeney, M. and T. Heidmann, Tumor cells expressing a retroviral envelope escape immune rejection in vivo. Proc Natl Acad Sci USA, 1998. 95(25): 14920-5). Despite being a different exogenous antigen, Env-transduced tumor cells still grow more rapidly. This observation is explained by the local immunosuppressive effect mediated by the Env protein. ISD affects both the innate and adaptive immune systems, as demonstrated by the suppression of macrophages, NK cells, and T cells (Lang, MS et al.). Immunotherapy with monoclonal antibodies directed against the immunosuppressive domain of p15E inhibits tumour growth. Clin Exp Immunol, 1995. 102(3): 468-75). Furthermore, the effects on regulatory T cell subsets have been proposed, which in turn inhibit other immune cells (Mangeney, M. et al., Endogenous retrovirus expression is required for murine melanoma tumor growth in vivo. Cancer Res, 2005. 65(7): 2588-91. The detailed mechanism of immunosuppression through ISD is not fully understood, but the effect appears to be primarily mediated by the CKS-17 peptide within ISD. CKS-17 has different effects on the immune system, mainly through 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.

[0012] One of the first therapeutic approaches targeting tumor cells expressing ERV involved the administration of monoclonal antibodies. Therefore, 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 through alterations in the cancer cell cycle and increased apoptosis. (This information was not provided by Wang-Johanning et al.) Immunotherapeutic potential of anti-human endogenous retrovirus-K envelope protein antibodies in targeting breast tumors. J Natl Cancer Inst, 2012. 104(3): 189-210) Another possible effect of such antibodies tested could be the prevention of immunosuppression. Like MelARV Env, HERV-K Env proteins also contain ISD and have immunomodulatory functions (Morozov, VA, VL 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): e70399). The method tested by Wang-Johanning et al. included xenograft tumors in immunodeficient athymic mice. Therefore, the effects of HERV-K can only affect innate immune cells, such as NK cells.

[0013] Another component of the adaptive immune response that can help eradicate tumors by targeting ERVs includes T cells. For example, adoptive T cells targeting the MuLV Env epitope in combination with IL-2 can eradicate lung metastases of melanoma cells (Yang, JC, 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): 177-83). Similar experiments were performed in a humanized mouse model of HERV-K. T cells were genetically modified to express a chimeric antigen receptor (CAR) on their surface that recognizes HERV-KEnv on cancer cells. Cytotoxic CAR + T cells can lyse tumor cells and prevent metastasis and tumor growth.

[0014] Besides direct injection of antibodies or T cells, a more practical, cheaper, and effective strategy is to induce an immune response through vaccination. A simple method is vaccination with virus-encoded antigens. However, this method is quite cumbersome because dendritic cells (DCs) must first be isolated and cultured, then pulsed with a defined HLA-restricted peptide, and then injected into mice or patients.

[0015] A more sophisticated vaccination strategy involves presenting antigens (such as viral envelope proteins) to the immune system on virus-like particles (VLPs), which are encoded by recombinant adenoviruses. Figure 1 These particles do not contain viral nucleic acid and are therefore non-infectious. Nevertheless, VLPs are highly immunogenic, and the proteins they exhibit are naturally occurring in the environment. For example, the viral Env protein integrated into VLPs is presented on a virus-like surface that promotes proper folding and conformation. In addition to the advantage of strong immunogenicity, vaccination strategies using VLPs also offer practical benefits. Therefore, VLPs are relatively easy to produce because they are constructed from only a single or a few proteins, and production can be performed in cell culture.

[0016] For vaccination against viruses or virus-related diseases (such as cancers expressing ERV), the entire Env protein should ideally be presented to the immune system to ensure an immune response against the intact protein target. However, because the Env protein contains an ISD (immunoassay serotype), the vaccine itself possesses an undesirable immunosuppressive capacity for the immunization approach. To overcome this drawback, mutations are introduced into the ISD to maintain the native conformation of the target protein while preventing immunosuppression.

[0017] One of the first to test inactivating mutations in ISDs of viral proteins was Schlecht-Louf et al. [Schlecht-Louf et al. (2010)]. Based on a comparative study between immunosuppressive syncytial 2 and non-immunosuppressive syncytial 1 [Mangeney et al. (2007)], Schlecht-Louf et al. identified mutations that inactivate ISDs without eliminating the general structure and function of the Env protein. This mutational strategy was applied to proteins of other viral origin (e.g., HTLV and XMRV) and tested more extensively for Fried mouse leukemia virus (F-MLV). This study not only revealed the suppression of both NK and T cells by ISDs but also showed that a live attenuated F-MLV virus containing a mutant ISD in the Env protein acts as a vaccine against the same virus with a WT ISD sequence. This protection is due to increased antibody levels against the F-MLV epitope and a T-cell response. Their findings were ultimately reflected in patent application WO 2011 / 092199, which focuses on heterophilic murine leukemia virus-associated virus (XMRV) already linked to human prostate cancer and chronic fatigue syndrome. Therefore, WO 2011 / 092199 specifically addresses ISD mutations in XMRV and the utility of such ISD-mutant viruses in vaccination strategies.

[0018] Another application of ISD mutations is described in patent application WO 2014 / 195510. In this case, a mutation of ISD is introduced into feline immunodeficiency virus (FIV) to reduce immunosuppression by the virus while maintaining its native conformation. WO 2014 / 195510 describes how a specific mutation increases antibody responses against FIV Env proteins when administered in a vaccination regimen, bound to MBP, or transduced in implanted tumor cells. Therefore, WO 2014 / 195510 relates to mutations in the ISD of FIV Env and the use of such mutant proteins in vaccination regimens against infection by FIV or other lentiviruses.

[0019] Another method for addressing a broader spectrum of ISD mutations in viral Env proteins is described in patent application WO 2013 / 050048. Specifically, WO 2013 / 050048 relates to antigen generation by first identifying ISDs in enveloped RNA viruses and then mutating these domains to reduce immunosuppression during vaccination. The ISD identification strategy is based on four parameters: 1) the peptide is localized in the fusion protein of an enveloped RNA virus; 2) the peptide is capable of interacting with the membrane; 3) high homology in the primary structure (sequence) of the peptide exists within the order, family, subfamily, genus, or species of the virus; and 4) the position of the immunosuppressive domain at the surface of the fusion protein in a given conformation is characterized by 3D structure or antibody staining. After identification of potential ISDs in the target viral Env, immunosuppressive function is verified, and mutations are subsequently introduced into the ISDs, confirming a reduction in immunosuppression of at least 25%. In summary, WO 2013 / 050048 describes the identification of ISD in enveloped RNA viruses, the generation of ISD mutant peptides, and the efficacy of said peptides as vaccines and the generation of antibodies.

[0020] Bayer et al. [Bayer et al. (2010)] demonstrated the importance of simultaneous antigen presentation encoded in adenoviral vectors and on the surface of the viral capsid. The benefit of presenting antigens through ordered structures that aid in cross-linking B cell receptors is previously known. However, by encoding different F-MLV proteins, such as the Gag and Env subunits gp70 and p15E, and simultaneously displaying such antigens on the adenoviral capsid protein pIX, Bayer et al. showed that only the combination of encoded and capsid-presented antigens could increase the level of functional antibodies. This observation is attributed to the fact that while presentation on the adenoviral capsid aids in cross-linking B cell receptors, the encoded antigen is a fundamental CD4 antigen that promotes affinity maturation of B cells. + The T-cell response is required. Using this vaccination strategy, Bayer et al. were able to reduce the viral load of F-MLV after challenge. However, no increase in CD8 targeting the target antigen was observed. + Indicators of T cell response.

[0021] Shoji et al. focused primarily on the optimization of adenovirus-based HIV vaccines. Despite codon optimization strategies and the use of different promoters, they co-encoded the Gag and Env proteins in the adenovirus via the cleavable furin protease site (F2A). This allowed for the simultaneous expression of both proteins and, consequently, in situ formation of Gag-based VLPs. In their study, this setup showed the highest immune response compared to other display strategies that did not promote in situ VLP formation [Shoji et al., 2012].

[0022] Duch et al. (2011, US20110305749A1) produced a VLP-based retroviral HIV vaccine and demonstrated increased immunogenicity of the ISD-mutated HIV envelope protein. The VLP immunogen was generated and purified in vitro.

[0023] US2012189647 relates to mutant envelope proteins resulting from mutations in the immunosuppressive domain of the transmembrane subunit of wild-type envelope proteins. US2009324553 relates to chimeric multi-directional viral envelope peptides suitable for targeted and controlled fusion of viral particles with other cell membranes.

[0024] Furthermore, the publication by Hohn et al. [Hohn et al., 2014] described alterations in viral assembly type and morphology when a codon-optimized version of HERV-K113 was expressed under the CMV promoter. Specifically, the VLP was retained at the cell surface and lacked Env.

[0025] Despite previous strategies involving ISD mutations in the viral Env protein and the use of adenovirus encoding and displaying viral antigens, past vaccination strategies employing ISD mutations were specifically designed to prevent viral infection [Schlecht-louf et al. 2010; WO 2011 / 092199; WO 2014 / 195510; US20110305749; WO 2014 / 195510]. Therefore, it remains necessary to break tolerance to self-antigens. Furthermore, in situ synthesis systems of virus-like particles have been used for HIV Env and malaria antigens prior to [Luo et al. (2003); Sohji et al. (2011); Andersson et al. (2016); Andersson & Holst (2016); Andersson et al. (2017)], but not for displaying ISD-mutated ERV on in situ synthesized VLPs. Moreover, given the findings of Hohn et al. It is also necessary to allow the production of VLPs, especially efficient systems for HERV-K VLPs.

[0026] This invention aims to produce an effective vaccine for the prevention and / or treatment of diseases caused by endogenous retroviruses. The vaccine of this invention exhibits an improved immune response derived from either a CD4 T cell or CD8 T cell-initiated response pathway. Invention Overview

[0028] This invention relates to vaccines for use in the prevention and / or treatment of diseases, comprising an adenovirus vector capable of encoding virus-like particles (VLPs) exhibiting an inactive immunosuppressive domain (ISD).

[0029] Many viral vectors used to generate VLPs are employed in vaccine development, including HIV, baculoviruses, lentiviruses, and adenoviruses. The inventors have shown that adenovirus vectors encoding ERVs with inactivated ISDs unexpectedly exhibit superior performance compared to, for example, HIV vectors combined with inactivated ISDs. Therefore, the present invention provides an unexpectedly high immune response, leading to the promotion of immunosuppression in tumors.

[0030] While any adenovirus vector is expected to perform satisfactorily in this invention, the current view is that optimal results are obtained when the adenovirus vector is derived from mammalian adenovirus types, human adenovirus types, chimpanzee adenovirus types, or gorilla adenovirus types. Human adenovirus vectors exist in at least 52 different serotypes, such as types 1, 2, 5, 19, 28, 35, and 40. When selecting human adenovirus, the adenovirus vector is derived from group D vectors, human adenovirus serotype Ad5, human adenovirus serotype Ad19a, human adenovirus serotype Ad26, or chimpanzee adenovirus serotypes. Due to favorable preclinical immunization results, the inventors have used adenovirus type 5 (Ad5) as the starting point for this vaccine vector. The reason why Ad5 induces a sufficiently strong immune response against the target protein is not only due to its efficient transport into antigen-presenting cells (APCs) but also to the adjuvant properties of the vector itself that stimulate innate immunity. In addition, transcription and release of immunostimulatory cytokines such as IFN, IL-6, IL-12, IL-15, and TNF-α are induced. These cytokines play important roles in the immune system and act as cellular activators of adaptive immune responses. A particular advantage of Ad5 is that the immune response against the vector is less intense, as this prevents transgene expression. Ad5 balances the innate immune response to a level that allows transgene expression while still activating the adaptive immune response. Considering the publication by Matthew J. Johnson et al. (J Immunol 2012; 188: 6109-6118), which showed that recombinant adenovirus serotypes 28 and 35 more effectively infected and led to in vitro maturation and activation of both human and mouse dendritic cells compared to recombinant adenovirus serotype 5, it is surprising that Ad5 showed the required response in the experiments reported in this paper. Additionally, another paper by Matthew J. Johnson et al. (Vaccine 32 (2014) 717–724) showed that, relative to rAd5 and control cells with simulated infection, recombinant adenovirus serotypes 28 and 35 increased apoptosis of antigen-presenting cells (APCs), such as monocytes.

[0031] Immunosuppressive domains (ISDs) can be viewed as a mechanism for balancing antitumor immune responses in tumors while preserving the tumor-promoted inflammatory environment induced by ERV activation, similar to natural infection. ISDs affect both the innate and adaptive immune systems through the suppression of macrophages, NK cells, and T cells. However, the detailed mechanisms of immunosuppression via ISDs are not fully understood. As demonstrated by this invention, inactivation of ISDs significantly increases the response.

[0032] The ISD region can be inactivated by mutation or deletion of one or more amino acids. In the case of inactivation by mutation, one or more amino acids are exchanged for different amino acids, typically selected from the other 19 naturally occurring amino acids. In the case of deletion, any one or more amino acids in the ISD region may be missing. Those skilled in the art have sufficient knowledge and experience to optionally determine which amino acids to exchange through preliminary experimental evaluation in order to obtain a satisfactory immune response.

[0033] In some embodiments of the 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), which has at least one amino acid deletion or exchange with a different amino acid. Preferably, the amino acid different from the original is selected from naturally occurring amino acids. The ISD segment of the ERV encoded in Ad5 used in the embodiments of this application has the following amino acid sequence: LQNRRGLDLLFLKEGGL (SEQ ID No. 7). The ISD can be inactivated by performing one or more mutations in the amino acid sequence. Although those skilled in the art can modify amino acid sequences by performing any number or form of mutations or deletions, it is currently suitable to exchange a single amino acid, i.e., the ISD preferred in this invention has the following sequence: LQNRRGLDLLFLKRGGL (SEQ ID No. 8).

[0034] Exchanging one or more amino acids in a region upstream or downstream of the ISD segment may be preferred. The mutation is a compensatory mutation, intended to preserve the structure of the domain so that it can still function against infectious viruses. Therefore, in one embodiment, at least one amino acid in a region of 10 amino acids upstream or downstream of the ISD is exchanged with a different amino acid. Figure 3In the specific implementation shown, the third amino acid flanking the ISD region is exchanged for an A->F mutation.

[0035] In order to inactivate ISD according to the present invention, the immunosuppressive capacity needs to be reduced by 70% or more compared to the immunosuppression performed by the original ISD. In a preferred embodiment of the invention, the ISD is inactivated by 80% or more, for example 90% or more, for example 95% or more, for example 99% or more, compared to the immunosuppression performed by the original ISD.

[0036] This invention provides a universal platform for displaying antigens against the body's immune system. Therefore, in principle, the encoding of any type of protein against which an immune response is desired can be incorporated into an adenoviral vector. In a preferred aspect of the 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 vaccines encoding virus-like particles direct ERV Env to dendritic cells (DCs), which present the antigen to cells of the adaptive immune system. Presentation on MHC class I induces activation and proliferation of CD8+ T cells. These cytotoxic T lymphocytes (CTLs) specific to the ERV Env antigen infiltrate tumors and kill cells displaying the corresponding antigen. Antigen presentation on MHC class II by specialized antigen-presenting cells (APCs) activates CD4+ T cells, which subsequently co-activate B cells. Activated B cells encountering ERV Env target proteins or antigens displayed on VLPs in circulation release antibodies specific to the ERV Env. These antibodies are able to bind to their targets on cancer cells, inducing destruction and phagocytosis of malignant cells. In this way, ERV-specific antibodies can prevent tumor growth and metastasis. The restored immunogenicity of tumor cells enables the induction of a diverse group of tumor-specific T cells that recognize different tumor-associated antigens and tumor-specific antigens. Newly induced and expanding CTLs infiltrate the tumor and kill malignant cells.

[0037] Although this vaccine can in principle be used to immunize many mammalian species, and has actually been developed using mouse models, in a preferred aspect of the invention, the ERV protein is a human endogenous retrovirus (HERV) protein or an immunogenic portion thereof. It is estimated that each human genome consists of approximately 8% endogenous retroviral DNA. However, most endogenous retroviral DNA is merely a remnant of previous retroviruses. ERV is evidence of ancient retroviral infection in ancient ancestors. After infection, viral RNA is reverse transcribed into proviral DNA, which integrates into the host genome. Eventually, the provirus integrates into the cells of the germline and becomes heritable, producing an endogenous retrovirus. Over millions of years, the viral DNA has been passed down through generations and become fixed in the population. As a result, a large portion of the human genome can potentially be used as the antigen-coding portion of an adenovirus vector. Currently, HERV is preferably selected from HERV-K, HERV-H, HERV-W, HERV-FRD, and HERV-E. More specifically, HERV-K can be selected from 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); HERV-H can be selected from HERV-H19 (=HERV-H_2q24.3) and HERV-H_2q24.1; HERV-W can be selected as ERVW-1 (=syncytokine-1); and HERV-FRD can be selected as ERVFRD-1 (=syncytokine-2).

[0038] This construction of the adenoviral vector allows for the presentation of the encoded ERV protein to the immune system to establish an appropriate immune response. In a suitable aspect of the invention, the ERV protein epitope or its immunogenic portion is positioned between the transmembrane domain and the ISD.

[0039] The experiments reported in this paper demonstrate the applicability of adenovirus-encoded ISD-mutated HERV-K VLPs not only in Ad5 but also in another adenovirus serotype, Ad19 (see Examples 15-17). Previously, HERV-K has been associated with cancer expression and has been shown to contain a functional enveloped ISD domain with in vitro activity similar to HIV (Morozov et al. 2013). In mice, HERV-K is a foreign antigen with similar ISD domain activity, and ISD mutations do not a priori predict an enhanced immune response. However, surprisingly, ISD mutations were found to increase antibody responses, T-cell responses, and anti-cancer protection against HERV-K Env p15E and SU domain proteins. The mutations in HERV-K differ from those disclosed in this paper for MelARV because the viral families differ in their ISD sequences. However, based on the information provided herein and general common sense, those skilled in the art can also identify suitable mutations that inactivate ISD in other viral families. The HERV-K mutation used in this study was inspired by the ISD mutation in HIV, which is shown to preserve viral infectivity and site-specific conservation between HERV-K and HIV-1 (Morozov et al. 2012). Analysis of vector-transfected cells revealed increased intracellular and cell surface expression of the HERV-K mutation (see Example 15 and...). Figure 24 This can help explain the increased immunogenicity and provides an additional mechanistic principle for the preparation of ISD mutations in HERV-K family Env proteins using any gene expression platform and construct that may or may not form VLPs.

[0040] Therefore, the present invention also relates toA nucleic acid molecule encoding an ERV envelope protein or its immunogenic portion, wherein the ISD of said protein contains a mutation causing inactivation of the ISD. Preferably, the ERV is a human endogenous retrovirus (HERV), more preferably, the HERV is HERV-K. Further preferably, the mutation in the ISD replaces Q525 with an alanine residue, such that the sequence of the mutated ISD becomes NSQSSIDQKLANAINDLRQT (SEQ ID No. 50) (instead of NSQSSIDQKLANQINDLRQT; SEQ ID No. 49). It should be understood that corresponding mutations with different sequences in the ISD are also contemplated. In a further preferred embodiment, the nucleic acid molecule is contained in an adenovirus vector. More preferably, the adenovirus vector is adenovirus vector type 19 (Ad19). Further preferably, the adenovirus vector containing the nucleic acid encodes a VLP. The invention further relates to a protein encoded by said nucleic acid molecule or said vector. The nucleic acid molecule, vector, or encoded protein is intended for use in the treatment or prevention of a disease, preferably cancer. The cancer to be treated is a cancer expressing the corresponding ERV. Preferably, the treatment comprises a "trigger-enhancement regimen," wherein a trigger containing an adenovirus or nucleic acid molecule is first administered, followed by a subsequent enhancement of MVA, adenovirus, or DNA. Preferably, the enhancement is an MVA enhancement. Different timings for triggering and enhancement are envisioned. Longer intervals between triggering and enhancement are possible, particularly in cancer patients with minimal residual disease. In a preferred regimen, the enhancement is administered 4 to 8 weeks after triggering.

[0041] In a preferred aspect of the vaccine according to the invention, the protein product of the adenovirus vector includes a gag protein, a 2A peptide, and an envelope protein (Env). Furthermore, the Env protein may include a surface unit (gp70), a cleavage site, and a transmembrane unit (p15E). Additionally, the transmembrane unit (p15E) may include a fusion peptide, an immunosuppressive domain (ISD), a transmembrane anchor, and a cytoplasmic tail.

[0042] To improve the immunosuppression of vaccines, conjugating p15E or its immunogenic moiety to the adenovirus capsid protein pIX may be suitable. To achieve this, the N-terminus of p15E is fused to the C-terminus of pIX. The highly ordered structure of pIX and its binding antigen on the adenovirus surface facilitate cross-linking of the B-cell receptor. As another advantage, pIX is typically displayed as a trimer and can also help present the bound p15E antigen in its natural trimer form. This modification has been shown to increase the induction of specific antibodies in CD1 mice.

[0043] In one aspect of the invention, a signal peptide encoded by an adenovirus vector interacts with a signal peptide derived from Gaussian (… GaussiaLuciferase signal peptide (LucSP) exchange. This signal peptide increases the transport of proteins to the extracellular membrane without altering their glycosylation state. Therefore, including this signal peptide, rather than the native sequence, has guided synthetic proteins to the membrane, where they are integrated into the VLP.

[0044] In another aspect of the invention, the transmembrane anchor and cytoplasmic tail encoded by the adenoviral vector are exchanged with the transmembrane domain and cytoplasmic tail of influenza A virus hemagglutinin. This insertion increases the expression of the recombinant protein on the cell surface and VLP, leading to a strong and broad antibody response. In a preferred embodiment, the transmembrane anchor and cytoplasmic tail encoded by the adenoviral vector are exchanged with the transmembrane domain and cytoplasmic tail of influenza A virus hemagglutinin H3N2 (HA-TMCT).

[0045] In another aspect of the invention, the trimerizing sequence is provided near the signal peptide. The trimerizing sequence can be incorporated into the protein to facilitate natural presentation. In a preferred aspect, the trimerizing sequence is GCN4.

[0046] The protein products of adenovirus vectors typically contain gag proteins, which are either exogenous or endogenous retroviral gag proteins.

[0047] Adenoviral vectors typically require cells to produce virus-like particles. Therefore, the adenoviral vector infects cells and produces components for VLPs. In one aspect of the invention, VLPs are produced in isolated cell lines. Suitable examples include Sf9 cells, Vero cells, HeLa cells, etc. However, it is currently desirable to produce VLPs in cells within a patient already infected with an adenoviral vector. This production is also known as virus-encoded virus-like particles (VE-VLPs) and has the advantage of avoiding an intermediate host for VLP production.

[0048] The present invention also relates to nucleic acid constructs encoding target proteins capable of forming virus-like particles (VLPs), wherein the target proteins comprise an immunosuppressive domain (ISD) that is inactive.

[0049] This invention is particularly suitable for the prevention and / or treatment of cancer. There are no particular limitations on the types of cancer that can be treated by this invention, and it includes prostate cancer, breast cancer, ovarian cancer, lymphoma, melanoma, leukemia, sarcoma, colorectal cancer, testicular cancer, ovarian cancer, breast cancer, lymphoma, lung cancer, and liver cancer.

[0050] Under certain conditions, treating patients with a priming-boost regimen may be advantageous. Therefore, in one embodiment of the invention, the use of a vaccine in the prevention and / or treatment of cancer includes a step of priming a patient with a nucleic acid construct at least 5 days prior to boosting with the vaccine disclosed above.

[0051] The present invention also relates to a vaccine for use in the prevention and / or treatment of cancer, comprising the step of post-treating a patient with a virus-encoded VLP different from that of an adenovirus vector, five days or more after exposure to the vaccine disclosed above. In one embodiment, the virus-encoded VLP different from that of an adenovirus vector is a VLP derived from Modified Vaccina Ankara (MVA).

[0052] Furthermore, surprisingly, contrary to what was reported by Hohn et al. (2014) regarding codon optimization of HERV-K113 under the CMV promoter, the expression cassette used, namely Gag-p2A-Env, where Env was again expressed in a 1:1 ratio with Eng under a strong promoter, did not result in retention at the cell membrane. Instead, VLP expression, which (again, contrary to the results reported by Hohn et al.), also contained Env. This shows that the genetic platform with Gag-p2a-Env performed better than constructs without p2a (or the corresponding operable adapter).

[0053] Therefore, the present invention further relates to nucleic acid molecules encoding Gag protein and ERV envelope protein (Env) or their immunogenic portions, wherein the native genomic structure connecting Gag and Env has been replaced with a operative linker. Preferably, the operative linker is p2A. In other words, the present invention also relates to nucleic acid molecules comprising a Gag operative linker-Env expression cassette, preferably a Gag-p2A-Env cassette. Preferably, the ERV is HERV-K. More preferably, the ERV is HERV-K113. Further preferably, the HERV-K sequence is a HERV-K common sequence, more preferably a codon-optimized common sequence. Even more preferably, the HERV-K codon-optimized common sequence is the following amino acid sequence (SEQ ID No. 55):

[0054]

[0055] Further preferred sequences contain mutations in their ISD (underlined and bolded in the sequence above). Particularly preferred sequences containing such mutations are shown in SEQ ID No. 48.

[0056] Further preferred nucleic acid molecules are adenovirus vectors. It is envisioned that nucleic acids could be used as gene vaccines, particularly in the prevention and / or treatment of disease-preferred cancers. Alternatively, nucleic acid molecules can also be used to generate VLPs in vitro, particularly HERV-K VLPs. The resulting VLPs can then be used in immunotherapy, particularly in the prevention and / or treatment of disease-preferred cancers. It should be understood that, also in this context, the cancer to be treated is an ERV-expressing cancer.

[0057] Furthermore, the present invention 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 natural genomic structure linking Gag and Env has been replaced with a operative linker. Preferably, the operative linker is p2A. More preferably, the ERV is HERV-K. More preferably, the ERV is HERV-K113. Preferably, the VLP contains a higher amount of Env compared to a HERV-K113 VLP produced according to the method described by Hohn et al. As mentioned above, the use of such VLPs in immunotherapy is envisioned. Furthermore, the present invention relates to nucleic acid molecules or VLPs for use in the prevention and / or treatment of a disease. Preferably, the disease is cancer. It should be understood that cancer is cancer expressing a corresponding ERV. Brief description of the attached diagram

[0059] In the following detailed sections of this disclosure, aspects, embodiments, and implementations are explained in more detail with reference to the exemplary embodiments shown in the accompanying drawings, wherein:

[0060] Figure 1 The mechanism of virus-like particle (VLP) encoded by a viral vector is disclosed. The vaccine comprises a recombinant adenovirus (Ad5) encoding the viral proteins Gag and Env. Upon injection, Ad5 infects cells and induces the expression of the encoded proteins. Gag and Env are coupled via a self-cleavable peptide (p2A), which ensures equimolar expression of the two proteins and their separation during translation. The structural protein Gag alone is sufficient to induce cell membrane budding and the formation of virus-like particles (VLPs). During VLP formation, Env binds to Gag and integrates into the released VLP. Thus, vaccination with the Ad5 vector induces the production of VLPs that display the target protein Env to the immune system on their surface.

[0061] Figure 2A schematic structure of the MuLV / MelARV envelope protein is shown. The envelope protein (Env) consists of two subunits. (Left) The transmembrane subunit p15E(TM) is anchored in the cell membrane and contains an immunosuppressive domain (ISD) and a fusion peptide. p15E is covalently coupled to the surface subunit gp70(SU) via a disulfide bridge. p15E, and especially ISD, is shielded by gp70 to prevent antibody binding. (Right) The protein subunit is expressed as a precursor protein that is cleaved and transported to the membrane during processing. This figure was modified by Mangeney et al. 2007.

[0062] Figure 3 The mutation in the ISD of the vaccine-encoded MelARV Env (p15E) is shown. Two amino acids in the ISD of p15E are mutated to inactivate the immunosuppressive mechanism. Therefore, the following amino acid change is performed: E 14 →R 14 and A 20 →F 20 .

[0063] Figure 4 The vector diagrams for 768tet and Capture-pBGH are shown. The DNA vectors 768tet (A) and Capture-pBGH (B) contain the relevant genes. Other genes present in the plasmids are not shown in the plasmid diagrams. First, the target protein was cloned into the expression vector 768tet (A). Subsequently, through homologous recombination, the expression cassette containing the target protein was cloned into the human Ad5 (hAd5) genome vector Capture-pBGH (B) to generate recombinant viruses in production cell lines.

[0064] Figure 5 The steps for generating recombinant Ad5 are disclosed. The protocol demonstrates the process of cloning the target protein into Capture-pBGH, followed by viral generation. The generation of recombinant Ad5 involves a series of steps to produce “viral lysis products,” “3-day lysis products,” and “large-scale lysis products” containing the recombinant virus.

[0065] Figure 6 : Peptide used for ELISA analysis of p15E-specific antibody response. The region of MelARV Env pointed to as TM (p15E) is synthesized as peptide and used for ELISA analysis of serum samples from vaccinated mice.

[0066] Figure 7: Antibody response 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 (pictured below), followed by booster doses of different Ad5 vaccines (legend). The vaccines used for booster doses were Ad5-MelARV (dark gray), Ad5-MelARV-ISD (light gray), or Ad5-GFP (white). Antibody binding to p15E was analyzed by ELISA. Bar graphs show mean absorbance relative to LEV76 control serum versus SEM. Sample size n = 5 in each group. (B) B16F10-GP-specific antibodies. B16F10-GP cells were incubated with serum from the same mice as in (A). Antibody binding was detected by flow cytometry using an APC-conjugated secondary antibody against mouse IgG. Mean fluorescence intensity for each vaccine group is shown as mean versus SEM. An asterisk indicates a significant difference between groups, where * (P ≤ 0.05); ** (P ≤ 0.01); *** (P ≤ 0.001).

[0067] Figure 8: Antibody response 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 the priming-boost regimen, according to vaccination timeline III. (A) Binding of cancer-specific antibodies to B16F10-GP cells. Antibodies in the serum of mice vaccinated for tumor cell specificity were analyzed by flow cytometry using an APC-conjugated secondary antibody against mouse IgG. Bar graphs show the mean fluorescence intensity of bound antibodies in each group. (B) Analysis of antibodies binding to p15E by ELISA. Values ​​show the mean for each group compared to SEM. (C) Vaccinated mice were challenged iv with B16F10-GP cells, and lung metastases were analyzed 14 days later. Horizontal lines indicate the mean number of metastases in each group. The groups consisted of n=7 (DNA + Ad5-MelARV) or n=8 (DNA + Ad5-MelARV-ISD) mice.

[0068] Figure 9ELISPOT analysis of T cell responses induced by Ad5-MelARV-ISD in Balb / C mice. Spleens from Balb / C mice were isolated 21 days after a single vaccination with Ad5 (Ad5-MelARV or Ad5-MelARV-ISD). Spleen cells were stimulated with AH1, and activated immune cells were detected by IFNγ production in the ELISPOT assay. Results were calculated as the number of spots per 10⁶ spleen cells (cells producing IFNγ). Bar graphs indicate the mean number of spots per group (n=5) versus SEM. Asterisks indicate significant differences from the PBS control, where * (P ≤ 0.05); ** (P ≤ 0.01); *** (P ≤ 0.001).

[0069] Figure 10: Intracellular staining (ICS) analysis of Ad5-MelARV-ISD-induced T cell responses in Balb / C mice. Intracellular staining (ICS) following AH1 stimulation was used to analyze the production of cytokines IFNγ and TNFα in T cells. Figure 9 The same spleen cells. This figure shows the total number of activated (CD44+) CD8+ T cells producing IFNγ or TNFα throughout the spleen. (A) IFNγ-positive CD8+ T cells. (B) TNFα-positive CD8+ T cells. (C) The integral geometric mean of IFNγ-producing CD8+ T cells per mouse was calculated by multiplying the number of IFNγ+ CD8+ T cells by the mean fluorescence intensity of IFNγ+ cells. (D) Double-positive CD8+ T cells. The horizontal line indicates the mean for each group. An asterisk indicates significant differences between groups, where * (P ≤ 0.05); ** (P ≤ 0.01); *** (P ≤ 0.001).

[0070] Figure 11 Titers of Ad5-specific antibodies in CD1 mice vaccinated with Ad5-MelARV versus Ad5-MelARV-ISD. CD1 mice were vaccinated with either Ad5-MelARV or Ad5-MelARV-ISD according to the IV vaccination timeline. Serum Ad5-specific antibodies were analyzed by ELISA using an ELISA plate coated with Ad5 particles. Serum from each mouse was tested at a 1:2 serial dilution to obtain antibody titers. The cutoff value for positive results was 4 times the background OD450. Bar plots show the mean titer for each group versus SEM. Groups contained n=5 mice. Asterisks indicate significant differences between groups, where * (P ≤ 0.05); ** (P ≤ 0.01); *** (P ≤ 0.001).

[0071] Figure 12: An excerpt from the amino acid sequence of p15E displayed on the adenovirus pIX protein. The complete sequence is represented in the sequence listing as: pIX-p15E (SEQ ID NO: 51; pIX-p15E-ISD (SEQ ID NO: 52), pIX-p15E-truc-wC (SEQ ID NO: 53); pIX-p15E-trunc-w / oC (SEQ ID NO: 54).

[0072] Figure 13: Characterization of the adenoviral vector for recombinant pIX. (A) The plasmid pcDNA3-pIX-Taglinker-xxx encoding recombinant pIX was transfected into HEK293 cells to verify correct expression. Cell lysis products of transfected cells were analyzed by Western blotting using an anti-pIX antibody. Row 1) pIX-p15E, Row 2) pIX-p15E-ISD, Row 3) pIX-p15E_trunc-wC, Row 4) pIX-p15E_trunc-w / oC, GFP row pIX-GFP. (B) Integration analysis of the generated and purified virus for recombinant pIX using Western blotting using an anti-pIX antibody. Row numbers indicate the same pIX modification as shown in (A) on the Ad5 vector, while row Ø indicates native Ad5 without pIX modification.

[0073] Figure 14: Antibody response in CD1 mice vaccinated with Ad5-pIX vaccine. (A) The pIX-modified Ad5 vaccine (striped stripes) was tested in CD1 mice (vaccination timeline IV) and compared with its unmodified counterpart (solid stripes). Adenovirus (Ad5-MelARV or Ad5-MelARV-ISD displaying natural or recombinant pIX) was tested on DNA-initiated vaccination with DNA-MelARV or DNA-MelARV-ISD. GFP-vaccinated mice served as negative controls. Binding of the antibody to the peptide of the MelARV Env transmembrane subunit p15E was evaluated at 450 nm and normalized to absorbance against standard LEV76 control serum. (B) The same serum sample as (A) was analyzed for binding to B16F10-GP cancer cells. The binding antibody was detected by flow cytometry with an APC-conjugated secondary antibody against mouse IgG and quantified by mean fluorescence intensity. LEV76 control serum and secondary antibody only (2.Ab only) served as positive and negative controls, respectively. Bar graphs show the mean versus SEM for each group (n=5). Asterisks indicate significant differences between groups, where * (P ≤ 0.05); ** (P ≤ 0.01); *** (P ≤ 0.001).

[0074] Figure 15Antibody response and metastasis counts in C57BL / 6 mice vaccinated with Ad5-MelARV_pIX-p15E. Mice were vaccinated with Ad5-MelARV_pIX-p15E or the natural form of this virus (Ad5-MelARV) according to vaccination timeline V. Mice vaccinated with GFP served as negative controls. (A) Antibody response of B16F10-GP tumor cells in the serum of vaccinated mice was analyzed by flow cytometry. LEV76 control serum was included as a positive control. Tumor cells incubated with only secondary antibody (2.Ab only) served as negative controls. (B) p15E-specific antibody response was analyzed by ELISA. Absorbance measured at 450 nm was normalized against LEV76 control serum. Each group in (A) and (B) contained n=5 mice. The values ​​shown are the mean for each group versus SEM. (C) Number of tumor metastases in vaccinated mice after challenge with B16F10-GP cells. The horizontal lines indicate the mean for each group. (D) Correlation between B16F10-GP specific antibody and transfer count. (E) Correlation between p15E specific antibody and transfer count. The negative control (GFP control) is not included in the correlation calculation.

[0075] Figure 16 Vaccine Improvement Strategy: Chimeric MelARV Env proteins with functional domains to improve display on VLPs. Two modified vaccines were produced using either the full-length MelARV Env (Ad5-LucSP_MelARV_Ha-TMCT) or the separate p15E (Ad5-LucSP_GCN4_p15E_Ha-TMCT). In Ad5-LucSP_MelARV_HA-TMCT, the native signal peptide of MelARV Env was replaced with the luciferase signal peptide (LucSP). Furthermore, the native transmembrane domain and cytoplasmic tail (TMCT) were altered to correspond to the sequences of influenza A virus hemagglutinin H3N2 (HA-TMCT). In Ad5-LucSP_GCN4_p15E_HA-TMCT, only p15E was encoded, instead of the full-length Env protein. p15E also contained HA-TMCT and had LucSP added at the N-terminus. Additionally, a trimerized sequence (GCN4) was included.

[0076] Figure 17MelARV Env expression on cells following infection with recombinant Ad5 encoding a chimeric MelARV Env protein. The expression of target proteins in infected Vero cells was tested using vaccine viruses with modified MelARV Env sequences (Ad5-LucSP_MelARV_Ha-TMCT and Ad5-LucSP_GCN4_p15E_Ha-TMCT). For comparison, Ad5-MelARV and Ad5-MelARV-ISD were also included. Vero cells were infected with the modified viruses, and target protein expression on cells was analyzed using different antibodies against MelARV Env: (A) 19F8 (anti-p15E, targeting ISD), (B) 4F5 (anti-p15E), (C) MM2-9B6 (anti-gp70), (D) MM2-3C6 (anti-gp70), and (E) MM2-9A3 (anti-gp70). The binding of antibodies to infected cells was detected by flow cytometry using separate fluorescently conjugated secondary antibodies. Each bar (where n=1) represents the average fluorescence intensity induced by the fluorescently conjugated antibody.

[0077] Figure 18 Analysis of target protein expression and VLP release in cells infected with Ad5 encoding chimeric MelARV Env (Western blot): Vero cells were infected with a modified virus. Cell lysis products and released VLPs were analyzed for target protein expression by Western blotting with different antibodies: (A) anti-p2A (MelARV Gag), (B) 4F5 (anti-p15E), (C) MM2-9B6 (anti-gp70). Additionally, the supernatant of infected cells was analyzed for the secretion of p15E (4F5) (D) and gp70 (MM2-9B6) (E) by Western blotting. Row 1) Ad5-MelARV, Row 2) Ad5-MelARV-ISD, Row 3) Ad5-LucSP_GCN4_p15E_Ha-TMCT, Row 4) Ad5-LucSP_MelARV_Ha-TMCT, Row Ø: Negative control virus. Expected band sizes are listed in Table 6.

[0078] Figure 19: Analysis of target protein expression and VLP release in cells infected with Ad5 encoding chimeric MelARV Env (ELISA): Vero cells were infected with the original virus and modified viruses: Row 1) Ad5-MelARV, Row 2) Ad5-MelARV-ISD, Row 3) Ad5-LucSP_GCN4_p15E_Ha-TMCT, Row 4) Ad5-LucSP_MelARV_Ha-TMCT, Row Ø negative control virus. ELISA plates were coated with cell lysates, supernatant (SN), or purified VLP from infected Vero cells. The presence of MelARV Env and Gag proteins was detected by binding to primary antibodies (anti-p2A, MM2-9B6, 4F5, and 19F8). (A) Anti-p2A antibody showed Gag expression. (B) MM2-9B6 binding revealed expression of the MelARV Env surface subunit gp70. (C)(D) 4F5 and 19F8 (ISD binding) bind to the transmembrane subunit p15E.

[0079] Figure 20 This shows a comparison with HIV ISD antibodies.

[0080] Figure 21 This shows a comparison of HIV ISD-T cells, in which...

[0081]

[0082] Figure 22 The strategy followed to improve vaccine design involves a point mutation at the ISD domain (p15E) of the HERV-K Env protein encoded in the vaccine. Glutamine (Q) (see “Ad19_HERV-K”; coding sequence shown in SEQ ID No. 43) is mutated to alanine (A) (“HERV-K-ISD”; coding sequence shown in SEQ ID No. 44) to inactivate the ISD domain that mediates immunosuppressive effects. This figure is modified from (Mangeney et al. 2007).

[0083] Figure 23Detection of HERV-K Env and Gag proteins (VLPs) in SN and cell lysis products from virus-transfected cells. The presence of functional Gag (A) and Env (B) proteins in SN and cell lysis products from A549 and VERO cells transfected with Ad19_HERV-K WT / ISDmut is highlighted by square boxes. Molecular weights of approximately 90, 80, and 40 kDa are equivalent to the values ​​of 80, 90, 80, and 42 kDa reported for HERV-K Gag, full-length unprocessed precursors of HERV-K Env (with and without signal peptide), and HERV-Kp15E (TM, Env), respectively, as shown in the table below (Opstelten DJ, et al., J Virol. 1998 Aug;72(8):6537-45).

[0084] .

[0085] Furthermore, the Ad5_MelARV_Gag protein (65 kDa) was also detected in the cell lysis products and SN of both cell lines (A1 and A2), indicating that both HERV-K and MelARV Gag proteins can be recognized by the same rabbit polyclonal anti-p2A antibody.

[0086] Figure 24 HERV-K Env expression inside and on the cell surface after transfection with Ad19_HERV-K_WT / ISDmut. The HERM-1811 antibody was used to visualize the production and presence of HERV-K Env protein both inside and on the cell surface of A549-infected cells. Cells infected with Ad19_HERV-K Env WT (medium gray transparent) / ISDmut (dark gray transparent) expressed high levels of HERV-K Env, while cells infected with the Ad5 vector encoding HERV-K Env (very light gray transparent) showed lower expression of the target protein, suggesting that Ad19 transfection may be more efficient than Ad5. Cells infected with an irrelevant antigen encoded by the Ad19 vector (light gray) were consistent with uninfected cells (dark gray) and therefore showed no signal.

[0087] Figure 25ICS analysis of CD8+ T cell responses induced by Ad19-HERV-K in BALB / c mice. This figure shows the total number of activated (CD44+) CD8+ T cells secreting TNFα contained in the spleen of each mouse. (A) Number of IFNγ-positive CD8+ T cells from mice immunized with different adv vaccines (priming-boost), followed by non-boost (Ø) and MVA_Env booster regimens. (B) Percentage of IFNγ and TNFα double-positive CD8+ T cells. (C) Mean fluorescence intensity (MFI) of IFNγ-positive CD8+ T cells. Mean values ​​for each group of mice are indicated by a horizontal line. An asterisk (*) indicates a significant difference where (P ≤ 0.05); ** (P ≤ 0.01); *** (P ≤ 0.001).

[0088] Figure 26 Survival curves of tumor-challenged mice that received therapeutic vaccines. The efficacy of our Ad19_HERV-K WT / ISDmut vaccine (medium and dark gray) in reducing or preventing tumor growth and metastasis was tested in BALB / c mice challenged with RENCA cells expressing the HERV-K Env target protein. Our vaccine was compared with an irrelevant vaccine that does not express HERV-K (black) and an MVA vaccine that does express the target protein (light gray). The lungs of euthanized mice at day 40 (final endpoint) were blinded to assess the progression of lung metastases. Kaplan-Meier estimators were used to analyze the survival of mice in different groups of vaccinated mice. Survival curves were compared using different statistical tests (time series, Wilcoxon, and Tarone-Ware), and a p-value < 0.05 was considered significant (*).

[0089] Figure 27 Gating strategy. Black gates with arrows indicate which groups are used for gating in the diagram below. This image was prepared from positive results from BALB / c mice immunized with an Adv-based vaccine (initiator) + MVA Env (booster).

[0090] Figure 28HERV-K staining of human breast cancer tissue (H841). Tissue samples were obtained from human breast tumors. They were sectioned at 4 μm and stained with a 1:1000 dilution of a primary antibody derived from (A) unimmunized mice (pre-bleeding serum) and (B) Ad5_HERV-K_Env-induced mice boosted with Ad19_HERV-K_ISD (8 weeks post-immunization) and MVA_Env (2 weeks post-immunization) vaccination regimens. Subsequently, biotin-labeled anti-mouse secondary antibody was used at a 1:500 dilution, and cancer cells were finally stained with hematoxylin / eosin. HERV-K-specific staining (dark gray) is clearly visible in the right-hand histological section, confirming that high-titer HERV-K antibodies from vaccinated mice can stain cancer tissue expressing HERV-K target proteins.

[0091] Figure 29 Morphology of VLPs secreted by transfected cells. A549 cells were transfected with Ad19a-HERV-KISDmut encoding the Gag_p2A_Env protein. Cells were fixed after 24 hours, and the released VLPs (circles of approximately 100 nm) were observed using transmission electron microscopy. Invention Details

[0093] The following shows the natural sequence, where the individual elements of the sequence are indicated as follows:

[0094] signal peptide

[0095] Surface subunits

[0096] transmembrane subunits

[0097] Immunosuppressive domain (ISU / ISD)*

[0098] Transmembrane domain

[0099] Cytoplasmic tail

[0100] This invention covers sequences in which one, two or more amino acids of an immunosuppressive domain are exchanged with another naturally occurring amino acid.

[0101] 1. HERV-K108 (=ERVK-6) possessing the amino acid sequence of the Env protein (SEQ ID No. 9):

[0102]

[0103] And the Gag protein with the amino acid sequence (SEQ ID No. 10):

[0104]

[0105] 2. Contains the amino acid sequence of the Env protein (SEQ ID No. 11). ERVK-19 :

[0106]

[0107] And the Gag protein with the amino acid sequence (SEQ ID No. 12):

[0108]

[0109] 3. Contains the amino acid sequence of the Env protein (SEQ ID No. 13). HERV-K115 (=ERVK-8) :

[0110]

[0111] Furthermore, the Gag protein has the following amino acid sequence (SEQ ID No. 14):

[0112]

[0113] 4. Possesses Env protein The amino acid sequence (SEQ ID No. 15) ERVK-9 :

[0114]

[0115] And the amino acid sequence of the Gag protein (SEQ ID No. 16):

[0116]

[0117] 5. , possessing Env protein The amino acid sequence (SEQ ID No. 17) HERV-K113 :

[0118]

[0119] And the amino acid sequence of the Gag protein (SEQ ID No. 18):

[0120]

[0121] 6. Possesses Env protein The amino acid sequence (SEQ ID No. 19) ERVK-21 :

[0122]

[0123] have Env proteinThe amino acid sequence (SEQ ID No. 20):

[0124]

[0125] 7. Possesses Env protein The amino acid sequence (SEQ ID No. 21) ERVK-25 :

[0126]

[0127] 8. Possesses Env protein The amino acid sequence (SEQ ID No. 22) HERV-K102 = ERVK-7 :

[0128]

[0129] And has Gag protein The amino acid sequence (SEQ ID No. 23):

[0130]

[0131] 9. Possesses Env protein The amino acid sequence (SEQ ID No. 24) HERV-K101 = ERVK-24 :

[0132]

[0133] And has Gag protein The amino acid sequence (SEQ ID No. 25):

[0134]

[0135] 10. Possesses Env protein The amino acid sequence (SEQ ID No. 26) HERV-K110 = ERVK-18 :

[0136]

[0137] 11. Possesses Env protein The amino acid sequence (SEQ ID No. 27) HERV-H19 = HERV-H_2q24.3 :

[0138]

[0139] 12. Containing the amino acid sequence of the Env protein (SEQ ID No. 28) HERV-H_2q24.1 :

[0140]

[0141] And has Gag protein The amino acid sequence (SEQ ID No. 29):

[0142]

[0143] 13. Containing the amino acid sequence of the Env protein (SEQ ID No. 30) HERV-W = ERVW-1 = Syncytidine- 1 :

[0144]

[0145] 14. Contains the amino acid sequence of the Env protein (SEQ ID No. 31). HERV-FRD = ERVFRD-1 = combined Cytokin-2 :

[0146]

[0147] 15. Containing the amino acid sequence of the Env protein (SEQ ID No. 32 and 33) HERV-E :

[0148]

[0149] And has Gag protein The amino acid sequence (SEQ ID No. 34 to 38):

[0150]

[0151] 16. Containing the amino acid sequence of the Env protein (SEQ ID No. 339) HERV-E :

[0152]

[0153] And has Gag protein The amino acid sequence (SEQ ID No. 40):

[0154]

[0155] The target cancers of HERV-K are prostate cancer, breast cancer, ovarian cancer, lymphoma, melanoma, leukemia, and sarcoma. The target cancer of HERV-H is colorectal cancer. The target cancers of HERV-W are testicular cancer, ovarian cancer, breast cancer, lymphoma, and leukemia, and the target cancers of HERV-E are lung cancer and liver cancer. Example

[0156] The materials and methods described below are general for the following embodiments.

[0157] The prototype vaccines (DNA-MelARV and Ad5-MelARV) consist 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 expresses the MelARV proteins Gag and Env linked via a self-cleaving peptide p2A. When Gag induces the formation of virus-like particles (VLPs), the target protein Env integrates into the formed VLP.

[0158] Furthermore, the vaccines were designed to target the envelope (Env) protein of human endogenous retrovirus type K (HERV-K or HML-2) expressed in tumor cells, and their induction of cellular and humoral immune responses and anticancer efficacy were tested.

[0159] The designed vaccine comprises a DNA plasmid (768tet), adenovirus type 5 (Ad5), or adenovirus type 19 (Ad19a), each encoding a group-specific antigen (Gag) and an Env gene (HERV-KGag_p2A_Env) under the strong human cytomegalovirus immediate early promoter (CMV promoter). These two proteins are co-expressed with the self-cleavable peptide p2A, which acts as a linker and remains bound to the Gag protein involved in virus-like particle (VLP) formation. The Env protein is incorporated into the formed VLP and acts as a target for generating a specific immune response.

[0160] To improve the induction of immune response in vaccines, an inactivating mutation of ISD in the vaccine-encoded MelARV Env was prepared to prevent immunosuppressive effects through the vaccine itself. Two point mutations were induced in the sequence of the Env transmembrane subunit p15E. Glutamic acid at position 14 of the ISD was replaced by arginine, and alanine at position 20 was changed to phenylalanine. Figure 3 ).

[0161] In HERV-K vaccines, the immune response induced by the vaccine is enhanced by introducing a point mutation, p15E, into the immunosuppressive domain (ISD) of the transmembrane (TM) subunit of the HERV-K Env protein. This modification involves replacing glutamine at position 52 of the ISD with alanine (Schlecht-Louf G, et al., Proc Natl Acad Sci US A. 2010 Feb 23;107(8):3782-7) (see See also Figure 22 This change triggers the inactivation of the structural domain in order to prevent the vaccine itself from producing an immunosuppressive effect.

[0162] Cell culture

[0163] Various cell lines were used in different experiments. All cell lines were maintained in a humidified atmosphere at 37°C and 5% CO2.

[0164] HEK293 HEK293 is derived from human embryonic kidney culture and is generated by transformation with cleaved 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.]. The advantages of this cell line include ease of growth and efficient transfection. Another benefit is the expression of the Ad5 E1 gene [Kovesdi, I. and SJ Hedley, ]. Adenoviral producer cells. Viruses, 2010. 2(8): 1681-703. ] Recombinant Ad5 vaccines are usually administered with a replication defect, which means that they lack genes essential for viral replication, such as E1. In this case, the missing genes must be provided externally during viral production. HEK293 cells provide the proteins required for replication and can therefore be used as producer cells in viral production [Kovesdi, I. and SJ Hedley, Adenoviral Producer cells. Viruses, 2010. 2(8): pp. 1681-703. ] In the current experiment, HEK293 cells were maintained in Dalbeco modified Eagle medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS), L-glutamine (2 mM), sodium pyruvate (1 mM) and penicillin + streptovirin (Pen / Strep).

[0165] HEK293_T-REx_Avtoxic (Avtoxic cells) Avtoxic cells are modified HEK293 cells designed to prevent the expression of recombinant proteins encoded by Ad5 during viral production. Inhibition of these recombinant protein expressions is necessary because some of the encoded target proteins are toxic to HEK293 cells and interfere with viral production [Cottingham, MG, et al.]. Preventing spontaneous genetic rearrangements in the transgene cassettes of adenovirus vectors. Biotechnol Bioeng, 2012. 109(3): 719-28. ] HEK293 cells are modified in two steps to include different protein inhibitory mechanisms. The first mechanism includes inhibition via 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 modified to express a tetracycline repressor protein (Tet repressor), which binds to and inhibits the Tet operon. This resulted in the recombinant target protein being expressed under the control of a strong CMV promoter.

[0166] Because the T-REx system is not entirely effective in preventing target protein expression, the T-REx-293 cell line was further modified by Sirion Biotech GmbH (Martinsried, Germany). The new cell line HEK293_T-REx_Avtoxic (Avtoxic cells) expresses short hairpin RNA (shRNA) that targets a messenger RNA (mRNA) sequence called p2TS transcribed along with the target protein. shRNA promotes the degradation of p2TS containing mRNA, and thus further inhibits recombinant proteins. Avtoxic cells are maintained in DMEM supplemented with 10% heat-inactivated FBS, L-glutamine (2 mM), sodium pyruvate (1 mM), and Pen / Strep.

[0167] HEK293(CCS)-shmir-pIX_221-puro (pIX cells) pIX cells are modified HEK293 cells used to produce Ad5-pIX virus, which displays an antigen 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 to promote the incorporation of recombinant pIX, the HEK293-encoded pIX is inhibited by shRNA expression in pIX cells. ShRNA transcription during virus production is induced by doxycycline. Additionally, a pIX encoding puromycin N-acetyltransferase (PAC) is used. pac Gene-transduced cells were used to select cells expressing shRNA using puromycin. Therefore, cells were maintained in DMEM supplemented with 10% heat-inactivated FBS, L-glutamine (2 mM), sodium pyruvate (1 mM), Pen / Strep, and 0.5 μg / mL puromycin.

[0168] B16F10-GPThe B16 cell line is a mouse melanoma cell line derived 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. It was obtained through 10 rounds of sequential selection regarding lung metastasis after intravenous injection of B16 cells into mice [Fidler, IJ, Selection of successive tumor lines for metastasis. NatNew Biol, 1973. 242(118): pp. 148-9, Fidler, IJ and GL Nicolson, Organselectivity 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 the experiment also expressed the immunodominant epitopes of the lymphocytic choriomeningitis virus (LCMV) glycoprotein (GP33-41) [Prévost-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): pp. 2187-2194. ] Cells were maintained in DMEM supplemented with 10% heat-inactivated FBS, L-glutamine (2 mM), sodium pyruvate (1 mM) and Pen / Strep.

[0169] CT26 CT26 is a mouse colon cancer cell line derived from the Balb / C mouse strain and obtained by Dr. Anders Elm Pedersen. This cell line is used to test the growth of primary tumors 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.

[0170] 4T1-Luc 4T1 is a mouse mammary gland cell line derived from the Balb / C mouse strain. When injected into the mammary fat pads 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]. Cell lines were stably transfected with 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.

[0171] Vero cells Vero cells are derived from African green monkeys (black-tailed macaques). Cercopithecus aethiops )) primate kidney cell lines [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 can be highly transduced by human Ad5 infection without the need for the supportive production of new viral particles, and is therefore used to analyze protein expression and VLP release via Ad5-vaccine. Cells were maintained in DMEM supplemented with 10% heat-inactivated FBS, L-glutamine (2 mM), sodium pyruvate (1 mM), and Pen / Strep.

[0172] A549 cells are human lung epithelial cells adapted for host virus transfection. Therefore, A549 cells were used for adenovirus transfection containing target sequences for VLP production. VLP secretion was analyzed by Western blotting (WB), and its presence on the cell surface was detected using fluorescence-activated cell sorting (FACS), followed by visualization using electron microscopy (EM). These cells were maintained in Kaighn's Modification of Ham's F-12 Medium supplemented with 10% heat-inactivated FBS, Pen / Strep, and sodium pyruvate (1 mM).

[0173] Expressing Gag and Env proteins Renca cells Renca cells are mouse (Mus musculus) kidney epithelial cells. They are derived from renal adenocarcinoma in Balb / c mice. Tumor growth and progression are precisely similar to those observed in human renal cell carcinoma, particularly mimicking spontaneous metastasis to the liver and lungs. The cells used in the following examples were kindly provided by Prof. Dr. Barbara Schnierle (Langen, Germany). In some of the following examples, the cells were modified to express the human endogenous retrovirus type K (HERV-K) Env or Gag protein. This allows for the induction of tumors expressing the HERV-K protein in mice, generating suitable mouse models for testing our novel vaccination strategy against human cancers expressing the ERV protein. These cells were maintained in Roswell Park Memorial Institute medium (RPMI-1640) at pH 7.2, supplemented with 10% heat-inactivated FBS, 20 x 10⁶ 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).

[0174] Primary cultures of chicken embryo fibroblasts (CEF)Widely used for virus culture. Following a protocol from (Staib et al. 2004), 11-day-old eggs from Jens Toft, Lohmann (Denmark) were used to prepare CEF cultures. In this case, CEF cells were used to produce a modified vaccinia aniline (MVA) encoding the HERV-K Env and Gag exogenous antigens. The reason for using this specific cell type is that MVA replication is limited to avian cells, meaning that MVA does not replicate in most mammalian cells, making it unsuitable for this purpose (Altenburg et al. 2014). CEF cells were cultured in CEF medium consisting of 3.7 g / L sodium pyruvate, 10% heat-inactivated FBS, and 1% (v / v) antibiotic-antifungal agent RPMI (Gibco™, 15240062).

[0175] Kidney fibroblasts of young hamsters (BHK-21 cells) Originally derived from juvenile Syrian golden hamster kidney cells (Mesocricetus auratus). The specific cell lines used in the examples below were kindly provided by Prof. Allan Randrup Thomsen (University of Copenhagen, Denmark). BHK-12 cells were used for MVA Env and Gag titrations because they are known to be one of the few cell lines that allow MVA replication. They were maintained in CEF medium supplemented with 3.7 g / L sodium pyruvate, 10% heat-inactivated FBS, and 1% (v / v) antibiotic-antifungal agent RPMI (Gibco™, 15240062).

[0176] plasmid constructs

[0177] To generate recombinant adenovirus, the target protein was cloned into the modified adenovirus vector Capture-pBGH. This vector contains the Ad5 genome with deletions in the E1 and E3 genes. Furthermore, it contains a region homologous to the vector 768tet, which requires a CMV promoter and a 3' polyadenylated (polyA) tail, and expresses the recombinant protein under the Tet operon. Figure 4 [Becker, TC et al., Use of recombinant adenovirus for metabolic engineering of mammalian cells. Methods Cell Biol, 1994. 43 Pt A: 161-89.] Therefore, the target protein is first inserted into the 768tet via subcloning, PCR cloning, or Gibson assembly, and then via homologous recombination (… Figure 5Clone to Capture-pBGH ( Figure 4 )Inside.

[0178] For adenovirus pIX modification, the target protein is cloned into a co-expression vector pcDNA3, which further encodes pIX and an adapter sequence (containing a FLAG tag), followed by a restriction site for insertion into the target gene (pcDNA3_pIX_Taglinker_xxx, where xxx = target antigen). The expression vector is transfected into producer cells to induce expression of recombinant pIX in these cells.

[0179] Table 1 lists the different plasmid constructs used.

[0180] Table 1: List of plasmid constructs used for cloning, virus generation, and vaccination. The DNA plasmids used in the project are listed, including abbreviations used in this work. Additionally, the genes encoded by the vectors (“Description”) and the applications of the DNA plasmids (“Purpose”) are explained.

[0181]

[0182]

[0183]

[0184] clone

[0185] Different cloning strategies are used to construct new DNA constructs for the production and testing of adenovirus vaccines.

[0186] Subcloning

[0187] For subcloning, the target DNA sequence is transferred from one plasmid (donor vector) to another plasmid (target vector). The donor and target vectors are cut at the ligation site via restriction digestion. To prevent religation, the target vector is treated with calf intestinal alkaline phosphatase (CIP), an enzyme that catalyzes dephosphorylation at the 5' and 3' ends of the DNA. The digested DNA is separated on a 1% agarose gel containing GelGreen dye (#41004, Biotium). The desired DNA band is cut, and the DNA components are extracted using the EZNA Gel Extraction Kit (D2500; OMEGA bio-tek). In short, the gel is dissolved in one volume of binding buffer (XP2) and loaded onto a HiBind® DNA Mini column. After washing twice, the column is dried, and the DNA is double-eluted in elution buffer.

[0188] After purification, the vector and insert were mixed at a stoichiometric ratio of 1:3. The ligation of the two DNA fragments was catalyzed using Instant Sticky-end Ligase Master Mix (M0370; New England BioLabs). The ligation 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. Cells were then permeated by a 45-second heat shock at 42°C. After a 2-minute incubation on ice, Super Optimal Broth (SOC) medium was added, and the bacteria were incubated with shaking at 37°C for 1 hour. The bacterial suspension was streaked onto Lysogeny Broth (LB) agar plates containing antibiotics and incubated overnight at 37°C.

[0189] To screen for the correct construct, several bacterial colonies were amplified for microplasmid preparation (see “0 DNA Preparation” below). The isolated plasmid DNA was cut by restriction digestion and analyzed by gel electrophoresis.

[0190] For the HERV-K constructs (and corresponding controls), subcloning was performed to insert DNA constructs containing the target sequences (DNA_ISDmut_coHERV-K-P2TS and DNA_coHERV-K-P2TS) into the receptor plasmid 768(TetO)-SP-alb-CIDR. To achieve this, the inserts and receptors were first amplified using PIR1 and XL1-Blue cells, and Kan and Amp selection markers, respectively. Since the XbaI enzyme activity was only 75% when using NEBuffer 3.1, all constructs were digested at 37°C for 1 hour and 30 minutes using XbaI (New England Biolabs, R0145) and SwaI (New England Biolabs, R0604) along with NEBuffer™ 3.1 (New England Biolabs, B7203). DNA was separated by electrophoresis on a 1% agarose gel with GelGreen dye (100 V, 200 A, 1 hour). Following the manufacturer’s instructions, bands containing the insert fragment and bands containing 768 (TetO) were cut and purified using the EZNA® Gel Extraction Kit (Omega bio-tek, D2500) and eluted in 20 µL of ultrapure water (UPW).

[0191] To ligate the constructs, 40 ng of the recipient vector and 120 ng of each insert fragment were incubated at 37°C with a 1:2 diluted instant sticky end ligase master mix (2X) for 15 to 30 minutes. Transformation was performed using XL1-Blue cells, and DNA was obtained using mini-preparation (described below). A test cut was then performed to confirm that the target sequence was correctly inserted into the recipient vector. If so, new transformations and midi-preparation (described below) were performed to obtain higher DNA concentrations.

[0192] PCR cloning

[0193] In contrast to subcloning, PCR cloning is characterized by the generation of an insert fragment in polymerase chain reaction (PCR). Using specific extension primers, the target sequence is amplified from the donor vector via PCR to insert the enzymatic restriction site. 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 incubation at 95°C for 2 minutes to activate Taq polymerase and promote complete denaturation of the DNA template. The initial steps were 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.

[0194] DNA was isolated from the reaction mixture using the EZNA Gel 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. The DNA underwent enzymatic digestion at specific restriction sites and was purified using the EZNA Gel Extraction Kit. Digestion and ligation of the target vector were performed according to the previously described subcloning protocol.

[0195] In the context of the HERV-K construct (and its corresponding control), to continue homologous recombination, the NotI site contained within the HERV-KWT / ISDmut sequence must be removed so that the NotI can subsequently be used to properly linearize the plasmid, allowing for correct recombination. To achieve this, two sequences (768(TetO)-SP-alb-CSP-HERV-KWT / ISDmut) derived from the above subcloning procedure were digested at 37°C for 1.5 hours with XbaI (New England Biolabs, R0145) and BspEI (New England Biolabs, R0540) along with NEBuffer™ 3.1 (New England Biolabs, B7203) and separated by electrophoresis on a 1% agarose gel containing GelGreen dye. EZNA was used. ® The Gel Extraction Kit digests and elutes DNA bands containing NotI sites to be removed.

[0196] The forward primer used for 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), while 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 eliminate it. The primers were obtained from TAG Copenhagen A / S (Copenhagen, Denmark).

[0197] 10 ng template DNA (1 ng / µL), 10 µM of each primer, and a 1:2 dilution of PfuUltra II Hotstart PCR MasterMix (Agilent Technologies, 600850) were used to prepare the reaction mixture for each DNA construct. The PCR reaction consisted of an initial denaturation step (95°C, 5 min), followed by 35 cycles of looping, which included 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 (72°C, 10 min) was performed, and the samples were stored at 4°C.

[0198] The PCR products, along with the receptor plasmid, were separated by gel electrophoresis, and the desired bands were collected and processed as described in the section "Subcloning" above. This yielded the 768(TetO)-HERV-K-Gag-p2A-Env WT and ISDmut constructs, which currently do not contain NotI restriction sites in their sequences.

[0199] Gibson Assembly

[0200] Gibson assembly is used to combine several DNA fragments into a single construct. The fragments are amplified by PCR extension to add overhangs homologous to the target vector. The PCR product is treated and purified as described for PCR cloning. The target vector is opened via restriction digestion at the insertion site. To assemble the fragments, the opened target vector and purified insert fragments are mixed in a 1:3 stoichiometric ratio and incubated at 50°C for 1 hour with Gibson Assembly Master Mix (E2611; New England BioLabs). Three key enzymes in the Master Mix facilitate assembly. Exonucleases remove DNA from the 5' end of the fragments, producing single-stranded 3' overhangs that anneal in the homologous region along with the other fragments. Nucleotides are inserted into the remaining gaps by DNA polymerase. Finally, DNA ligase ligates the gaps in the assembled DNA. The assembled DNA is transformed into bacteria as described in the previously described cloning techniques, and the correct constructs are then screened.

[0201] Homologous recombination to generate recombinant adenovirus genome

[0202] The target gene was inserted into the adenovirus genome (Ad5) via homologous recombination in *E. coli*. The insert fragment (target gene) with homologous regions to the target vector was excised from 768 tet via restriction digestion and purified by gel electrophoresis. The recipient vector, Capture-pBGH (Ad5 genome), was similarly linearized by restriction digestion. To prevent religation, the excised vector was subjected to CIP treatment (see subcloning). Subsequently, the vector-DNA was purified by ethanol precipitation. Briefly, the DNA precipitate was placed in 0.3 M sodium acetate and 70% ethanol, frozen at -80°C for 20 min, and centrifuged at 16,000 g for 15 min (4°C). The clump was washed in 70% ethanol and centrifuged again for 5 min. 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). Subsequently, the DNA was purified by 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 DNA was extracted by ethanol precipitation as described above.

[0203] To combine the vector and insert via homologous recombination, the two components were mixed at a stoichiometric ratio of 1:3 and added to electroporated competent BJ5183 cells. The bacteria were transferred to electroporation cuvettes (#1652086; Bio-Rad) and permeated via electroporation using a gene pulse generator (Bio-Rad) at 25 µFD, 2.5 kV, and 200 Ω. Following electroporation, the cells were transferred to SOC medium and further treated as described in the heat shock protocol (see “Subcloning”).

[0204] The following plasmids were provided by Sirion Biotech:

[0205] cDNA_HERV-K (Gag_p2A_Env)

[0206] cDNA_HERV-K (Gag_p2A_Env-(Q6A)ISD-mut).

[0207] The same construct is also provided by Sirion, but encoded by the Ad19a carrier.

[0208] The cDNA constructs were amplified and used as DNA vaccines and insertion vectors for cloning strategies, with the ultimate goal of obtaining an Ad5 vector encoding the aforementioned sequence, which could then be used as a vaccine. Specifically, for the HERV-K constructs encoded in hAd5s (and corresponding controls), the target gene was cloned into a pBGH plasmid encoding the human Ad5 genome with deletions in the E1 and E3 genes. The transgene was inserted into the E1 position via homologous recombination with a 768tet plasmid encoding the target gene. This strategy was chosen because conventional cloning with restriction digestion and ligation is very inefficient, while the pBGH vector is a very large plasmid exceeding 38 kbp.

[0209] Homologous recombination between the 768tet and pBGH capture plasmids was performed in *E. coli*. The capture vector contained green fluorescent protein (GFP) as an insert fragment, which would be replaced with the target gene.

[0210] Because the pBGH plasmid encoding the human Ad5 genome is too large (< 38 kbp) to undergo a universal cloning strategy that uses restriction enzyme digestion to insert the desired construct, homologous recombination is used to insert it at the E1 position.

[0211] First, the pBGH receptor vector was linearized using the SwaI enzyme (New England Biolabs, R0604) at 37°C over a 2-hour period. Simultaneously, 768(TetO)-HERV-K-Gag-p2A-Env WT and ISDmut were digested with the NotI enzyme (New England Biolabs, R3189) over a 1-hour period. The reaction products were separated by electrophoresis on a 1% agarose gel containing GelGreen. HERV-K sequences flanking the desired homologous regions for recombination were collected from the gel, and DNA was isolated using the EZNA® Gel Extraction Kit (Omega bio-tek, D2500) following the manufacturer's instructions and eluted in UPW.

[0212] Following pBGH digestion, both the 3' and 5' ends were phosphorylated using calf intestinal alkaline phosphatase (30 min, 37 °C; M0290) to prevent re-ligation. The vector was then subjected to ethanol precipitation in 0.3 M sodium acetate and 70% (v / v) ethanol over a 20-minute period at -80 °C. The sample was immediately centrifuged (15 min, 4 °C, 16,000 g) and the clumps were washed with 70% (v / v) ethanol. The vector was subjected to another centrifugation (5 min, 4 °C, 16,000 g), and the resulting clumps were allowed to stand and dry at RT, and finally resuspended in UPW.

[0213] To prevent further religation of the pBGH vector, it was treated with TempaseHotStart DNA polymerase (Ampliqon, A230306) at 72°C for 30 minutes, which increases adenosine overhangs. Phenol / chloroform was added, and the DNA was purified by centrifugation (10 min, 4°C, 16,000 g). The supernatant containing the DNA was then transferred to a microcentrifuge tube. The DNA underwent ethanol precipitation as before for further purification, and was then diluted in UPW.

[0214] All plasmids were stored in water, not in elution buffer, as salt content interferes with electroporation efficiency. The pBGH vector and HERV-K WT / ISDmut insert were combined with electroporated competent BJ5183 cells (Agilent, 200154) at a 1:3 molar ratio. The mixture was then transferred to electroporation cuvettes (Bio-Rad, 1652086) used to infiltrate the cells using a gene pulse generator (Bio-Rad) at 25 µFD, 2.5 kV, and 200 Ω. SOC medium was then added to recover the E. coli competent cells after transformation. They were then incubated in a shaking incubator at 37°C for 1 hour. Finally, the mixture was plated on LB agar plates containing Kan and incubated at 37°C.

[0215] To determine whether homologous recombination was performed correctly, DNA was isolated using a mini-preparation method as described below. It was then digested with restriction enzymes and separated on a 1% agarose gel containing GelGreen dye. The bands corresponding to the correct sizes of pBGH and the insert were cut and transformed into *E. coli*, and finally, the DNA was isolated again using a midi-preparation method as described below.

[0216] DNA preparation

[0217] Transformation of Escherichia coli (E. coli)

[0218] For transformation, chemically competent *E. coli* XL1-Blue Supercompetent Cells (Agilent, 200236) and One Shot™ PIR1 Chemically Competent Cells (ThermoFisherScientific, C101010) were used. 20 μL of the latter was mixed with 10 ng of plasmid DNA and incubated on ice for 3 minutes. The mixture was then heat-shocked at 42°C for 45 seconds in a Waterbath TW80 (Julabo) and then placed on ice again for 3 minutes. Immediately afterwards, 200 μL of Super Optimal Broth (SOC) medium with catabolism repression (20 g trypsin, 5 g yeast extract, 0.58 g NaCl, 0.19 g KCl, 3.96 g glucose, and 5.04 g MgSO4·7H2O) was added to the sample, and the sample was incubated in a shaking incubator at 37°C for 1 hour. The final step involves platening the sample onto LB agar plates containing the corresponding antibiotics (ampicillin (Amp): 100 μg / mL, kanamycin (Kan): 50 μg / mL), our plasmid is resistant to the antibiotics, and placing the E. coli agar plates (Binder) in an incubator at 37°C.

[0219] Agarose gel electrophoresis

[0220] To verify that the transformation was performed correctly, the DNA-purified constructs were electrophoresed on a 1% (w / v) agarose gel containing ethidium bromide or GelGreen™ dye (Biotium, 41004) to allow visualization of the DNA under UV light. 1X loading buffer (6X) was added to the samples, along with the size marker GeneRuler 1 kb Plus DNALadder (Thermo Fisher Scientific, SM1331), and loaded onto the gel. The buffer used was ethylenediaminetetraacetic acid (EDTA) (TAE) buffer (4.86 g / L Trizma® base, 0.37 g / L Na₂EDTA·2H₂O, and 0.11% (v / v) acetic acid at pH 8.3). Electrophoresis was performed at 120V for 1 hour using an EPS 3501 XL (GE Healthcare) electrophoresis power supply.

[0221] mini preparation

[0222] To screen for the correct constructs after cloning, a small-scale amplification of DNA is performed. Bacterial colonies are transferred to 3 mL or 5 mL of LB medium (containing the corresponding antibiotic Amp 100 µg / mL or Kan 50 µg / mL, depending on the resistance gene in the target plasmid) and incubated overnight at 37°C. Plasmid DNA is isolated using the EZNA® Plasmid DNA Mini Kit I (D6943, Omega bio-tek). Briefly, bacteria are clumped by centrifugation and resuspended in Solution I (resuspending buffer) containing RNase. Solution II (lysis buffer) is added to release DNA from the cells. To terminate the reaction and allow cell debris containing genomic DNA to precipitate, Solution III (neutralization buffer) is added. The precipitate is clumped by centrifugation, and the supernatant is transferred to a HiBind® DNA Mini column. After the DNA binds to the column membrane by centrifugation and the addition of HB buffer, the column is washed twice with DNA washing buffer and then dried. Finally, the plasmid DNA is eluted in elution buffer.

[0223] Midi preparation

[0224] To obtain higher and more purified DNA yields, a midi preparation was prepared using the NucleoBond® Xtra Midi Kit (#740410, AH Diagnostics) from *E. coli* grown overnight in 100 mL LB medium (again containing appropriate antibiotics). The principle is similar to the mini preparation, starting with resuspension and lysis of the bacteria. After neutralization, the lysate was loaded onto a equilibrated NucleoBond® Xtra column and washed with equilibration buffer. Inserted column filters containing residual cell debris were removed, and the column was washed with wash buffer. DNA was eluted in elution buffer and then precipitated in isopropanol. The precipitated DNA was clumped by centrifugation and washed with 70% ethanol. After a further centrifugation step, the supernatant was removed, and the DNA clumps were dried at RT. The precipitate was then dissolved in 100 μL of 10 mM Tris-HCl buffer (pH 8.0) or 100 μL of EZNA. ® The elution buffer of the Plasmid DNA Mini Kit reconstructs DNA, and in NanoDrop TM Concentration was measured in 2000.

[0225] 2.6 Virus Production

[0226] Different viruses were generated and tested in the experiments (Table 2). In addition to the usual recombinant adenovirus, an Ad5 vector displaying recombinant pIX on its surface (Ad5-pIX) was also tested, and it had to be produced using a different procedure.

[0227] Table 2: List of viral constructs used for mouse immunization: This list shows the different recombinant adenoviruses used in the project, including abbreviations used in this work and genes encoded by the virus.

[0228] Virus abbreviation describe Ad5_MelARVgag_p2A_envSTOP Ad5-MelARV Ad5 encoding MelARVgag and MelARVenv Ad5_MelARVgag_p2A_envISDmutSTOP Ad5-MelARV-ISD Ad5 encoding MelARVgag and ISD-mutated MelARVenv Ad5_MelARVgag_p2A_ envSTOP_pIX-p15E Ad5-MelARV_pIX-p15E The viral pIX protein displays the encoding of p15E, MelARVgag, and the Ad5 of MelARVenv. Ad5_MelARVgag_p2A_envISDmutSTOP_pIX-p15E-ISD Ad5-MelARV-ISD_pIX-p15E-ISD The viral pIX protein displays MelARVgag, which encodes the ISD-mutant p15E, and Ad5, which encodes the ISD-mutant MelARVenv. Ad5_MelARVgag_p2A_envSTOP_pIX-p15E-trunc-wC Ad5-MelARV_pIX-p15E-trunc-wC The viral pIX protein displays the Ad5 encoding MelARVgag and MelARVenv, which is a truncated p15E with an additional cysteine ​​residue. Ad5_MelARVgag_p2A_envSTOP_pIX-p15E-trunc-w / oC Ad5-MelARV_pIX-p15E-trunc-w / oC The viral pIX protein displays the Ad5 encoding MelARVgag and MelARVenv, which is a truncated p15E without additional cysteine. Ad5_SIVgag_p2A_LucSP_MelARV_HA-TMCT Ad5-LucSP_MelARV_HA-TMCT Ad5, which encodes SIVgag containing the luciferase signal peptide and the influenza hemagglutinin transmembrane domain + cytoplasmic tail, and modified MelARVenv. Ad5_SIVgag_p2A_ LucSP_GCN4_p15E_HA-TMCT Ad5-LucSP_GCN4_p15E_HA-TMCT The Ad5 encoding an SIV gag containing a luciferase signal peptide, a trimerized sequence, and an influenza hemagglutinin transmembrane domain plus a cytoplasmic tail, and a modified MelARVenv, is also present. Ad5_eGFP Ad5-GFP Ad5 encoding GFP

[0229] The sequence of MelARV Env protein with modified ISD

[0230] The Env protein has the following sequence (SEQ ID No: 41):

[0231]

[0232] The sequence was modified by replacing the original E with R at the gray background letter in the ISD sequence and replacing the A with F at the third amino acid outside the ISD (also marked in gray).

[0233] Reorganization of Ad5 production

[0234] The starting point for Ad5 production is the adenovirus genome plasmid Capture-pBGH. This plasmid contains all the genes required for the formation of infectious Ad5 particles, but lacks genes E1 and E3. E1 is required for viral replication and is produced by the producer cell line HEK293 / Avtoxic (Kovesdi, I. and SJ Hedley, Adenoviral producer cells. Provided in Viruses, 2010. 2(8): pp. 1681-703. E3 is a non-essential gene for viral production and is missing from the genome to create space for recombinant target genes. These target genes are inserted into the vector via homologous recombination during the capture cloning process (see “0 homologous recombination to generate recombinant adenovirus genome”). Figure 5 The paper summarizes the process of cloning the target protein into Capture-pBGH and the subsequent viral production.

[0235] Avtoxic cells were transfected with the recombinant Capture-pBGH vector. Cells were seeded in T75 flasks and grown to 50–70% confluence. The vector DNA was linearized by restriction digestion with PI-SceI (#R0696S; New England BioLabs) in PI-SceI buffer at 37°C for 1 hour. 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 onto a 1% agarose gel to confirm proper plasmid cutting. The remaining DNA was mixed with polyethyleneimine (PEI) at a DNA:PEI ratio of 1:3. After incubation at room temperature for 15 minutes, the mixture was added dropwise to the culture medium for Avtoxic cells. Transfected cells were incubated under normal cell culture conditions (see Becker, TC., et al., Methods Cell Biol. 1994; 43Pt A:161-89.), with the culture medium changed after 16 hours and every 2-3 days thereafter. When visible cell lysis was achieved by cell detachment (after 2-3 weeks), the cell culture medium containing the lysed cells (referred to as "viral lysis product") was harvested and stored at -80°C.

[0236] In the next step, cells are re-infected with "viral lysate" to obtain "3-day lysate." For this, Avtoxic cells are grown in 6-well plates until 70% confluence, and then infected well by well with a 1:10 serial dilution of "viral lysate." Three days after infection, the supernatant from the well with the highest dilution and complete lysis is harvested and frozen at -80°C. This viral sample is referred to as the "3-day lysate."

[0237] To produce the virus on a large scale (“large-scale lysis products”), Avtoxic cells were inoculated into four Nunc™ Cell Culture Treated Triple Flask™ cells (500 cm²). 2 (#132913; Thermo Fisher). When cells reached 70% confluence, flasks were infected with 150 μL of “3-day lysis product”. After complete cell lysis (approximately three days), the supernatant was harvested and frozen at -80°C.

[0238] Recombinant Ad5 purification

[0239] In the first step of virus purification, 0.5% Igepal CA-630 (#56741; Sigma-Aldrich) was added to the harvested large-scale lysis products. During a 10-minute incubation at RT, detergent caused the remaining cells to break down and viral contents to be released into the culture medium. To remove cell debris, the lysis products were centrifuged at 12186g for 20 minutes at 4°C. The supernatant was recovered, and half the volume was added to 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 precipitates, allowing for virus concentration in the next step. The precipitated virus was clumped by centrifugation at 12186g for 20 minutes. The viral clumps were resuspended in 5 mL of cold phosphate-buffered saline (PBS) and transferred to 15 mL Falcon tubes. The sample was centrifuged at 784g for 5 minutes to remove any remaining cell debris. Transfer the supernatant to a new 15 mL Falcon tube and repeat the previous centrifugation steps several times until only a small amount of cellular residue clumps remain in the tube, which cannot be completely removed. Add nearly saturated CsCl solution to the virus-containing supernatant to achieve a final density of 1.34 g / mL. Transfer the resulting solution to an ultracentrifuge tube (#342413; Beckman Coulter), seal the tube, and centrifuge overnight at 257,300 g in a Beckman Coulter Ti70.1 rotor. Extract clearly visible viral bands using a needle and syringe and load them onto a equilibrated PD-10 desalting column (#17-0851-01; GE Healthcare). Collect the flow fraction in 70% glycerol, with a final glycerol concentration of 10%. Combine the fractions with the highest viral concentration (highest turbidity), aliquot, and store at -80°C. Virus aliquots are frozen and thawed more than twice.

[0240] Production and purification of the recombinant Ad5 vector for antigens on pIX.

[0241] Ad5-pIX virus production was performed using a different strategy than that used for normal recombinant Ad5 virus. The producer cell line was the previously described HEK293(CCS)-shmir-pIX_221-puro cell line (pIX cells). pIX cells were seeded at 175 cm⁻¹. 2Cells were grown in four flasks (four flasks / virus) until 70% confluence. To generate recombinant pIX protein, cells were transfected with the pcDNA3_pIX plasmid, in which pIX is coupled to the recombinant protein via genetic fusion. Doxycycline (0.5 μg / mL) was added to the culture medium prior to transfection, inducing transcription of pIX-specific shRNA that inhibits the translation of native pIX. The cell culture medium was changed 18 hours post-transfection, and doxycycline was added again. Subsequently, cells were infected with a basal adenovirus (encoding the target recombinant protein) at 5 MOI (multiple of infection). Viral replication was allowed under normal culture conditions for 48 hours until viral cytopathic effects were visible. Cells were harvested and formed into clumps by centrifugation at 750g for 10 minutes. The clumps were resuspended in PBS with 0.5% sodium deoxycholate and incubated at RT for 30 minutes to degrade the cells and release the virus. To digest genomic DNA from the production cell line, 0.2 M MgCl2 and 0.05 mg / mL DNase I (A3778, AppliChem) were added, and the mixture was incubated at 37°C for 1 hour. Cell debris was removed by centrifugation at 3000g 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 viral bands were transferred to a dialysis membrane (Spectra / Por® Dialysis Membrane, 300 kDa, #131450, Biotech CE Tubing) and dialyzed overnight in PBS at 4°C. Finally, the virus was aliquoted in 10% glycerol and stored at -80°C.

[0242] Viral titration

[0243] To ensure experimental reproducibility, the purified virus was titrated to obtain the number of infectious units / mL (IFU / mL). 96-well plates with a flat-bottomed Δ-treatment surface were coated with polylysine for 15 minutes and washed three times with PBS. HEK293 cells were inoculated with 5 x 10⁶ cells / mL of 100 μL culture medium. 4 Inoculate the cells into the wells at a concentration of 1:50. Start with a 1:50 dilution and serially dilute the virus in the culture medium 10-fold. Add 50 μL of dilution factor 5x10. 4 Up to 5x10 7Two copies of the cell suspension were added to the wells of a 96-well plate. Infected cells were incubated for 48 hours under normal cell culture conditions. After removing the culture medium, the wells were dried at RT and the cells were fixed in 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 hexagonal antibody (1E11; #sc-51746; Santa Cruz Biotechnologies) diluted 1:1000 in PBS + BSA was added and incubated at 37°C for 1 hour. 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 incubated in the wells at 37°C for 1 hour. The residual antibodies were washed away, and viral plaques were visualized at RT for 10 minutes using 3,3'-diaminobenzidine (DAB) substrate.

[0244] To determine the viral titer, plaques at appropriate dilutions were counted under a microscope at 20x magnification. Several fields of view were counted in each well until approximately 100 plaques were detected. The final number of IFU / mL was calculated using the following formula:

[0245]

[0246] =Average number of plaques per field of view (total number of plaques counted / number of fields of view counted); VF=20x magnification Number of fields of view per well at the specified rate (52.7 fields of view / well); DF = dilution factor for virus in the counting well (e.g., 500,000x); W = Number of infected wells / mL of virus dilution (1000μL / mL / 50μL / well = 20 wells / mL); P = number of plaques.

[0247] As an additional quality control measure, the measured infection unit concentration / mL (IFU / mL) was compared with the viral particle (VP) count. This was achieved by measuring absorbance at 260 nm using NanoDrop. TM The absorbance was measured at 2000 VP / mL. One unit of absorbance corresponds to 10... 12 The concentration of VP / mL. The ratio of IFU / mL to VP / mL indicates viral activity, with an ideal / typical ratio of 1:30 to 1:100.

[0248] Genomic DNA purification from recombinant Ad5

[0249] DNA isolation from the recombinant adenovirus was performed to ensure correct insertion of the recombinant gene into the adenoviral genome. DNA was extracted using a modified protocol with the GenElute™ Mammalian Genomic DNA Miniprep Kit (G1N70; Sigma-Aldrich). For this purpose, 100 μL of purified viral 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 a prepared GenElute Miniprep Binding column. Subsequent steps followed the original protocol, with two washing steps and subsequent column drying. Viral DNA was eluted in elution solution. For viral quality assurance, the DNA was sent for sequencing (GENEWIZ UK Ltd.) to exclude mutations in homologous recombination regions. Additionally, the viral DNA was digested with restriction enzymes to confirm the correct band size by gel electrophoresis.

[0250] VLP production and purification

[0251] The primary function is to produce and purify virus-like particles (VLPs) to test the function of vaccines encoding VLPs. VLP production is tested in Vero cells, with the Vero cells used at a density of 1 x 10⁻⁶ cells. 7 Individual cells were seeded at a density of 175 cm⁻¹ 2 In a culture flask, incubate for 2 hours to allow for adhesion. Then, use 50 MOI of Ad5 (5x10⁻⁶). 8 Cells were infected with IFU / flask for 5 hours. After removing the culture medium, the cells were washed twice with PBS and incubated in serum-free medium for 48 hours. The supernatant (SN) was centrifuged at 282 g for 10 min and then filtered through a 0.45 μM membrane to remove cellular contaminants. VLPs were purified by forming clumps at 82.700 g through a 20% sucrose pad using an open 32 mL thick-walled tube (#355631; Beckman Coulter) in a Beckman Coulter Ti 70 rotor. The SN was removed, and the clumps were resuspended in 100 μL PBS (160x of the original concentration).

[0252] The Adv vaccine encoding HERV-K-Gag-p2A-Env wild-type (WT) / ISDmut was translated into a functional protein capable of generating VLPs, cell lysis products were generated from infected cells, and VLPs were purified from cell culture supernatant (SN):

[0253] Vero, A549, and HEK293 cell lines were used to produce and purify VLPs. On day 1, 10 x 10 6Vero, 10x10 6 A549 or 10x10 5 HEK293 cells were seeded into T175 (175 cm) culture media containing appropriate culture medium. 2 ) flask or T25 (25 cm) 2 Flasks (in the case of HEK cells). After 2 hours, cells were infected with different viral vectors encoding our target sequence (see Table 2b) using a multiplicity of infection (MOI) of 50 or 20 (HEK293), where the MOI indicates the number of virus particles / cells for a given infection. After 5 hours, cells were washed twice with phosphate-buffered saline (PBS) at pH 7.4 containing 8 g / L NaCl, 0.2 g / L KCl, 1.15 g / L Na2HPO4·2H2O, and 0.2 g / L KH2PO4. The medium was then replaced with the appropriate cell culture medium, but without FBS. Cells were incubated at optimal maintenance conditions for 48 hours or 16 hours (when using HEK293 cells).

[0254] Subsequently, two different procedures were followed to obtain VLPs from cell cultures. On one hand, the SN (cell-storage unit) was retained for purification and analysis of VLPs secreted by the cells. On the other hand, cells were lysed to analyze the VLPs contained within the cells.

[0255] For the first procedure, cells were centrifuged at 12,000 rpm for 10 minutes at 4°C, and the supernatant was then filtered through a 0.45 µM membrane (Sartorius, 16555) to remove cellular impurities. In open 32 mL thick-walled ultracentrifuge tubes (Beckman Coulter, 355631), 13.5 mL of SN was added dropwise to 3 mL of 20% (w / v) sucrose dissolved in PBS. The tubes were weighed for equal volumes and then placed in a Ti 70 rotor (Beckman Coulter, 337922), which was then centrifuged at 82.700 g and 4°C for 2.5 hours. Upon completion, the SN was carefully removed, and any remaining clumps were resuspended in 100 µL of PBS and stored at -20°C.

[0256] The second procedure consists of a first step of cold PBS washing. Then, 10 mL of cold PBS is added to the flask, and cells are mechanically scraped away. 4 mL is transferred to a 15 mL conical tube and centrifuged at 12,000 rpm for 5 minutes at 4°C. The SN is discarded, and 1300 µL of a mixture containing NP40 cell lysis buffer (Invitrogen, FNN0021) and a 7 µL / mL protease inhibitor mixture (Sigma-Aldrich, P8340) is added to each tube. The tubes are then placed on ice for 30 minutes, vortexed every 10 minutes using a Shaker Vortex 3 (IKA). Finally, the tubes are centrifuged at 13,000 rpm for 10 minutes at 4°C to remove cell debris, and the SN is transferred to a new tube and stored at -20°C.

[0257] Table 2b. List of adenoviruses coded for analysis and comparison of different constructs generated and expressed by VLP.

[0258]

[0259] MVA production and titration

[0260] The procedures for MVA production, purification, and titration were performed using guidelines described by Staib et al. 2004. The initial MVA from the cleavage products of the HERV-K Gag or Env protein seed used to perform this experiment was provided by Prof. Dr. Barbara Schnierle (Langen, Germany). A 175 cm⁻¹ diaphragm was used. 2 Before using flasks for large-scale MVA production, 175 cm flasks were also used. 2 Flasks were used to increase the amount of virus on a small scale, and in both cases, CEF cells were inoculated.

[0261] In this case, MVA titration was performed in BHK-21 cells. A 1:1000 dilution of primary polyclonal rabbit anti-vaccinia virus (BioRad, 9503-2057) and a 1:500 dilution of secondary HRP-conjugated polyclonal goat anti-rabbit Ig antibody (Dako, P0448) were used to detect infected cells. To determine the titer (IFU / mL), the number of stained lesions was counted in diluted samples with approximately 20–100 viral lesions per well to maximize accuracy.

[0262] Animal experiments

[0263] Female C57BL / 6, Balb / C, and CD1 mice, aged 6–8 weeks, were obtained from Taconic (C57BL / 6) or Envigo (Balb / C and CD1). Mice were allowed one week to acclimatize before the start of experiments. All experiments were conducted in accordance with national guidelines and experimental protocols approved by the National Animal Laboratory Inspection Agency (Dyreforsøgstilsynet, Denmark).

[0264] Separate serum samples

[0265] To obtain serum samples, approximately 10% of the total blood volume was obtained from mice by puncturing facial veins with a Goldenrod lancet.

[0266] Alternatively, for final exsanguination (complete exsanguination) of mice, the animals were anesthetized with 1 mg / mL xylazine and 10 mg / mL ketamine in PBS administered intraperitoneally (ip). Maximum blood volume was obtained by puncturing a facial vein, followed by euthanasia of the mice via cervical dislocation.

[0267] In the HERV-K experiment, mice were anesthetized with isoflurane for complete exsanguination cardiac puncture. Subsequently, the mice were placed face up with a continuous supply of 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 via cervical dislocation.

[0268] Alternatively, the mice were subjected to complete anesthesia with isoflurane. They were then tested for involuntary reflexes, and maximum blood volume was collected from the eye specifically via the sinus only after it was ensured that no involuntary reflexes were present. The mice were then immediately euthanized by mild cervical dislocation.

[0269] Blood samples were stored overnight at 4°C to allow coagulation, and blood cells were removed from the serum by two centrifugations at 800g for 10 minutes. The serum was then stored at -20°C.

[0270] Injection: IV, SC, IM, IP

[0271] Different injection procedures were performed. For intravenous (iv) injection, mice were heated in a heating chamber to increase superficial venous blood flow. Up to 200 μL was injected into the tail vein. In HERV-K related experiments, a solution containing 10 μL was used. 6 Mice were intravenously injected with 100 µL of RLZ Gag and Env cells (from B. Schnierle) to induce lung metastasis.

[0272] Subcutaneous (sc) injection into the foot pad (fp) is performed under isoflurane anesthesia by injecting 30 μL subcutaneously into the skin of the foot pad. For the HERV-K experiment, this type of injection is used to inject 10 μL. 6 RLZ Gag and Env cells (from B. Schnierle) (in 100 µL) were used to grow subcutaneous tumors in mice and to establish a mouse tumor model expressing HERV-K Env.

[0273] For intramuscular (IM) injection, inject a maximum volume of 60 μL into the thigh muscle.

[0274] In the context of HERV-K experiments, this type of injection is primarily used for immunization (initiation) and booster mice with the targeted vaccine (see Table 2c below). 50 µL / mouse is administered for either adenovirus or MVA vaccination / boost, respectively. The injection is performed in the thigh muscle under isoflurane anesthesia, which provides both analgesia and muscle relaxation.

[0275] Table 2c. Virus-based vaccines used for immunization of IM mice.

[0276] Virus IFU / mouse Vaccine type Ad19a(II)-(TetO)-Hiso-MfPV3-P2TS (IP1321_A2953_V_7b) from Sirion <![CDATA[1x10 8 ]]> Trigger Ad5-(TetO)-CMV-SIVgag_p2A_HERV-K108env_P2TS <![CDATA[1x10 8 ]]> Trigger Ad19a(II)-(TetO)-CMV-coHERV-K-P2TS from Sirion <![CDATA[1x10 8 ]]> Trigger Ad19a(II)-(TetO)-CMV-ISDmut_coHERV-K-P2TS from Sirion <![CDATA[1x10 8 ]]> Trigger / Enhance HERV-K Env protein expressing MVA <![CDATA[1x10 7 ]]> Trigger / Enhance DNA-(TetO)-CMV-ISDmut_coHERV-K-P2TS 1 µg / µL Trigger / Enhance

[0277] Intraperitoneal (ip) injection is performed by administering up to 500 μL into the peritoneal cavity.

[0278] Vaccination

[0279] Five different vaccination trials were performed in mice:

[0280] Vaccination Timeline I. Balb / C mice were vaccinated in a priming-boost regimen with two DNA vaccinations, followed by a single 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. Subsequently, mice were attacked with CT26 tumor cells in the right flank, and tumor growth was measured.

[0281] Vaccination Timeline II. Balb / C mice were challenged with CT26 tumor cells (sc). Mice were vaccinated with Ad5-MelARV on day 2 post-challenge (d.2p.c.) or day 5 pc (previously induced with DNA). Another group was vaccinated on day 2p.c. and subsequently received four injections of anti-PD1 antibody when the tumor was just palpable (d.8 pc). As a control group, mice were injected with either PBS or anti-PD1 only.

[0282] Vaccination Timeline III. In the priming-boost regimen, C57BL / 6 mice were vaccinated twice with DNA-MelARV, followed by Ad5 vaccination. Blood samples were collected 3 weeks after the last vaccination and challenged in vitro with 2x10⁵ B16F10-GP cells. The number of metastases in the lungs was determined 2 weeks after challenge.

[0283] Vaccination Timeline IV. CD1 mice were first vaccinated with a DNA plasmid encoding either MelARVgag_p2A_env (DNA-MelARV) or the ISD mutant form 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 collected four weeks after vaccination, and serum antibodies were analyzed.

[0284] Vaccination timeline V: C57BL / 6 mice were vaccinated twice with adenovirus (Ad5-MelARV_pIX-p15E or Ad5-MelARV). Ad5-GFP was used as a control. Subsequently, blood samples were obtained, and mice were challenged iv with 2x10⁵ B16F10-GP cells. Lungs were isolated two weeks after challenge, and metastases were analyzed.

[0285] For DNA vaccination, 50 μg of DNA was injected intramuscularly into 50 μL of TRIS / PBS (142 mM). Adenovirus was then mixed with 2 x 10 μg of TRIS / PBS in 30 μL of PBS. 8 IFU was injected into the footpad along with the virus. In experiments involving pIX-modified viruses (vaccine timelines IV and V), 10 IFU were injected into 60 μL of PBS. 10 Individual viral particles. Due to the low concentration of pIX virus, it is impossible to inject a small volume into the footpad.

[0286] Another experiment involved administering an anti-PD1 antibody (RMP1-14; #BE0146; BioXCell) to mice with tumor-attacked mice (see “0 tumor attack”). Anti-PD1 was administered via intraperitoneal injection of 200 μg of antibody in 200 μL PBS. Treatment began on day 8 post-tumor attack when subcutaneous tumor growth was palpable. Mice were given four injections every four days (on days 8, 12, 16, and 20 post-tumor attack) (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): 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): 129-36.

[0287] In the HERV-K assay, Adv and / or MVA boosters were performed approximately 4 or 8 weeks after initiation (day 0) with an Adv or DNA vaccine. Blood samples were collected before and after initiation (day 14). Mice were exsanguinated on days 14 and 28 following the MVA / Adv / DNA booster. Blood samples were used to analyze the humoral response (antibody production against HERV-K Env) in vaccinated mice. Additionally, mice were euthanized 10 days after the MVA booster to test their cellular immune response (generation of CD8+ T HERV-K Env-specific T cells).

[0288] To test the efficacy of the novel vaccination strategy, only one dose of vaccine was administered 10 days after tumor attack.

[0289] Tumor attack

[0290] To evaluate the 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 min. Cells were then centrifuged at 282 g, washed with PBS, and diluted to a concentration of 2 x 10^6 cells / mL in PBS. 2 x 10^5 cells per 100 μL of PBS were intravenously injected into the tail vein of mice, resulting in tumor metastasis in the lungs. The attacked mice were euthanized 14 days later. The lungs were isolated and fixed overnight in 2% paraformaldehyde (PFA) solution in PBS, followed by storage in PBS at 4°C. Metastasis was counted as black nodules on the surface of the lungs under a dissecting microscope. Samples were blinded, and metastasis was counted by at least two individuals.

[0291] To analyze the primary growth of CT26 tumors, CT26 cells were prepared as described for B16F10-GP cells and diluted in PBS to a concentration of 5 x 10^6 cells / mL. Subcutaneous injection of 5 x 10^5 cells in 100 μL PBS into the right thigh resulted in tumor formation at the injection site. Tumor size was measured three times weekly in terms of length and width. Tumor volume was determined as follows: length * width 2 *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 the tumor exceeded 16 mm on either side, necrotic wounds were present, or the mice showed significant decreased activity. During tumor measurement, the different vaccination groups were blinded to prevent biased evaluation.

[0292] 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 mammary fat pad. To visualize tumor formation after 6 weeks, mice were injected intraperitoneally with luciferin (1.5 mg per 10 g mice), and imaging was performed 12 minutes post-injection using the IVIS Spectrum in vivo imaging system. IVIS imaging was performed by Andreea-Cornelia Udrea and Melanie Schwerdtfeger.

[0293] To analyze tumor growth and metastasis of RLZ Gag and Env cells in vivo, cells were cultured until 60-80% confluence was achieved. Once the desired confluence was reached, RLZ cells were washed three times with PBS, and then Versene was added at 37°C for 15 minutes to allow cell detachment. Subsequently, the cells were rotated downwards at 282 g, washed with PBS, and finally diluted to 10 in PBS. 7 Cells / mL. For lung metastases in each mouse, use 10... 6 Mice were administered intravenously at 100 μL / cell, and subcutaneously at sc for subcutaneous tumors. To evaluate lung metastasis, mice were weighed on days 0, 7, and 14, and every 2 days thereafter. Mice were euthanized if they lost approximately 15–20% of their body weight over several days. The endpoint for termination was set at day 40 post-tumor challenge. Mice bearing sc tumors were examined at the same time points as those challenged via IV, and euthanized when their tumor diameter exceeded 16 mm.

[0294] Both sc 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 4ºC. Samples were treated with high-titer serum from vaccinated mice, and HERV-K Env-specific staining of the tissues was analyzed.

[0295] Protein blot

[0296] For the detection of pIX protein, cell lysis products (~10 μg) or purified virus (10 μg) were used.10 (1 viral particle) was mixed with 6x SDS loading buffer containing DDT and heated at 95°C for 5 minutes. To visualize MelARV protein expression, cell lysate (5 μg), cell supernatant (15 μg), and purified VLP (~2 μg) were similarly mixed with loading buffer containing DDT, but the samples were not heated. The mixture was then loaded into NuPAGE. TM The protein was deposited on 4-12% Bis-Tris Protein Gel (#NP0322, Thermo Fisher) and run at 150 V for 1 hour in MOPS buffer. In a wet transfer system, the protein content of the gel was imprinted onto a nitrocellulose membrane at 30 V for 1 hour.

[0297] After transfer, the membrane was blocked for 1 hour with 5% skim milk in TBS-T buffered saline + Tween 20 (TBS-T). The membrane was then washed three times with TBS-T for 10 minutes each on a shaker and incubated overnight at 4°C with diluted primary antibody (Table 3) (in TBS-T + 3% skim milk). After three additional washes, HRP-conjugated secondary antibody in TBS-T was added, and the membrane was incubated at RT for 1 hour. Unbound secondary antibody was washed away, and the target protein was visualized using LumiGLO Reserve Chemiluminescent Substrate (54-61-00 or 54-71-02) in an ImageQuant LAS 4000.

[0298] Table 3: List of primary and secondary antibodies used for Western blotting and ELISA. This table lists the different primary antibodies used for Western blotting and their origins. Further details show the dilutions used and the secondary antibody chosen for detection. Some antibodies are also used in ELISA analyses at dilutions described later.

[0299] Primary Antibody Product Number / Origin Dilution Secondary antibody Anti-p2A #ABS31; Millipore 1：1000 Anti-rabbit Ig-HRP (#P0448, Dako) MM2-9B6 20x cell culture supernatant from hybridoma (provided by Tsuyoshi Takami, University of Arizona Health Sciences Center) 1：200 Anti-mouse Ig-HRP (#P0447, Dako) 4F5 Concentrated cell culture supernatant from hybridoma (provided by George Cianciolo, Duke University Medical Center) 1：200 Anti-mouse Ig-HRP (#P0447, Dako) 19F8 Concentrated cell culture supernatant from hybridoma (provided by George Cianciolo, Duke University Medical Center) 1：200 Anti-mouse Ig-HRP (#P0447, Dako) Anti-pIX Antibodies produced in rabbits (provided by David T. Curiel, Washington University in St. Louis) 1：1000 Anti-rabbit Ig-HRP (#P0448, Dako)

[0300] In HERV-K related experiments, VLP expression at the protein level was analyzed using Western blotting (WB). To ensure equal sample loading, the protein concentrations of both VLP (SN) and cell lysis products were measured using the Pierce™ Dioctyl-Kenoic Acid (BCA) Protein Assay Kit (Thermo Fisher Scientific, 23225) according to the manufacturer's guidelines. 6x sodium dodecyl sulfate (SDS) loading buffer containing dithiothreitol (DTT) was added to different samples, which were then placed in a block heater SBH130DC (Stuart) at 95°C for 5 minutes. Subsequently, 5 µg of protein and 7 µL of RunBlue were added.TM Prestained Marker (Expedeon, NXA05160), along with NuPAGE™ MOPS SDS Running Buffer (Thermo Fisher Scientific, NP0001), was loaded into NuPAGE™ 4-12% Bis-TrisProtein Gels (Thermo Fisher Scientific, NP0322). Samples were separated by SDS-PAGE at 180 V for 45 minutes.

[0301] The sample was then transferred to a 0.45 µm nitrocellulose blot membrane (Bio-Rad, 1620115) at 30 V for 45 minutes. For this step, a transfer buffer with 20% ethanol was used (with 3.75 g / L Trizma® base and 18.1 g / L p-hydroxyphenylglycine at pH 8.5).

[0302] To prevent nonspecific binding, the membrane was blocked at room temperature (RT) for 1 hour using 5% (w / v) skim milk powder in Tween-buffered saline (TBS-T) (6.06 g / L Trizma® base, 8.76 g / L NaCl, 0.25% (v / v) Tween-20 at pH 7.6). Subsequently, the membrane was washed with TBS-T for 10 minutes and incubated overnight (o / n) at 4°C on a CERTOMAT® MO II (Sartorius) shaker with the corresponding primary antibody in 3% (w / v) skim milk powder in TBS-T (see Table 3a).

[0303] Table 3a. List of specific primary antibodies used for VLP protein detection.

[0304] Antibody Dilution source Polyclonal rabbit anti-2A peptide (Gag) 1：1000 Millipore, ABS31 Monoclonal (IgG) mouse anti-human endogenous retrovirus type K (HERV K) envelope protein (p15E, TM) 1：8000 Austral Biologicals, HERM-1811-5 Monoclonal (IgG) mouse anti-human endogenous retrovirus type K (HERV K) envelope protein (gp70, SU) 1：2000 Austral Biologicals, HERM-1821-5

[0305] The membrane was then washed three times with TBS-T for 10 minutes each time. It was then incubated at RT for 1 hour with the corresponding secondary antibody diluted in TBS-T (see Table 3b).

[0306] Table 3b. List of secondary antibodies for HRP conjugation used in WB.

[0307] Antibody Dilution source Polyclonal goat anti-rabbit Ig antibody, HRP conjugated 1：2000 Dako, P0448 Polyclonal rabbit anti-mouse Ig antibody, HRP conjugated 1：2000 Dako, P0260

[0308] The membrane was then washed three times with TBS-T (10 minutes each time). Peroxidase chemiluminescent substrate (KPL, 54-61-00) was used for protein detection in an ImageQuant LAS 4000 camera (GE Healthcare Life Sciences).

[0309] Enzyme-linked immunosorbent assay (ELISA)

[0310] For the detection of MelARV-specific antibodies in vaccinated mice, the peptide of MelARV Env subunit p15E conjugated with BSA was purchased from Schafer-N (Copenhagen, Denmark).

[0311] Table 4: Description of peptides used in ELISA. This table describes the peptides used to coat ELISA plates for analyzing antibody responses in mice. Further details are provided regarding the originating protein (target protein), the location (region) of the peptide within the target protein, and its sequence.

[0312]

[0313] MaxiSorp (Thermo Fisher) plates were coated overnight at 4°C with 100 μL peptide solution (2 μg / mL in PBS) per well, followed by two washes with wash buffer (PBS + 2.07% NaCl + 0.1% Tween-20). The wells were blocked at 37°C for 2 hours with dilution buffer (PBS + 2.07% NaCl + 0.05% BSA + 0.05% Tween-20), washed once with wash buffer, and incubated at 37°C for 3 hours with diluted mouse serum (1:50 in dilution buffer). After two washes, the peptide-bound serum antibody was incubated at 37°C at a 1:2000 dilution for 2 hours with HRP-conjugated goat anti-mouse immunoglobulin antibody (Dako, P0447). After two additional washing steps, 100 μL of LTMB PLUS2 (Kem-En-Tec Diagnostics, 4395A) was added, and the mixture was incubated at RT for 8 minutes. The reaction was terminated with 100 μL of 0.2 M H₂SO₄, and quantification was performed by measuring the optical density at 450 nm.

[0314] Ad5 was inactivated by heat (30 minutes, 56°C) at 5 x 10 9 ELISA plates were coated with virus particles / mL to perform the detection of Ad5-specific antibodies in mouse serum. The assay was performed as described above, but with a shorter incubation time for blocking and antibody binding at RT for 1 hour. The primary antibody was mouse serum serially diluted 1:200 starting at 1:2.

[0315] MelARV protein in infected Vero cell lysates, supernatants, and purified VLPs was detected by coating ELISA plates with separate samples. Cell lysates were diluted 1:2 in PBS (100 μL), the supernatant was applied undiluted (100 μL), and the purified VLPs were diluted 1:25 in PBS (50 μL). Primary antibodies were used with anti-p2A (1:500), MM2-9B6 (1:100), 4F5 (1:100), and 19F8 (1:100), and the detection was performed using the same procedure as for the secondary antibodies described previously in Table 3.

[0316] Flow cytometry

[0317] In HERV-K related experiments, FACS was used to detect both extracellular and intracellular markers from activated immune cells of vaccinated mice, as well as the presence of the HERV-K Env protein on the surface of infected A549 cells. The cell sorting instrument was a BD LSR II flow cytometer (BD Biosciences).

[0318] The following buffer solutions are used for FACS:

[0319] Table 3c. Description of the components contained in the different buffers used for FACS.

[0320]

[0321] Extracellular staining with serum antibodies

[0322] In non-HERV-K experiments, flow cytometry was performed to detect the binding of serum antibodies to cancer cells. B16F10-GP cells or CT26 cells were resuspended (as described in “0 Tumor Attack”) and cultured in 96-well round-bottom plates at 4 x 10⁻⁶ cells / well. 5Cells were seeded per well. The plate was centrifuged at 784 g for 3 minutes (4°C) to fix the cells at the bottom of the wells. The culture medium was removed by gently tapping the plate inverted, 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 incubation at 4°C for 20 minutes, the plate was centrifuged at 784 g for 3 minutes (4°C), and the culture medium was removed. The cells were washed twice with 200 μL of washing 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 at 4°C for 20 minutes, washed twice with washing medium, and fixed at 4°C in 200 μL PFA solution (1% in PBS) for 15 minutes. Cells were resuspended twice in FACS medium, and fluorescence was analyzed using a BD LSR II flow cytometer.

[0323] Following the same protocol, MelARV Env assays were performed on the surface of infected Vero cells using monoclonal antibodies targeting 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).

[0324] Furthermore, this technique was performed using the Ad19 vector encoding the HERV-K wt and HERV-K ISD mut transgenes (Sirion) to characterize novel vaccine strategies and compare the use of different adenovirus 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.

[0325] 3x10 6 A549 cells were seeded at 75 cm⁻¹ 2 In 15 mL of Ham's F-12K medium in each flask, incubated at 37°C for 2 hours. Each flask was infected with 50 MOI of the following virus (1.5 x 10⁸ IFU / flask):

[0326] Ad5-(TetO)-CMV-SIVgag_p2A_HERV-K108env_P2TS

[0327] Ad19a(II)-(TetO)-CMV-ISDmut_MelARV-P2TS

[0328] Ad19a(II)-(TetO)-CMV-coHERV-K-P2TS from Sirion

[0329] Ad19a(II)-(TetO)-CMV-ISDmut_coHERV-K-P2TS from Sirion

[0330] They were then incubated at 37°C for 5 hours, after which the culture medium was replaced with Ham's F-12K FBS-free medium. The cells were then incubated at 37°C for 48 hours.

[0331] Cells were placed on ice inside the LAF workbench. Culture medium was aspirated, and cells were carefully washed with cold PBS, scraped off in cold PBS, and then separated by centrifugation (3 min, 4°C, 784 g). Cells were resuspended in PBS and aliquoted into 96-well round-bottom plates (Thermo Fisher Scientific, 163320). The plates were centrifuged (3 min, 4°C, 784 g), and the SN was removed by gently tapping the plates. Cells were resuspended at 4°C in 50 µL of FACS buffer containing 2 µg / mL mouse monoclonal (IgG) primary antibody targeting the p15E(TM) domain of the HERV-K Env protein (AustralBiologicals, HERM-1811-5) for 20 min. Cells were then washed with FACS wash buffer (150 µL initially, followed by 200 µL) and centrifuged three times (3 min, 4°C, 784 g). The plates were incubated with 100 µL FACS buffer, previously containing goat anti-mouse IgG APC secondary antibody (BioLegend, 405308) diluted 1:100. They underwent 20 minutes of incubation at 4°C in the dark. The cells were then centrifuged (3 min, 4°C, 784 g) and washed three times with 200 µL FACS wash buffer. Subsequently, they were incubated for 15 minutes at 4°C in the dark with 200 µL 1% (w / v) paraformaldehyde (PFA; Rigshospitalet, Copenhagen, Denmark). After this, they were centrifuged (3 min, 4°C, 784 g) and resuspended in 100 µL FACS buffer, and centrifuged again (3 min, 4°C, 784 g). Finally, they were resuspended in 200 µL and stored in the dark at 4°C. The following day, the fluorescence of the cells was analyzed using a BD LSR II flow cytometer, and the data were processed and analyzed using a FlowJo 10 (FlowJo LLC) instrument.

[0332] Intracellular staining (ICS) of stimulated spleen cells

[0333] Mice were euthanized and their spleens isolated 3–4 weeks after vaccination. The extracted spleens were transferred to HANKSB.SS and homogenized using a sterile mesh to obtain a single-cell suspension. After centrifugation and resuspending in complete RPMI, the concentration of spleen cells was determined, and the cells were diluted to the desired concentration.

[0334] Splenocytes were divided into 2.5 x 10⁻⁶ cells. 6 Cells were added per well to a 96-well round-bottom plate. Cells were centrifuged at 784g for 3 min and then resuspended in complete RPMI (+50 μM 2-mercaptoethanol) containing 3 μM monensin (a pathway inhibitor) and 1 μg / mL peptide (AH1), while the negative control did not accept the peptide. Cells were then incubated at 37°C for 5 h. After washing cells in FACS medium (PBS + 1% BSA + 0.1% NaN3 + 3 μM monensin), cells were incubated at 4°C for 20 min with fluorescently labeled surface antibodies (anti-CD4, anti-CD8, anti-CD44, anti-B220) diluted 1:100 in FACS medium. Cells were washed twice with PBS + 3 μM monensin and fixed in 1% PFA at 4°C for 15 min. After washing in FACS medium, cells were permeabilized with 0.5% saponins in PBS at RT for 10 min. Intracellular antibodies (anti-IFNγ, anti-TNFα) were added at a 1:100 dilution in PBS + 0.5% saponin and incubated at 4°C for 20 min. Cells were washed twice and finally resuspended in PBS + 1% BSA + 0.1% NaN3. Cell fluorescence was analyzed using a BD LSR II flow cytometer. Analysis of flow cytometry data is shown in the supplementary... Figure 5 .

[0335] Table 5: List of primary antibodies used in flow cytometry. This table lists the primary antibodies used in flow cytometry, their origin, working dilution, and the respective fluorescently conjugated secondary antibodies. Some primary antibodies are directly conjugated to fluorescence and do not require labeling with a secondary antibody.

[0336] Primary Antibody Product Number / Origin Dilution Secondary antibody mouse serum Isolate from vaccinated mice 1：50 Goat anti-mouse IgG_APC 19F8 (anti-MelARVEnv; p15E) Concentrated cell culture supernatant from hybridoma (provided by George Cianciolo, Duke University Medical Center) 1：50 Goat anti-mouse IgG_APC 4F5 (Anti-MelARV Env; p15E) Concentrated cell culture supernatant from hybridoma (provided by George Cianciolo, Duke University Medical Center) 1：50 Goat anti-mouse IgG_APC MM2-9B6 (anti-MelARVEnv; gp70) 20x cell culture supernatant from hybridoma (provided by Tsuyoshi Takami, University of Arizona Health Sciences Center) 1：50 Goat anti-mouse IgG_APC MM2-3C6 (anti-MelARVEnv; gp70) 20x cell culture supernatant from hybridoma (provided by Tsuyoshi Takami, University of Arizona Health Sciences Center) 1：50 Goat anti-mouse IgM_PE MM2-9A3 (anti-MelARVEnv; gp70) Cell culture supernatant from hybridoma (provided by Tsuyoshi Takami, University of Arizona Health Sciences Center) Undiluted Goat anti-mouse IgG_APC PerCP / Cy5.5-CD8 #100734, Biolegend 1：100 FITC-CD4 #317407, Biolegend 1：100 Pacific Blue-B220 #RM2628, Invitrogen 1：100 APC / Cy7-CD44 #103028, Biolegend 1：100 APC-IFN #505810, Biolegend 1：100 PE / Cy7-TNFα #506324, Biolegend 1：100 PE / Cy7-CD8 #100721, Biolegend 1：100 Pacific Blue-CD8 #100728, Biolegend 1：100 APC-CD8 #100711, Biolegend 1：100 APC / Cy7-CD8 #100713, Biolegend 1：100

[0337] In HERV-K related experiments, spleen cell ICS was performed to evaluate specific cellular responses derived from vaccinated mice. To enable this experiment, different strong-binding (SB) HERV-K peptides, consisting of 8-10 amino acids from both the C57BL / 6 and BALB / c mouse strains, were previously tested for their ability to stimulate CD8+ T cells in HERV-K-vaccinated mice. Only one BALB / c 10-amino acid peptide (TYHMVSGMSL; SEQ ID No. 47) responded at position 192 of the HERV-K Env sequence. Therefore, this peptide, named P-HKE, was used to stimulate spleen cells in BALB / c mice immunized with Ad5 and Ad19 vectors encoding the HERV-K Env, along with an improved Ad19 vaccine containing a mutation at Env ISD.

[0338] Table 5a. Antibodies used for extracellular and intracellular staining of spleen cells obtained from vaccinated mice to test their derived cellular responses.

[0339] Antibody source Monoclonal rat anti-mouse TNFα, PE / Cy7 conjugate BioLegend, 506324 Monoclonal rat anti-mouse interferon-γ (IFNγ), APC conjugated BioLegend, 505810 <![CDATA[Monoclonal rat anti-mouse B220, Pacific Blue TM conjugated]]> Invitrogen, RM2628 Monoclonal rat anti-mouse / human CD44, APC / Cy7 conjugate BioLegend, 103028 Monoclonal rat anti-mouse CD8a, PerCP / Cy5.5 conjugate BioLegend, 100734 Monoclonal rat anti-mouse CD4, FITC conjugate BioLegend, 100406 Monoclonal rat anti-mouse CD8a, APC / Cy7 conjugate BioLegend, 100713 Monoclonal rat anti-mouse CD8a, APC conjugate BioLegend, 100711 <![CDATA[Monoclonal rat anti-mouse CD8a, Pacific Blue TM conjugated]]> BioLegend, 10072 Monoclonal rat anti-mouse CD8a, PE / Cy7 conjugate BioLegend, 100721

[0340] Ad5 and Ad19 HERV-K / ISDmut vaccines (initiated) were used in this experiment to compare the efficacy of different vaccines containing different vectors and insertion improvement strategies. Ten days after a booster immunization with the MVA vector, mice were euthanized, and their spleens were collected in 5 mL of Hank's BSS medium. The spleens were homogenized using a sterile Corning® 70 µm cell filter (Sigma-Aldrich, CLS431751) to obtain a single-cell suspension. The number of cells was then counted to seed the required amount of cells / well, and the total number of cells / spleen provided was recorded for later calculation of the absolute number of IFNγ+ CD8+ and CD4+ T cells per spleen.

[0341] Approximately 3x10 6Cells / well were seeded into 96-well round-bottom plates, centrifuged (3 min, 4°C, 784 g), and resuspended in RPMI medium. The 10-meric peptide of HERV-KEnv, previously named P-HKE (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. Before adding the P-HKE peptide, 50 µL of the protein transport inhibitor monensin (3 µM) was added to the wells to prevent cytokine expulsion from the cells. Additionally, 30 μL / well of the aforementioned P-HKE peptide was added to the stimulated wells to induce T cell cytokine production. The remaining wells did not accept any peptide but only DMSO at the same concentration as the stimulated sample and served as a negative control. Incubate the cells at 37°C for 5 hours.

[0342] After the incubation period, the cells were centrifuged (3 min, 4°C, 784 g) and washed twice with 100 µL of FACS buffer containing monensin (3 µM). Surface antibodies (PerCP / Cy5.5-CD8, FITC-CD4, Pacific Blue) were also added. TM -B220, APC / Cy7-CD44) was diluted 1:100 in FACS buffer containing monensin (3 µM). Spleen cells were resuspended in 50 µL of the previous solution, and 50 µL of FACS / monensin (3 µM) containing a 1:100 dilution of the following antibodies was used for compensation: PerCP / Cy5.5-CD8, FITC-CD4, Pacific Blue. TM-CD8, APC / Cy7-CD8, APC-CD8, PE / Cy7-CD8. Incubate the plate at 4°C in the dark for 20 minutes. Wash the wells twice with 100 µL of PBS containing 3 µM monensin. Then, add 100 µL of PBS / monensin (3 µM) along with 100 µL of PFA (2%) to fix the cells in the dark at 4°C. Wash the cells twice again with FACS / monensin (3 µM) and resuspend them in 150 µL of 0.5% saponin in PBS at 20°C (in the dark) for 10 minutes. Once the cells have been permeated, the intracellular antibodies (APC-IFNγ, PE / Cy7-TNFα) are diluted 1:100 in 0.5% saponin / PBS, and 50 µL is added to the wells. Incubate the plate at 4°C in the dark for 10 minutes. Cells were washed with PBS containing 1% BSA and 0.1% NaN3 and finally resuspended in 200 µL of the same buffer. The plate was kept at 4°C.

[0343] Additionally, intracellular staining was performed on A549-transfected cells to confirm the presence of HERV-K Env protein within the cells. In this case, production (rather than secretion into the cell membrane) was evaluated. A subsequent protocol involved an incubation step at 4°C in the dark for 10 minutes with 150 µL of 0.5% (w / v) saponin (Sigma Aldrich, 47036) diluted in PBS. This additional step was necessary to permeate the cell membrane. The antibody was also diluted with 0.5% saponin.

[0344] Gating strategy

[0345] FlowJo 10 (FlowJo LLC) is used to analyze data from both extracellular and IC FACS staining (see [link]). Figure 27Initially, cells were plotted in forward scatter (FSC)-H and FSC-A maps and then gated. This gate was used to separate lymphocyte populations in the side scatter (SSC)-A and FSC-A maps. A subsequent population was gated for CD8+ CD4- cells, and then for CD8+ B220- cells to obtain a CD8+ T cell population, removing CD4+ T cells and B cells (B220 markers) from the analysis (Coffman & Weissman 1981). Cells were then gated for CD8+ CD44+ T cells to obtain only activated CD8+ T cells. These were further gated for IFNγ+ CD44+ cells, two markers expressed after T cell activation. Furthermore, IFNγ is known to be a more sensitive marker for activated CD8+ T cells when compared to the TNFα cytokine (Badovinac & Harty 2000) (Kristensen et al. 2004). In addition, CD8+CD44+ T cells gate IFNγ+TNFα+ cells because CD4+ T cells that produce multiple cytokines are known to have higher levels of activity, activation, and transformation into memory cells (Kannanganat et al. 2007).

[0346] To estimate the absolute number of IFNγ+CD44+B220-CD8+ T cells, the percentage of IFNγ+CD44+B220-CD8+ T cells was multiplied by the number of lymphocytes in each spleen. Additionally, the percentage of IFNγ+TNFα+ cells was calculated by dividing IFNγ+TNFα+ cells by the sum of IFNγ+TNFα+ and IFNγ+TNFα- cells.

[0347] Enzyme-linked immunospot (ELISPOT)

[0348] An ELISPOT assay was 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 in the MelARV Env subunit gp70 (Huang, AY et al.). The immunodominant major histocompatibility complex class I-restricted antigen of a murine colon tumor derives from an endogenous retroviral gene product. Proc Natl Acad Sci USA, 1996. 93(18): 9730-5).

[0349] Prepare spleen cells from vaccinated mice as described for ICS.

[0350] The assay was performed using the mouse IFN-γ T cell ELISPOT kit (CT317-PR5, U-CyTech). Briefly, membranes in 96-well polyvinylidene fluoride (PVDF) plates (MSIP S4510, Millipore) were activated with 70% ethanol and then coated overnight with anti-mouse IFN-γ antibody. After removing the coating antibody and blocking the membrane, spleen cells were seeded at 2 x 10^5 cells / well in complete RPMI medium containing 1 μg / mL AH1. As a control, spleen cells were either unstimulated or stimulated with an effective T cell activator, concanavalin A (ConA) (2 μg / mL). After 48 hours of incubation under normal cell culture conditions, cells were removed, wells were washed, and then incubated with a biotinylated detection antibody targeting IFN-γ. Streptomycin-HRP conjugate was added, and IFN-γ spots were visualized using AEC substrate solution. Spots were counted using a CTL ImmunoSpot analyzer.

[0351] Positive control (control serum LEV76)

[0352] The positive control serum LEV76 was used as a standard for flow cytometry and ELISA analysis of mouse serum samples. LEV76 serum originated from an earlier preliminary study in which C57BL / 6 mice were vaccinated against MelARV Env and showed protection against B16F10-GP lung metastases. Therefore, the antibody response in this serum corresponds to a level that potentially provides protection against tumor attack and thus serves as a reference value for a successful antibody response. Furthermore, using LEV76 control serum as a standard allows for comparisons between different experiments.

[0353] Statistical analysis

[0354] All statistical analyses were performed using GraphPad Prism software (v5.03). Two-tailed unpaired Menwynne test was used for comparisons between groups. Significance was indicated by an asterisk: * (P ≤ 0.05); ** (P ≤ 0.01); *** (P ≤ 0.001). When comparing different groups of vaccinated mice, results are shown as the mean versus the standard error of the mean (SEM) for each group.

[0355] The Kaplan-Meyer estimator was used to compare survival curves in mice. This test measures the portion of subjects that survive a given period of time after treatment. Significant results are indicated by an asterisk (*), where * (P ≤ 0.05); ** (P ≤ 0.01); *** (P ≤ 0.001).

[0356] To evaluate the correlation between responses, Spearman correlation coefficient was used, followed by p-value adjustment using the Holm-Sidak method.

[0357] Example 1

[0358] Mutations in the immunosuppressive domain (ISD) encoded by vaccines

[0359] As a first-line strategy for improvement, two point mutations were introduced into the MelARV Env sequence to inactivate the immunosuppressive domain (ISD). Figure 3 These specific mutations were previously tested and analyzed by Schlecht-Louf et al. (Schlecht-Louf, G. et al., Retroviral infection in vivo requires an immune escape virus factor encrypted in the envelope protein of oncoretroviruses. Proc Natl Acad Sci USA, 2010. 107(8): 3782-7). The virus encoding this modified form of MelARV Env is called Ad5-MelARV-ISD.

[0360] Effect of Ad5-MelARV-ISD on antibody response in CD1 mice

[0361] Outcrossed CD1 mice were induced with DNA-MelARV or DNA-MelARV-ISD and subsequently boosted with AD5-MelARV or Ad5-MelARV-ISD according to the vaccination timeline IV. Blood samples were collected four weeks after adenovirus vaccination and analyzed by ELISA.

[0362] like Figure 7A As shown, p15E-specific antibodies are increased in mice vaccinated with the Ad5-MelARV-ISD vaccine. In particular, the combination of DNA-MelARV-ISD and Ad5-MelARV-ISD (strip D) produces a high antibody response comparable to that of the LEV76 control serum.

[0363] In addition, compared with the GFP control (strip E), vaccination with Ad5-MelARV (strips A and C) and Ad5-MelARV-ISD (strips B and D) increased the level of tumor cell-specific antibodies. Figure 7B However, the levels of tumor-binding antibodies induced by Ad5-MelARV-ISD were significantly lower than those induced by Ad5-MelARV (A vs. B; and C vs. D, but not significantly).

[0364] The levels of p15E and B16F10-GP binding antibodies suggest that induction with DNA-MelARV-ISD generally increases antibody responses compared to DNA-MelARV-induced mice, although these results were not significant.

[0365] Example 2

[0366] The role of Ad5-MelARV-ISD in antibody response and transfer in C57BL / 6 mice

[0367] C57BL / 6 mice were vaccinated and challenged according to vaccination timeline III. Mice received either DNA-MelARV or DNA-MelARV-ISD, followed by the corresponding adenovirus. Analysis of antibody responses revealed that MelARV-ISD slightly increased the level of B16F10-GP cell-specific antibodies (…). Figure 8A However, this increase was not significant and was only slightly higher than the background level in mice vaccinated with the PBS vaccine. Figure 8B As shown, no effect of the p15E-specific antibody was observed. Corresponding to the tumor cell-binding antibody, metastasis was slightly reduced in mice vaccinated with MelARV-ISD, but the difference was not significant. Figure 8C ).

[0368] Example 3

[0369] Effect of Ad5-MelARV-ISD on T cell responses in Balb / C mice

[0370] In addition to antibody response, the effects of Ad5-MelARV-ISD on T cell initiation and activation were also analyzed. ELISPOT ( Figure 9 Both ICS and ICS (Figure 10) showed increased levels of AH1-specific T cells in mice vaccinated with the Ad5-MelARV-ISD vaccine compared to Ad5-MelARV. As observed by ICS, double-positive IFNγ cells were also increased compared to the natural form. + TNFα + CD8 + T cells were significantly increased in mice vaccinated with the Ad5-MelARV-ISD vaccine. IFNγ + The integrated geometric mean (IGM) of cells also showed a significant difference from that of native Ad5-MelARV. IGM combines the number of positive cells with the mean fluorescence intensity and therefore also takes into account the quality of activated immune cells. The IGM for TNFα remained insignificant (data not shown).

[0371] Example 4

[0372] Immunosuppressive Effects of Ad5-MelARV / Ad5-MelARV-ISD

[0373] To analyze the mechanism behind the increased immune response to Ad5-MelARV-ISD, immunosuppression through the vaccine was investigated. The immune response against the viral vector Ad5 was analyzed by ELISA using the same mouse sera shown in Figure 7 as mice vaccinated with either Ad5-MelARV or Ad5-MelARV-ISD. The ISD-inactivated MelARV Env vaccine (Ad5-MelARV-ISD) showed a significantly increased titer of Ad5-binding antibodies compared to the natural form of MelARV Env (Ad5-MelARV with a functional ISD).

[0374] Example 5

[0375] Antigens were displayed on the capsid protein pIX of the adenovirus vector.

[0376] In an attempt to increase protective antibody responses, p15E was conjugated to the adenovirus capsid protein pIX on a previously tested adenovirus vaccine. Figure 12 Different constructs tested are shown in the figure. Native p15E (excluding the transmembrane subunit and cytoplasmic tail) was added to pIX (1) or alternatively to the ISD mutant form (2). In addition, variants of p15E truncated to ISD, which showed an additional cysteine ​​(3) or not shown (4), were tested. The core of the viral vector matched the shown p15E: Ad5-MelARV for pIX-p15E, pIX-p15E-trunc-wC, and pIX-p15E-trunc-w / oC, and Ad5-MelARV-ISD for pIX-p15E-ISD.

[0377] Characterization of the Ad5 vector displaying p15E on capsid protein pIX

[0378] The novel pIX plasmid construct (pcDNA3-pIX-Taglinker-xxx, where xxx = p15E antigen) was tested for proper expression of recombinant pIX by transfecting HEK293 cells. The lysis products of transfected cells were analyzed using Western blotting with an anti-pIX antibody. Figure 13A All four constructs showed expression of recombinant pIX, with the expected lower bands in the truncated p15E form (rows 3 and 4). GFP conjugated with pIX was used as a positive control, showing a higher band of approximately 50 kDa. To verify integration of recombinant pIX into the viral vector, purified virus was analyzed by Western blotting using an anti-pIX antibody. Figure 13BAdjacent to the natural pIX band (approximately 10 kDa), all constructs showed expression of recombinant pIX. The negative control, unmodified Ad5(Ø), showed only the natural pIX band. Band intensity was quantified using ImageJ software (version 1.51n), and the percentage of recombinant pIX is shown in Table 8.

[0379] Table 8: Integration efficiency of recombinant pIX into the Ad5 vector. The integration efficiency of recombinant pIX into the viral vector was analyzed using recombinant Ad5 virus displaying proteins on viral pIX. Virus analysis was performed by Western blot (Figure 13), and band intensity was quantified. This table shows the percentage of recombinant pIX in the total pIX within the viral particle.

[0380] Virus % of total pIX recombined pIX Ø 0% Ad5-MelARV_pIX-p15E 32% Ad5-MelARV-ISD_pIX-p15E-ISD 29% Ad5-MelARV_pIX-p15E-trunc-wC 35% Ad5-MelARV_pIX-p15E-trunc-w / oC 35%

[0381] Example 6

[0382] Analysis of antibody responses induced by pIX-modified virus in CD1 mice

[0383] According to the vaccination timeline IV, CD1 mice were vaccinated with DNA-initiated vaccines (DNA-MelARV or DNA-MelARV-ISD), followed by adenovirus booster (normal virus relative to pIX modification). Serum p15E-specific antibodies were analyzed by ELISA. Figure 14A Because the p15E peptide sequence used in ELISA is not included in the truncated form modified with pIX, only Ad5-MelARV_pIX-p15E and Ad5-MelARV-ISD_pIX-p15E-ISD can be evaluated in this setting. In most cases, p15E display on pIX increases the level of p15E-specific antibodies (A vs. B; C vs. D; E vs. F). However, in these comparisons, significant differences were observed only for DNA-MelARV + Ad5-MelARV (A vs. B). In the case of DNA-MelARV-ISD + Ad5-MelARV-ISD (G vs. H), pIX-p15E-ISD display had a deteriorating effect and significantly reduced antibody response compared to the unmodified vaccine.

[0384] In addition, the binding of serum antibodies to B16F10-GP cells was analyzed. Figure 14B The display of native p15E on pIX does not affect antibody responses against tumor cells. On the other hand, Ad5-MelARV-ISD_pIX-p15E-ISD is able to restore the lack of B16F10-GP specific antibodies, which are reduced due to the ISD mutation of MelARV Env (compared to Figure 7).

[0385] Example 7

[0386] The role of Ad5-MelARV_pIX-p15E in antibody response and transfer in C57BL / 6 mice

[0387] In a preliminary study, antibody response and protection against metastasis of the pIX-modified virus Ad5-MelARV_pIX-p15E were tested in C57BL / 6 mice. Mice were vaccinated twice and challenged according to vaccination timeline V. Figure 15 As shown in A and 15B, neither vaccine significantly increased the activity against B16F10-GP cells (15A) or p15E (15B). Figure 15 Antibody response (B). Furthermore, vaccination did not significantly reduce the number of metastases (B). Figure 15 C). However, although no correlation was detected between tumor cell-specific antibodies and metastasis counts ( Figure 15 D), but a significant negative correlation was observed between the level of p15E-specific antibodies and the number of metastases (D). Figure 15 E).

[0388] Example 8

[0389] In attempts to improve the representation of MelARV Env on VLPs in terms of both quantity and quality (to a more natural conformation), functional structural domains are inserted into the natural sequence. These modifications apply to the full-length MelARV Env as well as the individual p15E ( Figure 16 Modifications include the signal peptide from Gaussian luciferase (LucSP), the transmembrane domain and cytoplasmic tail from influenza A virus hemagglutinin H3N2 (HA-TMCT), and the trimer sequence (GCN4). Figure 16 The chimeric Env or p15E protein is co-encoded with the Gag protein of SIV.

[0390] Characterization of vaccines encoding chimeric MelARV Env or p15E

[0391] The modified vaccine has not yet been tested in mice, but expression of the adenovirus was tested by flow cytometry in infected Vero cells. Figure 17 This experiment not only demonstrated protein expression but also characterized some anti-MelARV Env antibodies regarding their target epitopes. Compared to natural vaccines (Ad5-MelARV and Ad5-MelARV-ISD), 19F8 ( Figure 17 A) and 4F5 Figure 17B) Both showed a significantly increased binding to the modified forms of MelARV Env and p15E. Since binding to Ad5-LucSP_GCN4_p15E_Ha-TMCT was also observed, this experiment indicates that both antibodies bind to the transmembrane subunit p15E. Furthermore, no binding of 19F8 to Ad5-MelARV-ISD-infected cells was observed, but a clear signal was detected with respect to 4F5, confirming that ISD is a target epitope of 19F8. None of the MM2 antibodies showed binding to the p15E construct, confirming that all three antibodies target the surface subunit gp70. MM2-9B6 ( Figure 17 C) and MM2-3C6 ( Figure 17 D) showed a similar profile, with antibodies binding equally strongly to cells infected with Ad5-MelARV and Ad5-LucSP_MelARV_Ha-TMCT. On the other hand, cells infected with Ad5-MelARV-ISD showed much less antibody binding. MM2-9A3 ( Figure 17 The profile for E) is similar, except that Ad5-MelARV-infected cells bind fewer antibodies compared to Ad5-LucSP_MelARV_Ha-TMCT-infected cells.

[0392] The ability of the novel construct to produce and display target proteins post-infection was also tested. Western blot analysis was performed on the lysate of infected Vero cells and purified VLP (Vol. LP). Figure 18 The binding of anti-p2A antibodies ( ). Figure 18 A) The displayed bands indicate the expression of MelARV Gag (rows 1 and 2) and SIV Gag (rows 3 and 4) in both the cleavage products and VLP. (See also...) Figure 18 As shown in B, p15E (bound by 4F5) was detected only in the lysates of Ad5-MelARV-infected cells (row 1), exhibiting a low band of approximately 20 kDa and a high band of 70 kDa corresponding to p15E, showing the full-length Env (gp70 + p15E). The full-length Env was also detectable for Ad5-MelARV-ISD (row 2), while the single p15E band was not visible. p15E and the full-length Env were detectable only in Ad5-MelARV-induced VLPs. Other weak bands were also present for different constructs, but the corresponding proteins are unknown.

[0393] exist Figure 18 Similar results to 4F5 have been shown in C, where gp70 is revealed by MM2-9B6. Only the VLP induced by Ad5-MelARV (row 1) shows gp70 detected by MM2-9B6.

[0394] In addition to cell lysis products, the supernatant of infected cells was also analyzed to investigate whether proteins were secreted. Figure 18 D, E). Supernatant ( Figure 18 p15E (bound by 4F5) in D) was only detectable for Ad5-MelARV (row 1) and is likely due to VLP in the supernatant. On the other hand, MM2-9B6 (Figure E) revealed that Ad5-MelARV-ISD-infected cells (row 2) released large amounts of gp70, which was detected as conjugates of varying sizes. Figure 18 E). In contrast, Ad5-MelARV induced a small amount of gp70 release (line 1).

[0395] Although expression was detectable on cell surfaces under non-denaturing conditions, none of the novel modified MelARV Env proteins (rows 3 and 4) showed expression in lysate, supernatant, or purified VLP.

[0396] Since no bands for the new construct were observed in Western blot analysis, it was assumed that the synthesized protein could not bind to the nitrocellulose membrane. Therefore, ELISA analysis was performed using ELISA plates coated with cell lysis products, supernatant, or VLP. Figure 19 As expected, Gag protein, detected by anti-p2A, was present in all samples. Figure 19 A). In contrast, MelARV Env gp70 (bound by MM2-9B6) was detected only in the VLP (row 1) of cells infected with AD5-MelARV, but not in cells infected with modified MelARV virus. Figure 19 B). Similar results were observed for p15E expression (by the combination of 4F5 and 19F8). Figure 19 C, D). Ad5-MelARV (row 1) induced high expression of the transmembrane subunit, which successfully integrated into the VLP. On the other hand, for Ad5-MelARV-ISD (row 2), the protein was almost undetectable in either sample. The modified vaccines (rows 3 and 4) induced p15E expression and VLP integration to some extent, but at much lower levels than the natural MelARV Env vaccine. Figure 19 C, D).

[0397] Example 9 (Comparative)

[0398] The following constructs were used to immunize balb / c mice: HIV B gag P2A ConB gp140 G / CD (WT), 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). Immunization was performed 4 weeks post-immunization (d. 28 – Figure 20 A) and 7 weeks (d. 49 – Figure 20 (B) Antibody responses were analyzed against the HIV ConB gp140 WT protein. Another group of mice immunized with both IiGP-P2A-IFNalpha4 and HIV B gag P2A ConB gp140 G / CD (WT) (420B) served as a control group for type I interferon-induced responses.

[0399] The following constructs were used to immunize c57 / bl6 or c57 / bl6 IFN-g KO mice: an adenovirus encoding HIV B clade gagp2A followed by the gp140 sequence of the B clade common sequence (HIV B gag P2A ConB gp140 G / CD) (WT) and HIV B gag P2A ConB gp140 G / CD ISD#19 (L70Q). Immunization was performed 4 weeks post-immunization (d. 26 – Figure 20 C), to measure the antibody response against HIV ConB gp140 CF protein.

[0400] Example 10

[0401] The following constructs were used to immunize balb / c mice: HIV B gag P2A ConB gp140 G / CD (WT), 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 post-immunization (d. 114), mouse T-cell responses were analyzed in response to the following peptide libraries: a peptide library covering Gag genes (MA (p17, matrix) (peptides 1-31), CA (p24, capsid) (peptides 32-89), and a single library covering p2, NC (nucleocapsid), p1 and p6 (peptides 90-124), gp120 (1) (peptides 1-62), gp120 (2) (peptides 63-124), and gp41 (peptides 125-211).

[0402] Example 11

[0403] BALB / c mice were vaccinated with MVAs expressing gag, env, gag+env, or gag+envISDmut as VE-VLP, or adenoviruses expressing gag-env or gag+envISDmut VE-VLP, or combinations thereof, and peptide responses were measured against the expected 9-amino acid-long peptide bound to MHC by ELISPOT or intracellular cytokine staining.

[0404] In particular, Gag-env or gag+envISDmut VEVLP in adenovirus vectors are strongly expected to be superior to the previously described MVA vectors in inducing T cell responses.

[0405] Example 12:

[0406] BALB / c mice were vaccinated with MVAs expressing gag, env, gag+env, or gag+envISDmut as VE-VLP, or adenoviruses expressing gag-env or gag+envISDmut VEVLP, or combinations thereof, and peptide responses were measured against sequences of the extracellular portion of the p15E transmembrane domain derived from HERV-Kcon.

[0407] The Gag-env or gag+envISDmut VEVLP vectors are expected to be superior to the previously described MVA vectors in inducing T cell responses.

[0408] Example 13

[0409] Animals were subcutaneously attacked with RENCA renal cell carcinoma cells expressing HERVcon-gag and HERVcon-env, respectively. Subsequently, animals were vaccinated with MVAs expressing gag, env, gag+env, or gag+envISDmut as VE-VLP, or adenoviruses expressing gag-env or gag+envISDmut VEEVLP, or combinations thereof, and tumor growth was monitored.

[0410] With the use of the unique VE-VLP vaccine and gag-env VEVLP vaccine, which are capable of controlling tumor growth in two cell lines, tumor control is expected to improve.

[0411] Example 14

[0412] Animals were intravenously challenged with RENCA renal cell carcinoma cells expressing HERVcon-gag and HERVcon-env, respectively. Animals were then vaccinated with MVAs expressing gag, env, gag+env, or gag+envISDmut as VE-VLP, or adenoviruses expressing gag-env or gag+envISDmut VEEVLP, or combinations thereof. Tumor growth was monitored 30 days post-tumor challenge via in vivo dissection and metastasis counting.

[0413] With the use of the unique VE-VLP vaccine and gag-env VEVLP vaccine, which are capable of controlling tumor growth in two cell lines, tumor control is expected to improve.

[0414] Example 15

[0415] The translation of the immunotherapy strategies described in the previous embodiments used proteins encoding the common human endogenous retroviral type K (HERV-K) envelope (Env) and group-specific antigen (Gag) proteins. (Dewannieux, M., et al.) Genome Res. 2006 Dec;16(12):1548-56) The adenovirus vector (Ad5 / Ad19a) was engineered to produce a human-associated form of the vaccine, which is expected to lead to VLP formation in transduced cells. (Muster T, et al., Cancer Res. 2003 Dec 15;63) (24):8735-41） To improve vaccination strategies, ISD is contained in the p15E subunit of the HERV-K Env protein. (Morozov, VA., et al. PLoS One 2013 Aug 7; 8(8)) Inactivation via single-point mutation (see...) Figure 22 Its selection is based on Morozov, VA., et al., Retrovirology 2012 Aug 13; 9:67 and the conservative relationship between HERV-K and HIV (van der Kuyl AC., Retrovirology 2012 Jan 16;9:6) (Dewannieux, M., et al. J Virol. 2005 Dec;79(24):15573-7) .

[0416] HERV-K Gag-p2A-EnvISDmut has the following amino acid sequence (SEQ ID No. 48):

[0417]

[0418] The immunogenicity of these vaccines was tested in BALB / c, C57BL / 6, and CD1 mice, and their efficiency, as measured by mouse survival curves, was investigated in BALB / c mice challenged with mouse renal cancer (Renca or RLZ) cells expressing the HERV-K target protein. Immune responses were assessed for their ability to induce cellular and humoral responses, testing for the presence of INFγ+ CD8+ T cells (by FACS analysis) and for specific antibodies against the HERV-K target protein in mice immunized with the DNA / Adv-HERV-K WT / ISD vaccine and boosted with MVA Env.

[0419] The Ad19-HERV-K WT vaccine and its improved form containing the ISD mutation were tested, and their ability to induce VLP expression via the Gag_p2A_Env HERV-K Adv protein was compared. Pre-existing immunity in individuals leading to neutralizing antibodies (NAb) that block the immune response may be a drawback of using the Ad5 vector. Furthermore, the Ad19 vector is known to be more successful in transducing different cell types (Kiener R, et al., Sci Rep. 2018 Jan 24;8(1):1474). Therefore, the use of different adenovirus vectors (Ad19 versus Ad5) was also analyzed and compared.

[0420] To analyze the functionality of the novel strategy, the induction of HERV-K Gag and Env target proteins was analyzed in the vaccines. Therefore, VLP production and secretion were tested in VERO and A549 cell lines transfected with different virus-based vaccines containing different target sequences (see [link to relevant documentation]). Figure 23 The presence of HERV-K Gag and Env proteins was detected by Western blotting (WB) in supernatants (SN) and cell lysis products from the transfected cell lines described above. Monoclonal antibodies HERM-1811-5 and HERM-1821-5, targeting 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 p2A-linked Gag proteins. HRP-conjugated secondary antibodies were used for detection.

[0421] Western blot results indicated the presence of HERV-K Gag_p2A and HERV-K Env proteins in both the SN and cell lysis products of VERO and A549 cells transfected with Ad19_HERV-K WT / ISDmut. Higher expression of both Gag and Env proteins was observed in cells transfected with Ad19_HERV-K ISDmut compared to Ad19_HERV-K_WT and Ad5_HERV-K_Env vaccines. Figure 23 (As shown in lines 2 and 8), suggesting that the modified prototype vaccine has enhanced functionality and greater potential. Furthermore, the low concentrations of protein obtained after VLP purification of the corresponding samples can explain the absence of Gag and Env proteins in the SN of VERO cells transfected with Ad19_HERV-K.

[0422] To further validate the expression of the HERV-K Env target protein, A549 cells were transfected with a VLP-encoded adenovirus vaccine (see [link to relevant documentation]). Figure 24 Forty-eight hours post-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 and without prior fixation and infiltration. Intracellular and extracellular fluorescence of the antibody-bound cells, and consequently, HERV-K Env expression inside and outside the infected cells, were analyzed by FACS. Results suggested superior transfection efficiency when using the Ad19 vector compared to Ad5, as a higher signal was detected when using Ad19, despite both encoding the same target protein. When comparing the Ad19_HERVK WT and ISDmut vaccines, greater cell surface signaling and similar intracellular signaling were detected in cells transfected with Ad19_HERV-K_ISDmut, indicating improved cell surface sorting of the mutant sequence.

[0423] To visually confirm the generation of the structural protein Gag and the subsequent release of Env HERV-K, A549 cells were infected with 50 MOI of Ad19_HERV-K_ISDmut and fixed at 24 and 48 hours post-infection. Sprouting and secreted VLPs were then detected by electron microscopy (see [link to original text]). Figure 29 This indicates that the vaccine is fully capable of expressing HERV-KGag and the target protein Env, which is incorporated into the secreted VLP.

[0424] Example 16

[0425] To test T-cell responses induced by the Ad19_HERV-K WT / ISDmut vaccine, T lymphocyte responses to P-HKE (a decapitated peptide of HERV-K Env with the sequence TYHMVSGMSL) in BALB / c mice were analyzed. Since P-HKE is an MHC class I restricted epitope, intracellular staining (ICS) of cytokines using FACS was used to measure the activation of CD8+ T cells in BALB / c mice following peptide stimulation, and consequently the secretion of interferon-γ (IFNγ) and tumor necrosis factor-α (TNFα) cytokines.

[0426] BALB / c mice were induced with various vaccines consisting of different vectors (Ad5 / Ad19 / MVA) encoding the HERV-K protein. Half of them were then boosted with MVA Env to test whether it could increase the cellular response induced by the first immunization regimen. Mice were euthanized 10 days after the MVA booster, and their spleen cells were analyzed by FACS under P-HKE stimulation (see [link to FACS]). Figure 25 In both the booster (MVA-Env) and non-booster (Ø) regimens, the group receiving the Ad19_HERV-K_WT / ISDmut vaccine showed a higher number of INFγ-secreting specific CD8+ T cells. Furthermore, cellular responses induced by all adv vaccines appeared to increase after the MVA booster regimen. This booster appeared to exacerbate the differences between the vaccines used, particularly when studying the ratio of IFNγ / TNFα CD8+ T cells, which showed a significantly superior percentage in the mouse group receiving the improved adv vaccine (Ad19_HERV-K_ISDmut). This suggests that the Ad19 vector, encoding the target sequence, is best suited for inducing relevant CD8+ T cell responses in the priming-booster regimen when compared to Ad5 and MVA vectors. Additionally, the results suggest that the MVA vector can be used in booster regimens to increase cytotoxic T cell responses against the HERV-K Env target protein. These results together indicate a particularly effective vaccine design that generates an IFNγ+ CD8+ T cell-specific response against tumor cells expressing HERV-K Env, consisting of an immunization with an Ad19 vector and a booster regimen with an MVA vector encoding the HERV-K_Env protein, preferably encoding the HERV-K_Gag_p2A_Env-ISDmut protein.

[0427] Example 17

[0428] To test and compare the efficacy of the vaccine, mice were challenged and subsequently vaccinated, and their survival in relation to tumor progression was assessed (see [link to vaccine description]). Figure 26For this experiment, BALB / c mice were intravenously challenged with RENCA cells expressing HERV-K Env. Ten days after tumor challenge, mice were vaccinated with MVA Env, Ad19_HERV-K WT / ISDmut, and an unrelated vaccine as a control. The experiment was based on (Kraus et al., 2013 PLoS One. Aug 30; 8(8): e72756), which aimed to score metastatic tumor burden 40 days after injection, but animals were weighed longitudinally and mice were euthanized if any physical, behavioral, or physiological changes were observed in the animals, or if weight loss was greater than 10%. Once mice were killed, lungs were collected and stored in 4% PFA for further analysis of the presence of metastasis. Notably, all animals euthanized due to weight loss had a large visible tumor burden. Surprisingly, significant mortality was recorded during the execution of the experiment, and survival curves were established and compared between different groups. This indicates faster progression of RENCA-HERV-K tumors compared to those previously reported. In this fairly rigorous tumor attack model, mice receiving the Ad19_HERV-K_ISDmut vaccine showed a significant increase in life expectancy compared to controls. Three different statistical tests (time-series, Wilcoxon, and Tarone-Ware) showed significant p-values ​​(0.037, 0.046, and 0.040, respectively). This suggests that the Ad19_HERV-K_ISDmut vaccine delayed lung tumor progression and metastasis in BALB / c mice, consistent with the aforementioned results showing increased antibody and CD8+ T cell responses. None of the other vaccines prolonged survival.

[0429] Example 18

[0430] To further confirm the findings in the human system, tissue samples were obtained from human breast tumors. These were sectioned at 4 μm and stained with a 1:1000 dilution of primary antibody from non-immunized mice (pre-bleeding serum), boosted with Ad5_HERV-K_Env vaccines administered via Ad19_HERV-K_ISD (8 weeks post-immunization) and MVA_Env (2 weeks post-immunization). Figure 28 As shown, HERV-K antibodies from vaccinated mice can stain cancerous tissues expressing HERV-K target proteins.

[0431] Various aspects and implementation methods have been described in conjunction with the various embodiments herein. However, based on a study of the drawings, the disclosure, and the appended claims, those skilled in the art will understand and implement other variations to the disclosed embodiments when practicing the claimed subject matter. 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 perform the functions of several items recounted in the claims. The fact that certain measurements are recited in dissimilar dependent claims does not indicate that combinations of these measurements cannot be advantageously used. Computer programs may be stored / distributed on suitable media, such as optical storage media or solid-state media provided together with or as part of other hardware, but may also be distributed in other forms, such as via the Internet or other wired or radio communication systems.

[0432] Reference symbols used in the claims should not be interpreted as limiting the scope.

[0433] The sequences are disclosed in the body of the specification and in a separate sequence list according to WIPO Standard ST.25. The SEQ IDs designated by specific numbers in the body of the specification and in the separate sequence list should be the same. For example, SEQ ID no.:1 should define the same sequence in both the body of the specification and the separate sequence list. When there is a difference between the sequence definitions in the body of the specification and in the separate sequence list (e.g., if SEQ ID no.:1 in the body of the specification incorrectly corresponds to SEQ ID no.:2 in the separate sequence list), references to specific sequences in this application, particularly in specific embodiments, should be understood as references to sequences in the body of the application, not to sequences in the separate sequence list. In other words, differences between sequence definitions / designations in the body of the specification and in the separate sequence list are resolved by correcting the separate sequence list to the sequences and their designations disclosed in the body of the application, which includes the specification, embodiments, drawings, and claims.

[0434] Patent Project

[0435] 1. A vaccine for use in the prevention and / or treatment of a disease, comprising an adenovirus vector capable of encoding a virus-like particle (VLP) exhibiting an inactive immunosuppressive domain (ISD).

[0436] 2. The vaccine according to Project 1 is used for the prevention and / or treatment of cancer.

[0437] 3. The vaccine according to Project 1 or 2, wherein 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), having at least one amino acid deletion or exchanged with a different amino acid.

[0438] 4. According to Project 3, the amino acids that differ from the original are selected from naturally occurring amino acids.

[0439] 5. A vaccine according to any of the foregoing items, wherein at least one amino acid in a region of 10 amino acids upstream or downstream of the ISD is exchanged with a different amino acid.

[0440] 6. The vaccine according to any of the foregoing projects, wherein the VLP further demonstrates an endogenous retroviral (ERV) envelope protein or an immunogenic portion thereof.

[0441] 7. The vaccine according to any of the foregoing items, wherein the ERV envelope protein is a human endogenous retrovirus (HERV) protein or an immunogenic portion thereof.

[0442] 8. The vaccine according to any of the preceding items, wherein the HERV is selected from HERV-K, HERV-H, HERV-W, HERV-FRD, and HERV-E.

[0443] 9. A vaccine according to any one of items 1 to 8, wherein the HERV-K is selected from 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); HERV-H is selected from HERV-H19 (=HERV-H_2q24.3) and HERV-H_2q24.1; HERV-W is selected as ERVW-1 (=syncytokine-1); and HERV-FRD is selected as ERVFRD-1 (=syncytokine-2).

[0444] 10. The vaccine according to any of the foregoing items, wherein the adenovirus vector is derived from a mammalian adenovirus type, a human adenovirus type, a chimpanzee adenovirus type, or a gorilla adenovirus type.

[0445] 11. The vaccine according to any of the preceding projects, 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 chimpanzee adenovirus serotype.

[0446] 12. The vaccine according to any of the foregoing items, wherein the adenovirus vector is adenovirus, serotype 5 (Ad5).

[0447] 13. The vaccine according to any of the foregoing projects, wherein the protein product of the adenovirus vector includes gag protein, 2A peptide and envelope protein (Env).

[0448] 14. The vaccine according to any of the foregoing items, wherein the gag protein is an exogenous retroviral gag protein or an endogenous retroviral gag protein.

[0449] 15. The vaccine according to any of the foregoing projects, wherein the Env protein comprises a surface unit (gp70), a cleavage site, and a transmembrane unit (p15E).

[0450] 16. A vaccine according to any of the foregoing projects, wherein the transmembrane unit (p15E) comprises a fusion peptide, an immunosuppressive domain (ISD), a transmembrane anchor, and / or a cytoplasmic tail.

[0451] 17. A vaccine according to any of the preceding items, wherein p15E or its immunogenic portion is conjugated to the adenovirus capsid protein pIX.

[0452] 18. A vaccine according to any of the preceding items, wherein a signal peptide encoded by the adenovirus vector is exchanged with a signal peptide (LucSP) from Gaussian luciferase.

[0453] 19. A vaccine according to any of the preceding items, wherein the transmembrane anchor and cytoplasmic tail encoded by the adenovirus vector are exchanged with the transmembrane domain and cytoplasmic tail of the hemagglutinin from influenza A virus.

[0454] 20. A vaccine according to any of the preceding items, wherein the transmembrane anchor and cytoplasmic tail encoded by the adenovirus vector are exchanged with the transmembrane domain and cytoplasmic tail of influenza A virus hemagglutinin H3N2 (HA-TMCT).

[0455] 21. A vaccine according to any of the foregoing projects, wherein a trimerization sequence is provided near the signal peptide.

[0456] 22. The vaccine according to any of the foregoing projects, wherein the trimerized sequence is GCN4.

[0457] 23. The vaccine according to any of the foregoing projects, wherein the VLP contains gag protein.

[0458] 24. The vaccine according to any of the foregoing items, wherein the gag protein is an exogenous retroviral gag protein or an endogenous retroviral gag protein.

[0459] 25. The vaccine according to any of the foregoing items, wherein the VLP is produced in cells in a patient who has been infected with the adenovirus vector.

[0460] 26. The vaccine according to any of the foregoing items, wherein the VLP is produced in isolated mammalian cells.

[0461] 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) that is inactive.

[0462] 28. The nucleic acid construct according to Item 27, wherein 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), having at least one amino acid deletion or exchanged with a different amino acid.

[0463] 29. Based on the nucleic acid constructs in Item 27 or 28, the amino acids that differ from the original amino acids are selected from naturally occurring amino acids.

[0464] 30. According to the nucleic acid constructs of items 27 to 29, at least one amino acid in a region of 10 amino acids upstream or downstream of the ISD is exchanged with a different amino acid.

[0465] 31. Based on the nucleic acid constructs of items 27 to 30, the VLP further demonstrates an endogenous retroviral (ERV) envelope protein or its immunogenic portion.

[0466] 32. According to the nucleic acid constructs of items 27 to 31, wherein the ERV envelope protein is a human endogenous retrovirus (HERV) protein or its immunogenic portion.

[0467] 33. Nucleic acid constructs according to items 27 to 32, wherein the HERV is selected from HERV-K, HERV-H, HERV-W, HERV-FRD, and HERV-E.

[0468] 34. According to the nucleic acid constructs of items 27 to 33, wherein HERV-K is selected from 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); HERV-H is selected from HERV-H19 (=HERV-H_2q24.3) and HERV-H_2q24.1; HERV-W is selected as ERVW-1 (=syncytokine-1); and HERV-FRD is selected as ERVFRD-1 (=syncytokine-2).

[0469] 35. Nucleic acid constructs according to items 27 to 34, wherein the adenovirus vector is derived from a mammalian adenovirus type, a human adenovirus type, a chimpanzee adenovirus type, or a gorilla adenovirus type.

[0470] 36. Nucleic acid constructs according to 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 chimpanzee adenovirus serotype.

[0471] 37. Nucleic acid constructs according to items 27 to 36, wherein the adenovirus vector is adenovirus, serotype 5 (Ad5).

[0472] 38. According to the nucleic acid constructs of items 27 to 37, the protein products of the adenovirus vector include gag protein, 2A peptide and envelope protein (Env).

[0473] 39. According to the nucleic acid constructs of items 27 to 38, wherein the gag protein is an exogenous retroviral gag protein or an endogenous retroviral gag protein.

[0474] 40. Nucleic acid constructs according to items 27 to 39, wherein the Env protein comprises a surface unit (gp70), a cleavage site, and a transmembrane unit (p15E).

[0475] 41. According to the nucleic acid constructs 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.

[0476] 42. Nucleic acid constructs according to items 27 to 41, wherein p15E or its immunogenic portion is conjugated to the adenovirus capsid protein pIX.

[0477] 43. Nucleic acid constructs according to items 27 to 42, wherein the signal peptide encoded by the adenovirus vector is exchanged with a signal peptide (LucSP) from Gaussian luciferase.

[0478] 44. Nucleic acid constructs according to items 27 to 43, wherein the transmembrane anchor and cytoplasmic tail encoded by the adenovirus vector are exchanged with the transmembrane domain and cytoplasmic tail of hemagglutinin from influenza A virus.

[0479] 45. Nucleic acid constructs according to items 27 to 44, wherein the transmembrane anchor and cytoplasmic tail encoded by the adenovirus vector are exchanged with the transmembrane domain and cytoplasmic tail of influenza A virus hemagglutinin H3N2 (HA-TMCT).

[0480] 46. ​​Nucleic acid constructs according to items 27 to 45, wherein the trimerized sequence is provided near the signal peptide.

[0481] 47. Nucleic acid constructs according to items 27 to 46, wherein the trimerized sequence is GCN4.

[0482] 48. A protein comprising the expression product of a nucleic acid construct according to any one of items 27 to 47.

[0483] 49. A virus-like particle (VLP) comprising a nucleic acid construct according to any one of items 27 to 47.

[0484] 50. A vaccine according to any one of items 1 to 27, which is used for the prevention and / or treatment of cancer.

[0485] 51. A vaccine according to any one of items 1 to 27 for use in the prevention and / or treatment of cancer, comprising the steps of: priming a patient with a nucleic acid construct according to any one of items 27 to 47 at least 5 days prior to a booster dose of a vaccine according to any one of items 1 to 26.

[0486] 52. A vaccine according to any one of items 1 to 26 for use in the prevention and / or treatment of cancer, comprising the step of post-treating the patient with a virus-encoded VLP different from that of an adenovirus vector 5 days or longer after the patient has been exposed to a vaccine according to any one of items 1 to 26.

[0487] 53. According to Project 52, the VLP encoded by the virus, which is different from the VLP derived from the adenovirus vector, is a VLP derived from the modified vaccinia aniline (MVA).

[0488] 54. A vaccine for use in the prevention and / or treatment of a disease, comprising a viral vector capable of encoding a virus-like particle (VLP) exhibiting an inactive immunosuppressive domain (ISD).

[0489] 55. The vaccine according to Project 54, wherein the viral vector is derived from a modified vaccinia aniline (MVA), adeno-associated virus (AAV), or lentivirus.

[0490] 56. A method for the prevention and / or treatment of cancer, comprising administering a vaccine according to any one of items 1 to 26.

[0491] 57. A method for the prevention and / or treatment of cancer, comprising the steps of: priming a patient with a nucleic acid according to any one of items 27 to 47 at least 5 days prior to a booster dose of a vaccine according to any one of items 1 to 26.

[0492] 58. A method for the prevention and / or treatment of cancer, comprising the step of post-treating a patient with a virus-encoded VLP different from that of a VLP derived from an adenovirus vector, 5 days or longer after the patient has been exposed to a vaccine according to any one of items 1 to 20.

[0493] 59. According to the method of Project 58, the VLP encoded by a virus that is different from the VLP derived from the adenovirus vector is a VLP derived from the modified vaccinia aniline (MVA).

[0494] The following patent projects were also disclosed:

[0495] 60. A nucleic acid molecule for use in the prevention and / or treatment of a disease, said nucleic acid molecule encoding an endogenous retroviral (ERV) envelope protein or an immunogenic portion thereof, wherein the ISD of said protein contains a mutation that causes the ISD to be inactivated.

[0496] 61. A vector for use according to item 60, comprising a nucleic acid molecule according to item 60, said vector preferably being an adenovirus vector, more preferably being an adenovirus vector derived from a mammalian adenovirus type, a human adenovirus type, a chimpanzee adenovirus serotype, or a gorilla adenovirus serotype, wherein said 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 wherein said adenovirus vector is adenovirus serotype 5 (Ad5) or adenovirus serotype 19 (Ad19).

[0497] 62. A vector according to item 61 for use in accordance with item 61, wherein the vector encodes a virus-like particle (VLP) displaying an endogenous retroviral (ERV) envelope protein or an immunogenic portion thereof, having an inactive immunosuppressive domain (ISD).

[0498] 63. A protein for use in the prevention and / or treatment of a disease, comprising the expression product of the vector of item 62.

[0499] 64. A vaccine for use in the prevention and / or treatment of a disease, comprising a nucleic acid molecule or vector according to any one of items 60-62 above or a protein according to item 63.

[0500] 65. A nucleic acid molecule, vector, protein or vaccine according to any one of items 60-64 above, used in accordance with the preceding items, wherein the disease is cancer, more preferably cancer expressing ERV, and even more preferably cancer selected from prostate cancer, breast cancer, ovarian cancer, lymphoma, melanoma, leukemia, sarcoma, colorectal cancer, testicular cancer, lung cancer and liver cancer.

[0501] 66. A nucleic acid molecule, vector, or vaccine according to any one of the preceding items 60-65 for use in accordance with the preceding items 60-65, wherein the ISD has a 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), YQNRLALDYLLAAEGGV (SEQ ID No. 6) or NSQSSIDQKLANQINDLRQT (SEQ ID No. 49), having at least one amino acid exchanged with a different amino acid.

[0502] 67. A nucleic acid molecule, vector, or vaccine according to any one of items 60-66 of the preceding items for use in accordance with the preceding items, wherein at least one amino acid in a region of 10 amino acids upstream or downstream of the ISD is exchanged with a different amino acid.

[0503] 68. A nucleic acid molecule, vector, or vaccine according to any one of items 60-67 of the preceding items for use in accordance with the foregoing items, wherein the ERV protein is a human endogenous retrovirus (HERV) protein or its immunogenic portion, wherein the HERV is selected from HERV-K, HERV-H, HERV-W, HERV-FRD, and HERV-E, and wherein the HERV-K is selected from HERV-K108 (=ERVK-6), ERVK-19, HERV-K115 (=ERVK-8), and ERVK-9. HERV-K113, ERVK-21, ERVK-25, HERV-K102 (=ERVK-7), HERV-K101 (=ERVK-24), and HERV-K110 (=ERVK-18); HERV-H is selected from HERV-H19 (=HERV-H_2q24.3) and HERV-H_2q24.1; HERV-W is selected as ERVW-1 (=syncytokine-1); and HERV-FRD is selected as ERVFRD-1 (=syncytokine-2).

[0504] 69. A vector of any one of items 62 to 67 or a vaccine of any one of items 60 to 68 for use in accordance with items 60-68, wherein the protein product of the adenovirus vector comprises a gag protein, a 2A peptide, and an envelope protein (Env), the Env protein comprising a surface unit (gp70), a cleavage site, and a transmembrane unit (p15E), wherein the transmembrane unit (p15E) comprises a fusion peptide, an immunosuppressive domain (ISD), a transmembrane anchor, and a cytoplasmic tail, and wherein p15E or its immunogenic portion is coupled to an adenovirus capsid protein pIX, and / or wherein a signal peptide encoded by the adenovirus vector is exchanged with a signal peptide from Gaussian luciferase (LucSP), and / or wherein the transmembrane anchor and cytoplasmic tail encoded by the adenovirus vector are exchanged with a transmembrane domain and a cytoplasmic tail from influenza A virus hemagglutinin H3N2 (HA-TMCT).

[0505] 70. A vaccine according to any one of items 60-69 for use in the prevention and / or treatment of cancer, comprising the step of inducing a patient to develop the adenovirus vector at least 5 days prior to a booster dose of the vaccine according to any one of items 60-69.

[0506] 71. A vaccine according to any one of the preceding items 60-70 for use in the prevention and / or treatment of cancer, comprising the step of post-treating a patient with a virus-encoded VLP different from a VLP derived from an adenovirus vector, five days or more after the patient has been exposed to the vaccine, wherein the virus-encoded VLP different from the VLP derived from the adenovirus vector is a VLP derived from a modified vaccinia ani (MVA).

[0507] 72. A nucleic acid molecule encoding a Gag protein and an ERV envelope protein (Env) or an immunogenic portion thereof, wherein the native genomic structure linking Gag and Env has been replaced with an operable adapter, wherein the adapter is preferably p2A.

[0508] 73. A VLP encoded by the nucleic acid molecule of Item 72.

[0509] 74. Nucleic acid molecules according to Item 72 or VLPs according to Item 73, which are used in the prevention and / or treatment of disease.

[0510] 75. A nucleic acid molecule according to item 60, a VLP according to item 73, or a nucleic acid molecule or VLP according to item 74 for use according to item 74, wherein the ERV is HERV-K, preferably HERV-K 113.

Claims

1. A nucleic acid molecule encoding human endogenous retrovirus K (HERV-K) envelope protein and group specific antigen (Gag) protein, wherein the encoded human endogenous retrovirus K (HERV-K) envelope protein and group specific antigen (Gag) protein consists of a sequence according to SEQ ID NO:

48.

2. A vector comprising the nucleic acid molecule according to claim 1.

3. The vector according to claim 2, wherein the vector is an adenoviral vector.

4. A protein encoded by the nucleic acid molecule of claim 1 or the vector according to claim 2 or 3.

5. A vaccine comprising the nucleic acid molecule of claim 1, the vector according to claim 2 or 3 or the protein according to claim 4.

6. Use of the nucleic acid molecule of claim 1, the vector of claim 2 or 3, the protein of claim 4 or the vaccine of claim 5 for the manufacture of a medicament for the treatment or prevention of a disease, wherein the disease is an ERV-expressing cancer selected from the group consisting of breast cancer, kidney cancer and lung cancer.

7. The use according to claim 6, wherein the medicament is a virus-like particle (VLP).

8. The vector of claim 3 or the vaccine according to claim 5 comprising the vector according to claim 3, wherein the protein product of the adenoviral vector comprises Gag protein, 2A peptide and envelope protein (Env) of HERV-K.

9. A VLP encoded by the nucleic acid molecule according to claim 1.

10. Use of the nucleic acid molecule according to claim 1 for the manufacture of a virus-like particle (VLP).

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