Antigen-specific physiological dendritic cells for vaccination

Abstract

The present invention relates to a composition comprising physiological dendritic cells and at least one mRNA. The invention further relates to a composition comprising physiological dendritic cells and at least one mRNA for vaccination.

Description

Technical Field

[0001] This invention relates to the field of preventive vaccination against diseases based on physiological dendritic cells. The invention further relates to vaccine compositions comprising physiological dendritic cells and at least one mRNA, and vaccine compositions for use in vaccination against infectious diseases and hyperproliferative diseases such as cancer or tumors. Background Technology

[0002] Dendritic cells (DCs) possess the ability to take up antigens, process them, and present them to T and B cells on their cell surface. Because DCs thereby activate naïve immune cells, effector immune cells, and memory immune cells, they are promising therapeutic agents for diseases such as cancer or infectious diseases. The potent activation of de novo T and B cell responses also suggests that dendritic cells are promising agents for prophylactic purposes.

[0003] However, dendritic cells in the human body account for only about 0.3% of the total circulating white blood cells and are composed of a heterogeneous population with different levels of maturity and the ability to induce or stimulate a tolerable immune response.

[0004] Techniques for the isolation and maturation of dendritic cells are described in numerous documents, and various methods exist, including contacting monocytes with hematopoietic growth factors and cytokines such as IFN-α, GM-CSF, TNF-α, IL-3, or combinations thereof (see, for example, EP 922,758 or EP 663,930, WO 95 / 28479).

[0005] Extracorporeal photopheresis (ECP) has been successfully used to treat cutaneous T-cell lymphoma (CTCL) in a subset of patients. In classic ECP, peripheral blood mononuclear cells (PBMCs) are isolated from the patient and passed through a clear plastic plate while exposed to 8-methoxypsoralen and UVA (8-MOP UVA), before being returned to the patient. The underlying mechanism is presumed to involve dendritic cells taking up antigens released by malignant T cells exposed to 8-MOP UVA and presenting these antigens to the patient's immune system. Based on their mode of action, these DCs are termed immunostimulated DCs. In investigating the principles of ECP, it has been learned that ECP leads to the transformation of passed-through blood mononuclear cells into DCs.

[0006] However, it is unclear which antigens are precisely taken up and presented during this process. Therefore, there is a persistent need for the selective delivery of immunostimulatory dendritic cells (DCs) equipped with specific antigens to elicit a desired and targeted immunostimulatory response. Summary of the Invention

[0007] One object of the present invention is to provide a vaccine composition comprising phDC and at least one mRNA.

[0008] Another object of the present invention is to provide a vaccine composition comprising antigen-specific phDCs and at least one mRNA. Another object of the present invention is to provide a vaccine composition comprising antigen-specific monocytes and at least one mRNA. Another object of the present invention is to use the vaccine composition of the present invention for vaccination.

[0009] Another object of the present invention is to provide a vaccine composition and a method for preventing or reducing disease in a subject.

[0010] Finally, one object of the present invention is to provide a kit comprising phDC and at least one mRNA, wherein the at least one mRNA encodes at least one antigenic protein.

[0011] These and other objectives are addressed by the subject matter of the independent claims, as will become apparent from the following description. Some preferred embodiments of the invention form the subject matter of the dependent claims. Other embodiments of the invention will be obtained from the following description.

[0012] The invention described illustratively below may be practiced in the absence of any one or more elements or limitations not specifically disclosed herein.

[0013] The invention is described below with reference to specific embodiments and certain accompanying drawings, but is not limited thereto by the claims.

[0014] This invention is based in part on the data and experiments presented below, which lead to the understanding that dendritic cells loaded with selected disease antigens can be effectively used for vaccination against diseases such as cancer or infectious diseases. As a key prerequisite for obtaining antigen-specific DCs, the dendritic cells need to be (i) obtained and (ii) equipped with disease-specific antigens. Regarding (i), so-called physiological dendritic cells (phDCs) can be used. phDCs can be produced in vitro, in particular, by applying shear force to monocytes (e.g., from blood samples), see, for example, Ventura et al., 2018 “Extracorporeal Photochemotherapy Drives Monocyte-to-Dendritic Cell Maturation to Induce Anticancer Immunity”, WO 2014 / 106629, WO 2016 / 001405, or Hanlon et al., 2020 “Rapid Production of Physiologic Dendritic Cells (phDC) for Immunotherapy”. Physiological dendritic cells (phDCs) can be characterized by marker expression (including markers such as HLA-DR, CD83, CD86, ICAM, or PLAUR) and / or increased expression without GILZ (see, for example, Ventura et al., 2018, “Extracorporeal Photochemotherapy Drives Monocyte-to-Dendritic Cell Maturation to Induce Anticancer Immunity” or WO 2014 / 106629). Since monocytes do not require cytokine exposure to induce differentiation into dendritic cells, it is assumed that dendritic cells obtained by subjecting monocytes to physical forces are closer to the characteristics of naturally occurring dendritic cells. This is why dendritic cells obtained by subjecting monocytes to physical forces will be designated as physiological dendritic cells (phDCs) below.

[0015] As previously described (Ventura et al., 2018 “Extracorporeal Photochemotherapy Drives Monocyte-to-Dendritic Cell Maturation to Induce Anticancer Immunity” or WO 2014 / 106629), physical forces inducing monocyte differentiation into dendritic cells (phDCs) can be applied by passing monocytes through a flow chamber, which can take the form of, for example, a plate or, for example, a bag (e.g., a flexible bag or a plastic bag). Such bags are described, for example, in Buechler et al., 2004 “Generation of Dendritic Cells Using Cell Culture Bags - Description of a Method and Review of Literature”. It is assumed that passing monocytes through, for example, a plate or placing them in, for example, a flexible bag or a combination thereof (e.g., a mixture of bags and chambers as disclosed herein) exposes them to shear stress, which induces differentiation into dendritic cells.

[0016] The presence of platelets can improve the maturation process. As described above, this method allows monocytes to mature into dendritic cells without the need for the addition of expensive cytokine mixtures. Also, as mentioned, the process of passing monocytes through, for example, a plate or placing them in, for example, a bag or a mixture of a chamber and a bag, mimics some aspects assumed to occur in vivo; this is why dendritic cells generated by passing through a plate (or placing them in a bag and moving them) are referred to throughout this disclosure as “physiological dendritic cells” (phDCs). Although phDCs have been found to be particularly effective for loading with disease-specific antigens (e.g., with at least one mRNA encoding at least one antigenic protein), in principle any population of DCs can be used to generate the antigen-specific DCs of the present invention, such as DCs obtained after incubating blood-derived monocytes with cytokines. As previously shown (Ventura et al., 2018 “Extracorporeal Photochemotherapy Drives Monocyte-to-Dendritic Cell Maturation to Induce Antiticancer Immunity” or WO 2016 / 001405), phDCs generated under conditions where apoptotic signals are absent and subsequently loaded with antigens will effectively stimulate an immune response. It should be understood that transfection (using, for example, at least one mRNA encoding at least one antigenic protein) can also occur at the monocyte stage when monocytes are subjected to physical forces. Therefore, monocytes can be combined with disease-specific antigens (in the form of, for example, at least one mRNA encoding at least one antigenic protein) and subjected to physical forces such as shearing forces. As described in WO 2016 / 001405, monocytes activated by physical forces (so-called globally activated monocytes) can be identified, in particular, by increased expression of at least HLA-DR, PLAUR, or ICAM-1.

[0017] Regarding (ii), the inventors have discovered that phDCs can internalize mRNA-containing LNPs and express encoded antigens, such as disease-related proteins or peptides. Antigen-specific phDCs loaded with these molecules (e.g., vaccine compositions containing phDCs and at least one mRNA) have been found to be useful for targeted immunotherapies against various diseases, such as cancer or infectious diseases. It appears that the antigen-specific phDCs of the present invention can elicit a disease-preventive immune response through their unique ability to present specific antigens on MHC molecules, leading to T cell activation, proliferation, and differentiation into armed effector T cells. An important function of effector T cells is to drive humoral immune responses through the killing of infected cells by CD8+ cytotoxic T cells and the activation of macrophages by Th1 cells (which together constitute cell-mediated immunity), and the activation of B cells by both Th2 and Th1 cells to produce different classes of antibodies. Importantly, T cells (such as cytotoxic T cells) can spread to the brain, resulting in superior vaccination efficacy compared to classical vaccines or, particularly, mRNA vaccines, which rely primarily on evoking a B cell response, including antibody production, which cannot cross the blood-brain barrier. Direct use of antigen-specific phDCs (e.g., vaccine compositions comprising phDCs and at least one mRNA) can be considered an additionally safer vaccination method because, compared to mRNA vaccination (which randomly disperses antigenic peptides from muscle tissue into systemic circulation, providing a greater probability of loss of target cells and triggering unwanted side effects), the immune system is activated by a very specific set of cells. Furthermore, the use of adjuvants may be lower for the vaccines of this invention compared to conventional vaccines (including mRNA vaccines). Since the antigen-specific phDCs of this invention (e.g., vaccine compositions comprising phDCs and at least one mRNA) are highly effective, this invention also provides the possibility of significant dose savings.

[0018] Furthermore, direct use of antigen-specific phDCs (e.g., vaccine compositions containing phDCs and at least one mRNA) allows for the induction of an immune response with a lower LNP dose. Another advantage of antigen-specific phDCs (e.g., vaccine compositions containing phDCs and at least one mRNA) is the faster initiation of immune protection after administration. For vaccines such as mRNA vaccines, the clonal expansion of memory B cells requires approximately 10 to 14 days. However, antigen-specific phDCs can directly trigger the expansion of pre-existing T cell clones in the subject into effector cytotoxic T cells, independent of the 10–14 day maturation lag. Unlike naive T cells, memory T cells can trigger cytotoxic function upon encountering an antigen. Therefore, vaccination with antigen-specific phDCs (e.g., vaccine compositions containing phDCs and at least one mRNA) corresponds to a vaccination method with immediate potency, supported by the subsequent humoral immune response it induces as additional potency.

[0019] First aspect: Vaccine composition

[0020] In a first aspect, the present invention relates to a vaccine composition comprising a pharmaceutically effective amount of phDC and at least one RNA, the RNA comprising a coding sequence encoding at least one antigenic protein.

[0021] In one embodiment, the RNA is single-stranded RNA, mRNA, circular RNA, self-amplifying RNA, and / or synthetic RNA. In a preferred embodiment, the RNA is self-amplifying mRNA. In a preferred embodiment, the RNA is mRNA. Therefore, the present invention relates to a vaccine composition comprising a pharmaceutically effective amount of phDC and at least one mRNA (e.g., one or more mRNA molecules), the mRNA containing a coding sequence encoding at least one antigenic protein.

[0022] In one embodiment, the RNA (e.g., mRNA) contains at least one chemical modification. If more than one RNA (e.g., mRNA) is used, each RNA (e.g., gmRNA) independently contains at least one chemical modification (i.e., the chemical modification from one RNA to another can be different). For the purposes of this disclosure, chemical modification means that one of the four naturally occurring standard nucleosides present in the RNA (adenosine (A), guanosine (G), uridine (U), and cytidine (C)) is replaced by its modified form, wherein the modification affects the base moiety within the nucleoside. The modified nucleoside can be naturally occurring or non-naturally occurring modified nucleosides. Naturally occurring modified nucleosides are preferred.

[0023] It is assumed that replacing A, C, U, and G with naturally occurring or non-naturally occurring modified nucleosides reduces Toll-like receptor (TLR)-mediated immune responses in recipient dendritic cells and / or increases the expression of antigens encoded by RNA (e.g., mRNA).

[0024] In one embodiment, chemical modification is the substitution of one or more nucleosides of RNA (e.g., mRNA) with one or more modified nucleosides.

[0025] Naturally occurring modified nucleosides include 1-methyladenosine (m 1 A), N 6 -Methyladenosine (m 6 A), 2'-O-methyladenosine (Am), 5-methylcytidine (m) 5 C), 2'-O-methylcytidine (Cm), 2-thiocytidine (s) 2 C), N 4 - Acetylcytidine (ac) 4 C), 5-formylcytidine (f) 5 C), 2'-O-methylguanosine (Gm), inosine (I), pseudouridine (Ψ), 5-methyluridine (m) 5 U), 2'-O-methyluridine (Um), 1-methylpseudouridine (M1Ψ), 2-thiouridine (s2U), 4-thiouridine (s4U), 5-methoxyuridine (mo5U) and 3-methyluridine (m3U).

[0026] Of the four nucleosides A, U, C, and G, it is preferred to replace at least one or more uridine nucleosides of RNA (e.g., mRNA) with naturally occurring modified uridine nucleosides. It is assumed that the above-described effects on TLR-mediated immune responses and / or the expression levels of antigens encoded by RNA (e.g., mRNA) depend on the degree to which uridine is replaced by naturally occurring modified uridine nucleosides. In a preferred embodiment, all uridine nucleosides within the RNA (e.g., mRNA) encoding the antigen are thus replaced with naturally occurring modified uridine nucleosides. However, the invention also considers the replacement of less than 90%, less than 80%, less than 70%, less than 60%, or less than 50% of the uridine nucleosides present in the RNA (e.g., mRNA) encoding antigenic proteins with naturally occurring modified uridine nucleosides. When uridine nucleosides are replaced by their naturally occurring modified forms, pseudouridine is preferred, and N1-methylpseudouridine or N1-ethylpseudouridine is more preferred. N1-methylpseudouridine is the most preferred. Therefore, the present invention considers a particularly preferred embodiment to be an RNA (e.g., mRNA) encoding an antigen, wherein all uridines are replaced N1-methylpseuuridines.

[0027] The present invention also contemplates replacing or substituting A, C, and / or G nucleosides with modified forms of A, C, and / or G nucleosides (and preferably this is in addition to uridine nucleosides). Naturally occurring modified forms of A, C, and / or G are preferably used for such substitutions. If such additional substitutions are considered, it is preferable that all A, C, and / or G nucleosides are replaced with their modified forms.

[0028] In one embodiment, the RNA (e.g., mRNA) comprises structural elements including a 5' untranslated region (UTR), a 3' UTR, a 5' cap, and / or a multiple (A) tail. In one embodiment, the RNA (e.g., mRNA) comprises all of these elements. In one embodiment, the 5' cap is a Cap1 structure or an m7GpppG cap. Preferably, the 5' cap is a Cap1 structure. In one embodiment, the sequence of the RNA (e.g., mRNA) is optimized. In one embodiment, the sequences of the 5' UTR, 3' UTR, and / or the coding sequence of the antigenic protein are optimized. In one embodiment, the sequence of the RNA (e.g., mRNA) has optimized codon usage or optimized G / C content. In one embodiment, codon usage, G / C content, and structural elements are optimized. Optimization of the 3' UTR and / or 5' UTR sequences may additionally include the use of one or more of heterologous UTRs, Kozak sequences, FI elements, removed AURES elements, and enzymatically added tails (e.g., multiple (A) tails). In one embodiment, an enzymatically added multiple (A) tail is used.

[0029] At least one RNA (e.g., mRNA) may be contained in the nanoparticles. The nanoparticles include lipid nanoparticles, poly(amine-co-ester) particles (PACE), poly-β-amino-ester particles, PACE multi-complex particles, lipid complexes, and poly(N,N-cystamine bis(acrylamide)-co-4-amino-1-butanol) (pABOL) particles. In one embodiment, the nanoparticles are lipid nanoparticles (LNP).

[0030] In a preferred embodiment, RNA (e.g., mRNA) is contained in lipid nanoparticles (LNPs). The lipid nanoparticles may contain cationic lipids, PEG-modified lipids, cholesterol, DSPE-PEG-maleimide, DSPN-PEG-azide, and / or non-cationic lipids. In one embodiment, the lipid nanoparticles contain cationic lipids, PEG-modified lipids, cholesterol, and / or non-cationic lipids. In another embodiment, the lipid nanoparticles contain cationic lipids, PEG-modified lipids, cholesterol, and non-cationic lipids. Cationic lipids include cKK-E12, cKK-E14, LP01, SM102, lipid 5, etc. In one embodiment, the cationic lipid is cKK-E12 lipid. In another embodiment, the cationic lipid is SM102 lipid. In yet another embodiment, the cationic lipid is MC3 lipid (DLin-MC3-DMA). In one embodiment, the lipid nanoparticles contain cKK-E12, cholesterol, C14-PEG 2000-PE, and DOPE. In one embodiment, the lipid nanoparticles comprise cKK-E12, cholesterol, C14-PEG2000-PE, and DOPE in a ratio of 30-40:41-51:1.0-4.0:12-21. In one embodiment, the lipid nanoparticles comprise cKK-E12, cholesterol, C14-PEG 2000-PE, and DOPE in a ratio of 33-37:44-48:2.0-3.0:14-18. In one embodiment, the lipid nanoparticles comprise cKK-E12, cholesterol, C14-PEG 2000-PE, and DOPE in a ratio of 34-36:45-47:2.2-2.8:15-17. In one embodiment, the lipid nanoparticles comprise cKK-E12, cholesterol, C14-PEG 2000-PE, and DOPE in a ratio of 35:46.5:2.5:16.

[0031] In one embodiment, the lipid nanoparticles comprise SM-102, cholesterol, C14-PEG 2000-PE, and DOPE. In another embodiment, the lipid nanoparticles comprise SM-102, cholesterol, C14-PEG 2000-PE, and DOPE in a ratio of 30-40:41-51:1.0-4.0:12-21. In yet another embodiment, the lipid nanoparticles comprise SM-102, cholesterol, C14-PEG 2000-PE, and DOPE in a ratio of 33-37:44-48:2.0-3.0:14-18. In yet another embodiment, the lipid nanoparticles comprise SM-102, cholesterol, C14-PEG 2000-PE, and DOPE in a ratio of 34-36:45-47:2.2-2.8:15-17. In one embodiment, the lipid nanoparticles comprise SM-102, cholesterol, C14-PEG 2000-PE, and DOPE in a ratio of 35:46.5:2.5:16. In some embodiments, the lipid nanoparticles comprise SM102, cholesterol, DMG-PEG-2K, and DSPC. In one embodiment, the lipid nanoparticles comprise SM102, cholesterol, DMG-PEG-2K, and DSPC in a ratio of 30-50:35-45:1.0-4.0:10-20.

[0032] In one embodiment, the lipid nanoparticles comprise MC3, cholesterol, C14-PEG 2000-PE, and DOPE. In one embodiment, the lipid nanoparticles comprise MC3, cholesterol, C14-PEG 2000-PE, and DOPE in a ratio of 30-40:41-51:1.0-4.0:12-21. In one embodiment, the lipid nanoparticles comprise MC3, cholesterol, C14-PEG 2000-PE, and DOPE in a ratio of 33-37:44-48:2.0-3.0:14-18. In one embodiment, the lipid nanoparticles comprise MC3, cholesterol, C14-PEG 2000-PE, and DOPE in a ratio of 34-36:45-47:2.2-2.8:15-17. In one embodiment, the lipid nanoparticles comprise MC3, cholesterol, C14-PEG 2000-PE, and DOPE in a ratio of 35:46.5:2.5:16.

[0033] In one embodiment, at least one mRNA encoding at least one antigenic protein is heterologous or exogenous to phDC. In one embodiment, the antigenic protein is exogenous to phDC. The terms "heterologous" or "exogenous" as used herein with respect to a particular cell type (e.g., phDC) can refer to a nucleic acid sequence (e.g., mRNA) or protein derived from a source other than that particular cell type (e.g., the phDC). Thus, heterologous or exogenous mRNA to phDC refers to mRNA that is not endogenous to phDC but has been introduced into phDC, for example, through transfection or other means.

[0034] In one embodiment, the antigenic protein is an infectious disease-associated antigen or a tumor-associated antigen. In another embodiment, the antigenic protein is an infectious disease-associated antigen. Therefore, the vaccine composition comprises phDC and at least one mRNA containing a coding sequence encoding at least one infectious disease-associated antigenic protein. In one embodiment, the infectious disease-associated antigen is a viral antigen, bacterial antigen, fungal antigen, or parasitic antigen. In one embodiment, the infectious disease-associated antigen is a bacterial antigen. In one embodiment, the bacterial antigen is derived from a species of *Borrelia* or *Mycobacteria*. In another embodiment, the infectious disease-associated antigen is a fungal antigen. In one embodiment, the fungal antigen is derived from *Cryptococcus neoformans*, *Histoplasma capsulatum*, *Coccidioides immitis*, *Blastomyces dermatitidis*, *Chlamydia trachomatis*, or *Candida albicans*. In another embodiment, the infectious disease-associated antigen is a parasitic antigen. In one embodiment, the parasitic antigen is derived from Plasmodium malariae.

[0035] In one embodiment, the infectious disease-associated antigen is a viral antigen. Therefore, the vaccine composition comprises phDC and at least one mRNA containing a coding sequence encoding at least one viral antigenic protein. In one embodiment, the viral antigen is a coronavirus antigenic protein or an HIV antigenic protein. In one embodiment, the viral antigen is a coronavirus antigenic protein. In one embodiment, the viral antigen is a β-coronavirus antigenic protein. In another embodiment, the viral antigen is a SARS-CoV-2 antigenic protein. In one embodiment, the coronavirus antigenic protein or β-coronavirus antigenic protein is an antigenic protein of the spike protein, envelope protein, nucleocapsid protein, membrane protein, and / or Orf1ab polyprotein, or a fragment of each of the foregoing. In one embodiment, the SARS-CoV-2 antigen is an antigenic protein of the spike protein, envelope protein, nucleocapsid protein, membrane protein, and / or Orf1ab polyprotein, or a fragment of each of the foregoing. In one embodiment, the SARS-CoV-2 antigen is an antigenic protein of the spike protein or a fragment thereof. The fragment contains at least 10, at least 50, at least 100, at least 200, at least 400, or at least 800 amino acid residues.

[0036] In one embodiment, the RNA (e.g., mRNA) comprises at least 75%, 85%, 90%, 95%, 98%, 99%, or 100% identical to a sequence selected from the group consisting of: SEQ ID NO: 1 (spike protein transcript), SEQ ID NO: 2 (enveloping protein transcript), SEQ ID NO: 3 (nucleocapsid protein transcript), SEQ ID NO: 4 (membrane protein transcript), and / or SEQ ID NO: 5 (Orf1ab polyprotein transcript). In one embodiment, the RNA (e.g., mRNA) comprises at least 75%, 85%, 90%, 95%, 98%, 99%, or 100% identical to a sequence or a portion thereof of SEQ ID NO: 1. In one embodiment, the antigenic protein encoded by RNA (e.g., mRNA) comprises at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1200, at least 2000, or at least 3000 amino acids. In one embodiment, the antigenic protein encoded by RNA (e.g., mRNA) comprises at least 50 amino acids. In one embodiment, the antigenic protein encoded by RNA (e.g., mRNA) comprises at least 500 amino acids. In one embodiment, the antigenic protein encoded by RNA (e.g., mRNA) comprises at least 1000 amino acids.

[0037] In one embodiment, the SARS-CoV-2 antigen is a protein or peptide comprising at least 75%, 85%, 90%, 95%, 98%, 99%, or 100% identical sequences to or a portion thereof selected from the group consisting of: SEQ ID NO: 6 (spike amino acid sequence), SEQ ID NO: 7 (enveloping amino acid sequence), SEQ ID NO: 8 (nucleocapsid amino acid sequence), SEQ ID NO: 9 (membrane amino acid sequence), and / or SEQ ID NO: 10 (Orf1ab amino acid sequence). In one embodiment, the protein or peptide comprises at least 75%, 85%, 90%, 95%, 98%, 99%, or 100% identical sequences to or a portion thereof of SEQ ID NO: 6.

[0038] In one embodiment, at least one antigenic protein is the SARS-CoV-2 hexapro spike protein (which has a foldon domain in its extracellular domain directly above the transmembrane domain (its RNA sequence is depicted in bold and underlined in Table 3)) or RNA (e.g., mRNA) encoding it. The corresponding RNA (e.g., mRNA) may be modified with one or more of N1-methylpseuuridine, a cap1 structure, and a multi-A tail. In one embodiment, the RNA (e.g., mRNA) comprises N1-methylpseuuridine (e.g., each uridine is replaced by N1-methylpseuuridine), a cap1 structure, and a multi-A tail. In one embodiment, codons may be optimized for GC enrichment of the RNA (e.g., mRNA), and optimized 5' UTRs and 3' UTRs may be used. In one embodiment, the RNA (e.g., mRNA) comprises a sequence corresponding to or a portion of SEQ ID NO: 19, or a sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity with SEQ ID NO: 19. In one embodiment, the RNA (e.g., mRNA) comprises a sequence or a portion thereof corresponding to SEQ ID NO: 19 or a sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity with SEQ ID NO: 19; and a multi-A tail (e.g., about 200 to 400 nucleotides).

[0039] In one embodiment, the viral antigen is derived from HIV. In one embodiment, the HIV antigen is derived from the envelope protein (env), the swarm antigen polyprotein (gag), the reverse transcriptase (pol), and / or the negative factor protein (nef). In one embodiment, the RNA (e.g., mRNA) encoding the antigenic protein contains at least 75%, 85%, 90%, 95%, 98%, 99%, or 100% identical sequences to or a portion thereof selected from the group consisting of: SEQ ID NO: 11 (env protein transcript), SEQ ID NO: 12 (gag protein transcript), SEQ ID NO: 13 (pol protein transcript), and / or SEQ ID NO: 14 (nef protein transcript).

[0040] In one embodiment, the HIV antigen is a protein or peptide that contains at least 75%, 85%, 90%, 95%, 98%, 99%, or 100% identical sequences to or a portion thereof selected from the group consisting of: SEQ ID NO: 15 (env amino acid sequence), SEQ ID NO: 16 (gag amino acid sequence), SEQ ID NO: 17 (pol amino acid sequence), and / or SEQ ID NO: 18 (nef amino acid sequence).

[0041] The vaccine composition of the present invention may comprise a pharmaceutically effective amount of physiological dendritic cells (phDCs) and at least one mRNA containing a coding sequence encoding at least one antigenic protein, wherein the mRNA may be at least partially contained in the phDC. In some embodiments, substantially all of the mRNA is contained in the phDC. Unincorporated mRNA may be removed by washing the phDC or by other means known in the art. Thus, the resulting vaccine composition may comprise phDCs in which the phDCs have taken up mRNA encoding at least one antigenic protein. The mRNA may be translated into the antigenic protein. In some embodiments, the phDCs express the antigenic protein. The antigenic protein may be exogenous or heterologous to the phDCs. In some embodiments, free, i.e., unincorporated mRNA is not removed from the vaccine composition, and therefore the composition comprises unincorporated mRNA. In another embodiment, the vaccine composition of the present invention may comprise a pharmaceutically effective amount of physiological dendritic cells (phDCs) and at least one mRNA containing a coding sequence encoding at least one antigenic protein, wherein the mRNA is at least partially unincorporated in the phDC. In some embodiments, only a subset of the mRNA contained in the vaccine composition is taken up by phDCs present in the composition. In some embodiments, more than 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the mRNA is not incorporated into the phDC. In some embodiments, more than 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the mRNA contained in the vaccine composition of the present invention is incorporated into the phDC.

[0042] In one embodiment, at least one antigenic protein is a tumor-associated antigen. Therefore, the vaccine composition comprises phDC and at least one mRNA containing a coding sequence encoding at least one tumor-associated antigenic protein. In one embodiment, the tumor-associated antigen is a leukemia antigen, melanoma antigen, lymphoma antigen, endometrial cancer antigen, kidney cancer antigen, brain cancer antigen, cervical cancer antigen, liver cancer antigen, head and neck cancer antigen, gastrointestinal cancer antigen, lymph node cancer antigen, pancreatic cancer antigen, ear, nose, and throat (ENT) cancer antigen, breast cancer antigen, prostate cancer antigen, ovarian cancer antigen, or lung cancer antigen. In one embodiment, at least one antigenic protein is a blood cancer-associated antigen. The blood cancer antigenic protein may be a leukemia antigenic protein, a lymphoma antigenic protein, or a myeloma antigenic protein.

[0043] In one embodiment, phDCs can be obtained by subjecting monocytes (e.g., obtained from a donor) to physical forces. These physical forces can be applied to the monocytes by passing them through a flow chamber. The flow chamber can be a plate, bag, or flow chamber of a device, such as a large ECP device, for example a clinical ECP device (e.g., the THERAKOS® CELLEX® device; a combination of Terumo's Spectra Optia and UVA PIT; an apheresis device from Fresenius and a combination of Macopharma's Macogenix; a single-needle option for the Amicus® in vitro photodissociation protocol), or a miniaturized ECP device, such as a transimmunization plate as described in WO2017 / 005700 A1, or a combination thereof (e.g., a hybrid of bags and chambers as disclosed herein). In one embodiment of the invention, phDCs are available by passing a plate of monocytes obtained using a method derived from in vitro photodissociation (ECP). Therefore, in one embodiment, the flow chamber is a plate. Monocytes (e.g., obtained from a donor) are passed through a plate, exposing them to shear forces. Preferably, platelets are present in the plate, which may be derived from a donor blood sample or a fraction thereof or provided separately. Additionally or alternatively, plasma components may be present in the plate, which may be derived from a donor blood sample or a fraction thereof or provided separately. Methods for preparing phDCs and the terminology itself have previously been described, for example, in Hanlon et al., 2020 (“Rapid Production of Physiologic Dendritic Cells (phDC) for Immunotherapy”). In one embodiment, all method steps are performed in vitro.

[0044] In another embodiment, the flow chamber is a bag. Optionally, the bag is a flexible bag or a plastic bag. In one embodiment, the flow chamber is a flexible bag. In one embodiment, the bag is a plastic bag. In one embodiment, the material of the flow chamber (e.g., a plate, a flexible bag, or a plastic bag) is plastic. In one embodiment, the material of the flow chamber is non-plastic, such as glass, ceramic, or silicone. If plastic materials are considered, a technician may use acrylics, polycarbonates, polyetherimides, polysulfones, polyphenylene sulfones, styrene-based materials, polyurethanes, polyethylene, Teflon, or any other suitable medical-grade plastics. In a preferred embodiment of the invention, the flow chamber is made of acrylic plastic. If the bag (e.g., a flexible bag) is considered, the material may be plastic, rubber, or silicone. In a preferred embodiment, the material is plastic. Plastic materials include polyolefins, polyethylene, fluoropolymers, polyvinyl chloride, ethylene-vinyl acetate copolymers, ethylene vinyl alcohol, polyvinylidene fluoride, and / or other plastics containing materials approved for medical use.

[0045] In some embodiments, the flow chamber comprises or is composed of a plate. The plate can be made of a variety of materials, including but not limited to plastic materials. In one embodiment, the plate is made of plastic. In another embodiment, the plate is made of a non-plastic material, such as glass, ceramic, or silicone. Non-limiting examples of materials used for the plate include acrylics, polycarbonates, polyetherimides, polysulfones, polyphenylene sulfones, styrene-based materials, polyurethanes, polyethylene, Teflon, or any other suitable medical-grade plastics. The plate can be rigid or flexible. In some embodiments, the plate material can comprise or consist of plastics, rubber, or silicone. In some embodiments, the plate is elastic, i.e., made of an elastic material. Elastic materials can include cyclic olefin copolymers (COCs), polyolefins, polyethylene, fluoropolymers, polyvinyl chloride, ethylene-vinyl acetate copolymers, ethylene vinyl alcohol, polyvinylidene fluoride, polydimethylsiloxane (PDMS), dimethicone, and / or other plastics containing materials approved for medical use. In one embodiment, the plate is made of PDMS (e.g., PDMS RTV-615 or PDMS Sylgard 184). In another preferred embodiment of the invention, the plate is made of acrylic plastic.

[0046] In some implementations, the flow chamber is a hybrid flow chamber. A hybrid flow chamber may include a chamber and a bag, a chamber and a plate, or a bag and a plate. The individual components of a hybrid flow chamber, namely the bag, plate, or chamber, are as defined herein.

[0047] In one embodiment, human AB serum, autologous (e.g., human) serum, autologous (e.g., human) plasma, allogeneic (e.g., human) serum, allogeneic (e.g., human) plasma, mouse serum, mouse plasma, or FBS is additionally added to the composition. In another embodiment, autologous (e.g., human) serum, autologous (e.g., human) plasma, allogeneic (e.g., human) serum, allogeneic (e.g., human) plasma, mouse serum, mouse plasma, or FBS is additionally added to the composition. In yet another embodiment, mouse serum, mouse plasma, or FBS is additionally added to the composition. Alternatively, before adding monocytes, the flow chamber, bag, plate, or mixing flow chamber may be coated with human AB serum, autologous (e.g., human) serum, autologous (e.g., human) plasma, allogeneic (e.g., human) serum, allogeneic (e.g., human) plasma, mouse serum, mouse plasma, or FBS. In one embodiment, the flow chamber, bag, plate, or mixed flow chamber is coated with autologous (e.g., human) serum, autologous (e.g., human) plasma, allogeneic (e.g., human) serum, allogeneic (e.g., human) plasma, mouse serum, mouse plasma, or FBS. In another embodiment, the flow chamber, bag, plate, or mixed flow chamber is coated with mouse serum, mouse plasma, or FBS.

[0048] In one embodiment, phDCs are incubated with at least one antigenic protein or at least one mRNA encoding at least one antigenic protein. Incubation can be performed under standard conditions for culturing human cells, such as in a standard medium such as RPMI-1640 at 37°C and 5% CO2. Alternatively or additionally, an incubation step can be inserted after phDCs are generated from monocytes. Incubation can be performed for 0.5 h, 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 8 h, 12 h, or 24 h. In another embodiment, the incubation step can be performed for at least 0.5 h, at least 1 h, at least 2 h, at least 3 h, at least 4 h, at least 5 h, at least 6 h, at least 8 h, at least 12 h, or at least 20 h. In one embodiment, incubation is performed for 6 h. In one embodiment, incubation is performed for 12 h. In one embodiment, incubation is performed for 20 h. In one embodiment, incubation is performed for 6 to 20 hours. In one embodiment, incubation is performed for 8 to 20 hours. Generally, phDCs have a higher transfection capacity compared to other DCs. The incubation step increases transfection efficiency.

[0049] Monocytes can be obtained by any suitable means, for example, from a blood sample or fraction thereof obtained from a donor. The blood sample or fraction thereof may be, for example, a erythrocyte sedimentation rate (ESR) layer containing leukocytes and platelets. Alternatively, the blood sample or fraction thereof may be isolated peripheral blood mononuclear cells (PMBCs). In one embodiment, the monocytes are autologous.

[0050] In one embodiment, the vaccine composition further comprises human AB serum, autologous (e.g., human) serum, autologous (e.g., human) plasma, allogeneic (e.g., human) serum, allogeneic (e.g., human) plasma, mouse serum, mouse plasma, or FBS. In one embodiment, the vaccine composition further comprises autologous serum, autologous plasma, allogeneic serum, allogeneic plasma, mouse serum, mouse plasma, or FBS. In one embodiment, the vaccine composition further comprises mouse serum, mouse plasma, or FBS.

[0051] In one embodiment, the vaccine composition further comprises human AB serum, autologous (e.g., human) serum, autologous (e.g., human) plasma, allogeneic (e.g., human) serum, allogeneic (e.g., human) plasma, mouse serum, mouse plasma, or FBS. In one embodiment, the vaccine composition further comprises autologous serum, autologous plasma, allogeneic serum, allogeneic plasma, mouse serum, mouse plasma, or FBS. In one embodiment, the vaccine composition further comprises mouse serum, mouse plasma, or FBS.

[0052] In one embodiment, the present invention relates to a vaccine composition comprising phDC and at least one mRNA, the mRNA comprising a coding sequence encoding at least one infectious disease-associated antigenic protein or at least one tumor-associated antigenic protein.

[0053] In one embodiment, the present invention relates to a vaccine composition comprising phDC and at least one mRNA, the mRNA comprising a coding sequence encoding at least one viral antigenic protein, optionally a coronavirus (e.g., β-coronavirus) antigenic protein or an HIV antigenic protein.

[0054] In one embodiment, the present invention relates to a vaccine composition comprising phDC and at least one mRNA, the mRNA comprising a coding sequence encoding at least one SARS-CoV-2 antigenic protein.

[0055] In one embodiment, the present invention relates to a vaccine composition comprising phDC and at least one mRNA, the mRNA comprising a coding sequence encoding at least one tumor-associated antigenic protein (optionally a blood cancer antigenic protein).

[0056] In one embodiment, the present invention relates to a vaccine composition comprising phDC and at least one mRNA, the mRNA comprising a coding sequence encoding at least one infectious disease-associated antigenic protein or at least one tumor-associated antigenic protein, wherein the at least one mRNA is contained in nanoparticles, and wherein the at least one mRNA is optionally modified with one or more naturally occurring modified nucleosides.

[0057] In one embodiment, the present invention relates to a vaccine composition comprising phDC and at least one mRNA, the mRNA comprising a coding sequence encoding at least one infectious disease-associated antigenic protein or at least one tumor-associated antigenic protein, wherein the at least one mRNA is contained in lipid nanoparticles, and wherein the at least one mRNA is optionally modified with one or more naturally occurring modified nucleosides.

[0058] In one embodiment, the present invention relates to a vaccine composition comprising phDC and at least one mRNA, the mRNA comprising a coding sequence encoding at least one viral antigenic protein, optionally a SARS-CoV-2 antigenic protein, wherein the at least one mRNA is contained in lipid nanoparticles, and wherein the at least one mRNA is optionally modified with one or more naturally occurring modified nucleosides. In a preferred embodiment, the at least one mRNA encodes a SARS-CoV-2 spike protein or a fragment thereof. In one embodiment, the fragment has a length of at least 100, at least 200, at least 400, at least 600, or at least 800 amino acids. In one embodiment, the fragment has a length of 400 to 1200 amino acids, 600 to 1200 amino acids, or 800 to 1200 amino acids.

[0059] In a preferred embodiment, at least one mRNA encoding a SARS-CoV-2 antigenic protein comprises the sequence of SEQ ID NO: 19 or at least 75%, 85%, 90%, 95%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 19.

[0060] In one embodiment, the present invention relates to a vaccine composition comprising phDC and at least one mRNA, the mRNA comprising a coding sequence encoding at least one blood cancer antigenic protein, wherein the at least one mRNA is contained in lipid nanoparticles, and wherein the at least one mRNA is optionally modified with one or more naturally occurring modified nucleosides.

[0061] For the above embodiments, the relevant embodiments regarding phDC, lipid nanoparticles, mRNA, and at least one antigenic protein are applied with necessary modifications as described above and below. For example, the lipid nanoarticles may comprise cationic lipids, polyethylene glycol (PEG)-modified lipids, cholesterol-based lipids, and / or non-cationic lipids. Optionally, the cationic lipids are present at a molar ratio between 30% and 40%, the PEG-modified lipids at a molar ratio between 1.5% and 4.0%, the cholesterol-based lipids at a molar ratio between 40% and 52%, and the non-cationic lipids at a molar ratio between 11% and 21%, wherein all molar ratios are relative to the total lipid content of LNP. Furthermore, at least one mRNA optionally modified with one or more naturally occurring modified nucleosides may be fully modified with N1-methyl-pseudouridine instead of each uridine. In one embodiment, the vaccine composition comprises antigen-specific phDC (e.g., at least a portion of antigen-specific phDC). The antigen-specific phDC can be obtained by combining the phDC with at least one mRNA encoding at least one antigenic protein or by combining a monocyte with at least one mRNA encoding at least one antigenic protein, and then subjecting the mixture of the monocyte and the at least one mRNA encoding at least one antigenic protein to physical force.

[0062] The present invention further relates to a vaccine composition comprising antigen-specific mononuclear cells and at least one mRNA encoding at least one antigenic protein. The antigen-specific mononuclear cells can be obtained by:

[0063] - Combine a monocyte (obtained as described above) with at least one mRNA encoding at least one antigenic protein;

[0064] - To subject monocytes and at least one mRNA encoding at least one antigenic protein to physical forces.

[0065] In another embodiment, the present invention relates to a vaccine composition comprising antigen-specific mononuclear cells, phDCs, and at least one mRNA encoding at least one antigenic protein. Physical forces (e.g., shear forces) can be applied by passing the mononuclear cells through a flow chamber, which may take the form of, for example, a plate or a bag (e.g., a flexible bag or a plastic bag) or a combination thereof (e.g., a mixture of plates and bags). The at least one mRNA may be contained in nanoparticles, particularly lipid nanoparticles. All embodiments, such as those relating to at least one mRNA encoding at least one antigenic protein, or nanoparticles (particularly lipid nanoparticles), are applied with necessary modifications as disclosed herein.

[0066] The second aspect: vaccine compositions containing phDCs used for vaccination.

[0067] The vaccine composition according to the first aspect can be used for vaccination. Therefore, in one embodiment, the present invention relates to a vaccine composition for a method of preventing or reducing a disease in a subject, the method comprising administering the vaccine composition to the subject. In one embodiment, the present invention relates to a vaccine composition for a method of preventing a disease in a subject, the method comprising administering the vaccine composition to the subject.

[0068] The vaccine compositions of the present invention are suitable for prophylactic vaccination against diseases caused by pathogens listed in the first aspect or against tumors / cancers listed in the first aspect. Furthermore, the vaccine compositions of the present invention are suitable for prophylactic vaccination against diseases caused by pathogens listed in the detailed description of the invention's "Pathogens" section or against tumors / cancers listed in the detailed description of the invention's "Tumors / Cancer" section. For example, based on at least one antigen or at least one mRNA (encoding at least one antigenic protein) present in the composition, the vaccine compositions of the present invention are suitable for vaccination against AIDS, hepatitis A, hepatitis B, hepatitis C, poliomyelitis, gastroenteritis, dengue fever, yellow fever, encephalitis, meningitis or meningoencephalitis caused by West Nile virus, influenza, Ebola, rabies, mumps, measles, herpes, pox, Middle East Respiratory Syndrome (MERS), Severe Acute Respiratory Syndrome (SARS), Covid-19, borreliosis, Pseudomonas, Burkholderia, tuberculosis, and / or malaria. In one embodiment, the vaccine composition of the present invention is used for vaccination against AIDS and / or Covid-19. In one embodiment, the vaccine composition of the present invention is used for vaccination against Covid-19. In one embodiment, the vaccine composition of the present invention is used for vaccination against Covid-19 and is administered intranasally.

[0069] In one implementation, the subject and the donor are the same. In this implementation, the subject is inoculated with autologous (antigen-specific) phDCs. In one implementation, the subject and the donor are related. In another implementation, the subject and the donor are different (i.e., unrelated). In this implementation, the subject is inoculated with allogeneic (antigen-specific) phDCs.

[0070] The vaccine composition of the present invention can be administered subcutaneously, intravenously, intramuscularly, inhaled, intra-articularly, intrasynovially, intrasternally, intrasheathically, intrahepatically, intralesionally, intracranially, transdermally, intradermally, intrapulmonaryly, intraperitoneally, intracardiacly, intra-arterially, intranasally, or sublingually. In one embodiment, administration is inhaled, intravenously, intradermally, intranasally, or intramuscularly. In one embodiment, administration is intranasal. In one embodiment, administration is intradermal or intramuscular. In one embodiment, administration is intravenous. In some embodiments, the vaccine composition of the present invention is administered as a primary and / or booster dose. In one embodiment, the vaccine composition of the present invention is administered as a primary dose. In one embodiment, the vaccine composition of the present invention is administered as a booster dose. In one embodiment, the vaccine composition of the present invention is administered as both a primary and booster dose.

[0071] The vaccine compositions of the present invention are capable of stimulating an immune response against disease-associated antigens or antigenic proteins in subjects. In one embodiment, the vaccine compositions of the present invention are capable of stimulating an immune response against infectious disease-associated antigens or infectious disease-associated antigenic proteins. In one embodiment, the vaccine compositions of the present invention are capable of stimulating an immune response against tumor-associated antigens or tumor-associated antigenic proteins.

[0072] In one embodiment, the disease is caused by pathogenic particles, which optionally comprise viruses, bacteria, fungi, parasites, and / or tumor cells. In one embodiment, the pathogenic particles comprise pathogens such as viruses, bacteria, fungi, and parasites. In one embodiment, the pathogenic particles comprise viruses. In one embodiment, the pathogenic particles comprise tumor cells (or cancer cells).

[0073] In some embodiments, the vaccine compositions provided herein are characterized in that, compared with a suitable control (e.g., comparable subjects or populations that have not been vaccinated and have been exposed to pathogenic particles or comparable subjects or populations that have been vaccinated with different vaccines such as mRNA vaccines at a predetermined expected level), subjects vaccinated with such vaccine compositions (e.g., with at least one dose, at least two doses, etc.) may show a reduced and / or more transient presence of pathogenic particles in the relevant sites (e.g., nose and / or lungs, and / or any other tissue susceptible to the corresponding disease or condition) at the time of disease outbreak.

[0074] In one embodiment, the concentration of pathogenic particles in a subject after a disease outbreak is effectively reduced compared to the concentration of pathogenic particles in a second subject (where an outbreak of the same disease is observed (e.g., diagnosed) but who has not yet been administered the vaccine composition of the present invention). In one embodiment, the concentration of pathogenic particles in a second infected subject who has not been administered the vaccine composition of the present invention is effectively reduced compared to the concentration of pathogenic particles in a second infected subject who has been vaccinated with a different vaccine (preferably a vaccine that does not contain dendritic cells) targeting the same pathogenic particles; and even more preferably, the concentration of pathogenic particles in a second infected subject who has been vaccinated with an mRNA vaccine targeting the same pathogenic particles.

[0075] In one embodiment, the concentration of pathogenic particles (e.g., tumor cell count or tumor volume) at the time of tumor outbreak in the subject is effectively reduced compared to the concentration of pathogenic particles in a second subject in which the same tumor outbreak was observed and who has not yet been administered the vaccine composition of the present invention; or compared to the concentration of pathogenic particles in a second subject in which the same tumor outbreak was observed and who has been vaccinated with a different vaccine (preferably one that does not contain dendritic cells) targeting the same pathogenic particles; and even more preferably, compared to the concentration of pathogenic particles in a second subject in which the same tumor outbreak was observed and who has been vaccinated with an RNA vaccine targeting the same pathogenic particles. In some embodiments, the subject is in remission following conventional antitumor therapeutic intervention. In one embodiment, the vaccine composition can be administered to such subjects in remission to act as an antitumor vaccine and reduce the risk of relapse.

[0076] In one embodiment, the anti-antigen antibody titer in subjects vaccinated with the vaccine composition of the present invention is lower compared to the anti-antigen antibody titer in a second subject who has been vaccinated against the same pathogenic particle with a different vaccine (preferably one that does not contain dendritic cells). In one embodiment, the anti-antigen antibody titer in post-vaccination and post-infection subjects is lower compared to a second infected subject who has been vaccinated against the same pathogenic particle with a different vaccine (preferably one that does not contain dendritic cells). Importantly, subjects receiving the vaccine composition receive equivalent protection compared to second subjects exhibiting higher anti-antigen antibody titers. This effect may be due to the fact that the antigen-specific phDCs of the present invention favor T-cell-based cell-mediated immunity, while conventional vaccines, especially RNA-based vaccines, elicit strong antibody responses.

[0077] In one embodiment, the concentration of pathogenic particles in the subject is effectively reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99%. This reduction can be measured by comparison with an appropriate control, such as the concentration of pathogenic particles before the onset of disease or with the concentration of pathogenic particles in a second subject as described above. In one embodiment, in subjects who have been vaccinated with the vaccine composition of the present invention, the time period during which the subject exhibits disease symptoms is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99%. This can be compared with an appropriate control, such as subjects who have not received antigen-specific phDC or who have received different vaccines such as RNA vaccines.

[0078] In one embodiment, the concentration of pathogenic particles is effectively reduced systemically in the subject upon infection. In one embodiment, the concentration of pathogenic particles is effectively reduced locally. In one embodiment, the concentration of pathogenic particles in the brain is effectively reduced. In one embodiment, the concentration of pathogenic particles in mucosal tissue is effectively reduced (i.e., the vaccine composition of the present invention induces mucosal immunity). The reduction may be at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%. Comparison can be made with the concentration of pathogenic particles in a second subject who has not received the vaccine composition of the present invention or who has received a different vaccine such as an RNA vaccine.

[0079] In one embodiment, after vaccination with the vaccine composition of the present invention, compared with a second subject vaccinated against the same pathogenic particle with a different vaccine (preferably a vaccine without dendritic cells), and even more preferably with a second subject vaccinated against the same pathogenic particle with an RNA vaccine, the subject showed an increased proportion of central memory T cells (Tcm) and / or stem cell-like T cells specific to at least one disease-associated antigen (or at least one antigenic protein). In one embodiment, after vaccination with the vaccine composition of the present invention, compared with a second subject vaccinated against the same pathogenic particle with a different vaccine (preferably a vaccine without dendritic cells), and even more preferably with a second subject vaccinated against the same pathogenic particle with an RNA vaccine, the subject showed a decreased proportion of effector-dominant T cells (Teff) specific to at least one disease-associated antigen (or at least one antigenic protein). In one embodiment, after vaccination with the vaccine composition of the present invention, compared with a second subject vaccinated against the same pathogenic particle with a different vaccine (preferably a vaccine without dendritic cells), and even more preferably with a second subject vaccinated against the same pathogenic particle with an mRNA vaccine, the subject showed an increased proportion of natural killer (NK) cells. In one embodiment, after vaccination with the vaccine composition of the present invention, compared with a second subject vaccinated against the same pathogenic particle with a different vaccine (preferably a vaccine without dendritic cells), and even more preferably with a second subject vaccinated against the same pathogenic particle with an mRNA vaccine, the subject showed an increased level of IFN-γ. In one embodiment, after vaccination with the vaccine composition of the present invention, compared with a second subject vaccinated against the same pathogenic particle with a different vaccine (preferably a vaccine without dendritic cells), and even more preferably with a second subject vaccinated against the same pathogenic particle with an mRNA vaccine, the subject showed a decreased proportion of exhaustion-effect T cells. These effects can be observed some time after the initial and / or booster administration (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks after the initial and / or booster administration). Comparisons can also be made with other suitable controls, such as the same subject prior to administration of the vaccine composition of the invention (rather than with a second subject). The increase in the proportion of central memory T cells and / or stem cell-like T cells and / or natural killer cells can be at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%.The reduction in the proportion of effector-dominant T cells and / or exhausted effector T cells can be at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%. The increase in IFN-γ secretion levels can be at least 2-fold, at least 10-fold, at least 50-fold, at least 100-fold, at least 500-fold, or at least 5000-fold. The proportion of Tcm can be determined, for example, by measuring markers associated with the central memory T cell phenotype, such as CD44 / CD62L. Compared to Tcm, Teff shows reduced CD62L expression. The proportion of stem cell-like T cells can be determined, for example, by measuring markers associated with the stem cell-like T cell phenotype, such as IL7Ra / SCA-1. For stem cell-like T cells, the expression of both markers is increased. NK cells can be determined, for example, by positive selection using an NK1.1 cell isolation method. The proportion of exhausted effector T cells can be determined, for example, by measuring expression markers associated with exhaustion, such as PD1. Vaccination can refer to initial vaccination and / or booster administration, i.e., one or more doses. In one implementation, vaccination refers to initial administration and booster administration.

[0080] As described above, in one embodiment, the present invention relates to a vaccine composition for a method of prophylactic vaccination. Through prophylactic vaccination, the present invention relates to administering the vaccine composition described herein to a subject who has not yet been diagnosed with the aforementioned condition (e.g., an infectious disease (e.g., Covid-19) or tumor) targeted by the vaccine composition. Therefore, prophylactic vaccination relates to the prevention or reduction of undiagnosed conditions.

[0081] In one embodiment, the present invention relates to a vaccine composition for a first aspect of a preventive method for preventing or reducing disease in a subject, the method comprising administering the vaccine composition to the subject.

[0082] In one embodiment, a prophylactic method for using a vaccine composition to prevent or reduce an infectious disease or tumor in a subject includes administering the vaccine composition to the subject.

[0083] In one embodiment, a preventive method for using a vaccine composition to prevent or reduce viral diseases in a subject includes administering the vaccine composition to the subject.

[0084] In one embodiment, a preventive method of using a vaccine composition to prevent or reduce Covid-19 (or one of the symptoms of Covid-19) in a subject includes administering the vaccine composition to the subject.

[0085] In one embodiment, a preventive method for using a vaccine composition to prevent or reduce blood cancers in a subject includes administering the vaccine composition to the subject.

[0086] In one embodiment, a prophylactic method for using a vaccine composition to prevent or reduce solid tumors in a subject includes administering the vaccine composition to the subject.

[0087] Third aspect: Methods of preventing or reducing disease

[0088] In a third aspect, the present invention relates to a method for preventively preventing or reducing a disease in a subject, the method comprising administering to the subject a vaccine composition of the first aspect (including all embodiments of the first and second aspects as described above).

[0089] Fourth aspect: Use of the pharmaceutical composition for vaccination

[0090] In a fourth aspect, the present invention relates to the use of a pharmaceutical composition for vaccination, wherein the pharmaceutical composition is the vaccine composition of the first aspect. Therefore, the corresponding embodiments of the first, second, and third aspects are applied with necessary modifications regarding the pharmaceutical composition, phDC, nanoparticles, RNA (e.g., mRNA), and at least one antigenic protein.

[0091] Fifth aspect: Kits containing phDC and mRNA

[0092] In a fifth aspect, the present invention relates to a kit comprising phDC and at least one RNA (e.g., mRNA) encoding at least one antigenic protein. The kit may further comprise nanoparticles (e.g., lipid nanoparticles). In the case where the kit comprises nanoparticles, the nanoparticles comprise RNA (e.g., mRNA) encoding at least one antigenic protein. The corresponding embodiments of the first aspect are applied with necessary modifications regarding phDC, nanoparticles (e.g., lipid nanoparticles), RNA (e.g., mRNA), and at least one antigenic protein.

[0093] The present invention also relates to a kit comprising a monocyte and at least one RNA (e.g., mRNA) encoding at least one antigenic protein. The kit may further comprise nanoparticles (e.g., lipid nanoparticles). In the case where the kit comprises nanoparticles, the nanoparticles comprise RNA (e.g., mRNA) encoding at least one antigenic protein. With regard to the monocyte, nanoparticles (e.g., lipid nanoparticles), RNA (e.g., mRNA), and at least one antigenic protein, the corresponding embodiments of the first aspect are applicable with necessary modifications. Attached Figure Description

[0094] Figure 1. Specific internalization and expression of spike protein encoded by LNP containing mRNA in mouse phDCs. A, B, FACS images showing spike protein positivity in CD11b-positive phDCs without LNP transfection (A) and with LNP transfection (B). C, D, confocal microscopy analysis using the same samples, showing spike protein expression in CD11b+Ly6G- phDCs (projected Z-stack images). E. Z-planar slice image of LNP-transfected phDCs.

[0095] Figure 2A, Subcutaneous tumor implantation of EG7-OVA (3x10 per mouse) 6 Two days after treatment (within cells), C57BL / 6 mice were treated intravenously with phDCs transduced with OVA mRNA LNPs, or via immediacy with OVA mRNA LNPs (without phDCs). Approximately 10 ng of LNPs was used to transduce phDCs (per mouse), and the same amount (0.5 ug / kg LNP, equivalent to the dose of Pfizer's COVID-19 mRNA LNP vaccine in the current human vaccine context) was administered via immediacy. Treatment was given twice weekly, with a total of 5 treatments per mouse. At the start of treatment, tumors were visible and palpable (>50 mm). 3 B, Monitor EG7-OVA tumor growth throughout the experiment.

[0096] Figure 3 A, from 3*10 per mouse 6 Two days after subcutaneous inoculation of individual cells with EG7-OVA (with) Figure 3 (Same experiment), phDC[ova] or IM[ova] mice were treated a total of five times with 10 ng [ova] per mouse. BC, Tumor growth was monitored during the experiment, which presents the cumulative tumor growth for each experimental group (B) and the individual mouse tumor growth curve (C). DE, CD8+ spleen T cells were isolated from the treated mice on day 30 and immediately injected with 1*10 μg / mL SIINFEKL peptide, with or without the presence of 10 μg / mL. 5 Cells / well were placed in an 18-hr IFN-g Elispot assay. Representative Elispot well images (D) and IFN-g spot quantification (E) for each experimental group are provided. *[ova] = LNP - mRNA encoding ovalbumin.

[0097] Figure 4A, from 3*10 per mouse 6 Two days after subcutaneous inoculation of individual cells with EG7-OVA (with) Figure 3(Same experiment), phDC[ova] or IM[ova] mice were treated a total of five times with 10 ng [ova] per mouse. At the end of the 28-day observation period, spleen cells from the treated mice were harvested for T cell analysis and characterized by flow cytometry for: A, antigen (SIINFEKL) specific CD8 T cells, via dextramer analysis; Tem (effective) / Tcm (central memory) type phenotype analysis, via CD44 / CD62L expression; detection of stem cell-like T cells, via IL7Ra / SCA-1 expression; and B, evaluation of T cell exhaustion markers, via PD-1 expression. *[ova] = LNP - mRNA encoding ovalbumin.

[0098] Figure 5 PhDC[ova] or IM[ova] mice were vaccinated on day -7 and day 0, respectively (primary / booster). Undifferentiated spleen cells were harvested immediately after vaccination and placed in 18-hr IFN-g Elispots (A, B, and C) without additional antigen stimulation. To identify the cells responsible for spleen IFN-g, isolated CD8+ or NK1.1+ spleen cells were collected from phDC[ova] mice after vaccination, and IFN-g spots were evaluated (D). *[ova] = LNP-encoding ovalbumin mRNA.

[0099] Figure 6 A, Subcutaneous tumor implantation with EG7-OVA (1x10⁶ per mouse) 6 On days -14 and -7 prior to the treatment, C57BL / 6 mice were treated with phDCs transduced with 1 μg / mL OVA mRNA LNP, or with soluble OVA protein (50 μg / mouse, without phDCs) via IV. EG7-OVA tumor growth was monitored throughout the experiment.

[0100] Figure 7 Mouse phDCs were transduced with 1 μg / ml OVA mRNA LNP (CKK), and intracellular OVA protein was stained after overnight culture. phDCs transduced with SP LNP (1 μg / ml) served as controls. (n=3) OVA was detected using Rockland OVA ab (FITC conjugated).

[0101] Figure 8. Mouse phDCs were incubated overnight with various antigenic forms of OVA; these antigenic forms were soluble LNPs expressed in tumor cells or containing OVA-encoding mRNA. Cells were then harvested and stained for surface CD11b, Ly6G, and 25.D1 (an ab against MHC I binding SIINFEKL in the H-2Kb strain). Ly6G-positive cells were selected for neutrophil exclusion. FACs plots show 25.D1 positivity on CD11b+ phDCs.

[0102] Figure 9. phDCs (PP PBMCs from B6 mice) were transduced with OVA mRNA LNP (1 μg / ml) in a standard overnight culture protocol. Cells were harvested at different time points, washed, fixed (using the Intraprep kit), and stained for surface CD11b and the SIINFEKL-MHC I complex (25.D1 ab). The 25.D1 expression level in CD11b+ cells at different time points is shown in the FACs plot (top). The percentage of 25.D1-positive cells in the CD11b+ subset and the MFI level of 25.D1 are shown in the bar chart (bottom).

[0103] Figure 10. In the standard overnight protocol, enriched phDCs (monocytes purified from mouse PBMCs via plate) or BMDCs (cytokine-induced DCs from mouse bone marrow) were incubated overnight with / without 0.1 μg / ml LNP containing OVA mRNA. Cells were harvested and surface staining for CD11b and 25.D1 (ab targeting MHC I binding SIINFEKL in the H-2Kb strain) was performed on phDC samples, and CD11c and 25.D1 were performed on BMDC samples. FACs plots show 25.D1 positivity on CD11b+ phDCs and CD11c+ BMDCs.

[0104] Figure 11. Mouse phDCs were transduced using SIINFEKL peptide mRNA LNP or OVA protein mRNA LNP. After overnight incubation, cells were harvested and stained with 25.D1 ab to detect SIINFEKL MHC I complex signal. FACS plots show 25.D1 signaling on the CD11b+ phDC subset.

[0105] Figure 12. PhDCs from mice were pulsed with different concentrations of OVA mRNA LNP and then co-cultured with OT1 T cells. The proliferation of OT1 CD8 T cells (CFSE-labeled) was evaluated as indicated by the reduction in CFSE signaling.

[0106] Figure 13. PhDCs from mice (B6) were pulsed with 1 μg / ml OVA mRNA and then cultured with 100 K OT1 T cells at different phDC numbers (96-well U-plate). The proliferation of OT1 CD8 T cells (CFSE-labeled) was evaluated as readings.

[0107] Figure 14. Four days after EG7 tumor implantation (3 x 10⁶ cells per mouse), C57BL / 6 mice were treated intravenously with phDCs transduced with OVA mRNA LNP*, or via im* route with mimic (SP) mRNA LNP* or OVA mRNA LNP in mice (without phDCs). Treatment was administered twice weekly (on days 4, 8, 11, 15, 18, and 25). At the start of treatment, tumors were visible and palpable (>50 mm³). N=10 per group.

[0108] Figure 15 (a) SIINFEKL tetramer positivity was analyzed in spleen cells from mice in Experiment 21-1008 (therapeutic experiment, HB) at the end of the in vivo tumor surveillance period (day 32). H-2Kb OVASIINFEKL tetramer positivity on the CD8+ subset is shown in the FACs plot. Spleen cells from 10 mice were pooled prior to analysis. The last treatment was administered 1 week prior to analysis. (b) T cells isolated from spleen cells from the three experimental groups (15M T cells / mouse) were transferred to mice bearing EG7 tumors, and tumor development was monitored over a 19-day period. Invention Details

[0110] I. Exemplary preparation of phDC

[0111] In one instance, phDCs can be prepared from monocytes (e.g., obtained from a donor). For example, phDCs can be generated by subjecting monocytes (e.g., obtained from a donor) to physical forces. These physical forces can be generated, for example, by passing the monocytes through a flow chamber. In one embodiment of the invention, phDCs are obtained by passing a plate of monocytes through a method derived from in vitro photodisplacement (ECP). Thus, in one embodiment, the flow chamber can be a plate. Plates used for preparing pHDCs have previously been described in the literature, see, for example, Durazzo et al., 2014 (“Induction of Monocyte-to-Dendritic Cell Maturation by Extracorporeal Photochemotherapy: Initiation via Direct Platelet Signaling”) or Ventura et al., 2018 (“Extracorporeal Photochemotherapy Drives Monocyte-to-Dendritic Cell Maturation to Induce Anticancer Immunity”). Methods and apparatus for activating monocytes in vitro and generating dendritic cells therefrom are described in WO2014 / 106629 A1, WO2014 / 106631 A1, WO2016 / 001405 A1, and WO2017 / 005700 A1, each of which is incorporated herein by reference in its entirety. The ECP describes a method in which monocytes derived from a blood sample or fraction thereof are exposed to mechanical stress (physical forces in the form of shear forces) and plasma components (e.g., platelets) or their derivatives or mimics, thereby activating the monocytes to differentiate into healthy, physiological dendritic cells, also referred to herein as phDCs. However, providing monocytes and subjecting them to physical forces is sufficient to activate the monocytes and cause them to differentiate into phDCs. ECP and ECP-derived processes (including monocyte differentiation into phDCs) can be performed in a flow chamber (e.g., a THERAKOS® CELLEX® device); or in a miniaturized ECP device, such as a Transimmunization plate as described in WO2017 / 005700 A1; or in a bag (e.g., a flexible bag or a plastic bag); or a combination of the above.

[0112] The bag can be made of any material that does not leak liquid, such as rubber, silicone, or plastic. Preferably, the material is flexible enough to bend easily without breaking.

[0113] Optionally, the bag or plate is made of a plastic material. Suitable plastic materials include polyolefins, polyethylene, fluoropolymers, polyvinyl chloride, ethylene-vinyl acetate copolymers, ethylene vinyl alcohol, polyvinylidene fluoride, and / or other plastics containing materials approved for medical use. A preferred plastic material is an ethylene-vinyl acetate copolymer. The bag or plate may be made of a material that provides a degree of transparency, allowing the sample or cell mixture to be illuminated with visible or UV light.

[0114] In one embodiment, the flow chamber may be a hybrid flow chamber or a combination of chambers and bags, wherein each component is as described herein.

[0115] The inventors have discovered that phDCs obtained by the above method are advantageous compared to DCs obtained by other methods (such as incubating blood monocytes with cytokines or directly isolating them from a donor) because phDCs are physiologically generated with greater reproducibility and controllability under precise in vitro laboratory conditions (without requiring chemicals such as cytokines and / or apoptosis agents). The benefits of the above method for generating phDCs compared to other methods (such as incubation with cytokines) include one or more of the following: higher yield, faster process, better intracellular antigen processing, and more effective initiation of disease-specific cytotoxic T cells.

[0116] Donor-derived phDCs are obtained by subjecting the monocytes contained in a blood sample (obtained from the donor) or a fraction thereof to shear stress through a flow chamber. Preferably, platelets are present in the flow chamber, which may be derived from the donor's blood sample or a fraction thereof or provided separately. Additionally or alternatively, plasma components may be present in the flow chamber, which may be derived from the donor's blood sample or a fraction thereof or provided separately. After obtaining phDCs, they can be cryopreserved until further use, for example, in combination with at least one disease-associated antigen or at least one mRNA encoding at least one antigenic protein.

[0117] Donor monocytes can be obtained by any suitable means, for example, from a blood sample or fraction thereof. A fraction of the blood sample can be, for example, an erythrocyte sedimentation rate (ESR) layer comprising leukocytes and platelets. Alternatively, a fraction of the blood sample can be isolated peripheral blood mononuclear cells (PMBCs). PMBCs can be isolated from a blood sample using, for example, centrifugation on a Ficoll-Hypaque gradient (Isolymph, CTLScientific). In another instance, a fraction of the blood sample can be a purified or enriched monocyte preparation. Monocytes can be enriched from PBMCs using, for example, plastic adhesion; CD14 magnetic bead positive selection (e.g., from Miltenyi Biotec); and Monocyte Isolation Kit II (Miltenyi Biotec).

[0118] Any suitable volume of blood can be used. The blood sample (e.g., a fractionated blood sample) can be between about 1 μL and about 500 mL, for example, between about 1 μL and about 10 mL, between about 1 μL and about 5 mL, between about 1 μL and about 1 mL, between about 1 μL and about 750 μL, between about 1 μL and about 500 μL, between about 1 μL and about 250 μL, between about 10 mL and about 450 mL, between about 20 mL and about 400 mL, between about 30 mL and about 350 mL, between about 40 mL and about 300 mL, between about 50 mL and about 200 mL, or between about 50 mL and about 100 mL. In some embodiments, the blood sample or fraction thereof, or additional blood samples or fractions thereof, are less than or equal to about 100 mL (e.g., from about 50 mL to about 100 mL). In one embodiment, a mononuclear cell-derived blood sample is obtained from a donor. However, monocytes can be derived from other sources that provide monocytes, such as bone marrow, spleen, or other peripheral tissues. In one embodiment, monocytes are derived from peripheral blood mononuclear cells (PBMCs) obtained from a donor.

[0119] Those skilled in the art are familiar with methods for distinguishing dendritic cells (including phDCs) from monocytes, such as by assessing gene expression. For example, CD80, CD83, and CD86 are markers expressed at higher levels by mature dendritic cells compared to monocytes. In one embodiment, the donor is a mammal. Mammals include, but are not limited to, humans, non-human primates, pigs, dogs, cats, and rodents. In a preferred embodiment, the donor is a human.

[0120] II. Antigenic proteins

[0121] PhDCs are combined with at least one mRNA encoding at least one antigenic protein. The antigenic protein can be a disease-associated antigen or a fragment thereof. If it is referred to as a "disease-associated antigen" or "antigenic protein," then "fragment thereof" is included in that meaning. After combining phDCs with a disease-associated antigen, the phDCs are able to take up, process, and present the antigen on their surface. This process is also known as "loading." Such loaded phDCs can elicit an effective immune response against disease (e.g., against infectious diseases or tumors).

[0122] At least one disease-associated antigen can be loaded as the antigen itself (e.g., protein, peptide, epitope, cell, cell or tissue lysate, virus, viral particle, etc., including fragments of each of the foregoing) or as a nucleic acid encoding the antigen (loaded in the sense of phDC taking up the antigen or antigenic protein). Thus, in one embodiment, the phDC comprises a nucleic acid encoding an antigenic protein or disease-associated antigen, preferably RNA, more preferably mRNA. In one embodiment, at least one disease-associated antigen is loaded as a nucleic acid encoding the antigen. At least one disease-associated antigen can be provided directly or stored in freeze-dried or lyophilized form until use. Methods of loading dendritic cells with antigens are known to those skilled in the art. In one example, phDC is loaded by combining it with at least one disease-associated antigen (e.g., incubation). In some embodiments, phDC can be loaded with different antigens to produce multivalent antigen-specific phDC. In other examples, phDC is loaded by electroporation, polymer- or lipid-based nanoparticles, or cell extrusion methods. In one embodiment, phDC is loaded by encapsulating at least one disease-associated antigen (e.g., in the form of mRNA) in lipid nanoparticles and then combining the phDC with the encapsulated at least one disease-associated antigen. In cases where at least one antigen is encoded by RNA, particularly mRNA, for the purposes of this invention, such at least one disease-associated antigen is referred to as an antigenic protein.

[0123] Regarding the cell extrusion method, phDCs are flowed in a solution containing at least one disease-associated antigen or a fragment thereof. For loading, phDCs are extruded by microfluidic compression smaller than the cell diameter. This results in temporary disruption of the cell membrane, allowing at least one disease-associated antigen to enter the cytosol of the phDCs.

[0124] Once phDCs have been obtained and combined with at least one disease-associated antigen, the mixture can be incubated, for example, under standard conditions. Culture can be performed under standard conditions (e.g., 37°C and 5% CO2) in a standard medium for culturing human cells, such as RPMI-1640 medium (available from, for example, GIBCO) supplemented with 15% AB serum (available from, for example, Gemini Bio-Products). An incubation step can be added after combining phDCs with at least one disease-associated antigen. Alternatively or additionally, an incubation step can be inserted after phDCs are generated from monocytes. The incubation step can be performed for 0.5 h, 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 12 h, 20 h, or 24 h. In another embodiment, the incubation step can be performed for at least 0.5 h, at least 1 h, at least 2 h, at least 3 h, at least 4 h, at least 5 h, at least 6 h, at least 12 h, at least 20 h, or at least 24 h. It should be understood that all method steps are performed in vitro.

[0125] II.A Pathogenic Particles

[0126] At least one disease-associated antigen or at least one antigenic protein can be derived from a pathogenic particle, which includes pathogens and tumor cells. Pathogens are viruses, bacteria, fungi, prions, and parasites. Thus, in one embodiment, the pathogenic particle includes viruses, bacteria, fungi, prions, parasites, and tumor cells. In the following sections concerning pathogens and tumors / cancer, the term "antigen" and the term "antigenic protein" are used interchangeably. For example, if an infectious disease antigen or tumor antigen is referred to, it also refers to an infectious disease-associated antigenic protein or a tumor-associated antigenic protein. If a viral antigen is referred to, it also refers to a viral antigenic protein. If an HIV or SARS-CoV-2 antigen is referred to, it also refers to an HIV or SARS-CoV-2 antigenic protein.

[0127] II.A.1 Pathogens

[0128] In some embodiments, the disease-associated antigen is an infectious disease-associated antigen. In some embodiments, the disease-associated antigen is a viral antigen. In some embodiments, the disease-associated antigenic protein is a viral antigenic protein.Examples of viruses that can derive disease-associated antigens or antigenic proteins include: Retroviridae (e.g., human immunodeficiency virus, such as HIV-1 (also known as HTLV-III, LAV, or HTLV-III / LAV or HIV-III; and other isolates such as HIV-LP, HIV-2); Picornaviridae (e.g., poliovirus, hepatitis A virus; enterovirus, human coxsackievirus, rhinovirus, echovirus); Calciviridae (e.g., strains causing gastroenteritis); Togaviridae (e.g., equine encephalitis virus, rubella virus); Flaviviridae (e.g., dengue virus, encephalitis virus, yellow fever virus); Coronaviridae (e.g., coronavirus); Rhabdoviridae (e.g., vesicular stomatitis virus, rabies virus); and Filoviridae. (e.g., Ebola virus); Paramyxoviridae (e.g., parainfluenza virus, mumps virus, measles virus, respiratory syncytial virus); Orthomyxoviridae (e.g., influenza virus); Bungaviridae (e.g., Hantavirus, bungalow virus, sandfly virus, and Nairovirus); Arena viridae (hemorrhagic fever viruses); Reoviridae (e.g., reovirus, circovirus, and rotavirus); Birnaviridae; Hepadnaviridae (hepatitis B virus); Parvoviridae (parvovirus); Papovaviridae (papillomavirus, polyomavirus); Adenoviridae (Most adenoviruses); Herpesviridae (herpes simplex virus (HSV) 1 and 2, varicella-zoster virus, cytomegalovirus (CMV), herpesviruses); Poxviridae (smallpox virus, vaccinia virus, poxviruses); and Iridoviridae (e.g., African swine fever virus); and unclassified viruses (e.g., the agent of spongiform encephalopathy, the agent of delta hepatitis (considered a defective accessory of hepatitis B virus), the agent of non-A, non-B hepatitis (enteral transmission; extra-gastrointestinal transmission (i.e., hepatitis C); norovirus and related viruses and astroviruses).

[0129] Preferably, the disease-associated antigen is a viral antigen, more preferably a retroviral or coronavirus antigen, and most preferably an HIV or SARS-CoV-2 antigen. In even more preferred embodiments, the disease-associated antigen is derived from SARS-CoV-2.

[0130] In some implementations, the disease-associated antigen is a bacterial antigen. Examples of bacteria that can derive disease-associated antigens include: *Helicobacter pylori*, *Borrelia* spp. (e.g., *Borrelia burgdorferi*), *Legionella pneumophila*, *Mycobacteria* spp. (e.g., *Mycobacterium tuberculosis*, *Mycobacterium avium*, *Mycobacterium intracellulare*, *Mycobacterium kansasii*, *Mycobacterium gordonae*), *Staphylococcus aureus*, *Neisseria gonorrhoeae*, *Neisseria meningitidis*, *Listeria monocytogenes*, and *Streptococcus pyogenes*. (Group A Streptococci), *Streptococcus agalactiae* (Group B Streptococci), *Streptococcus* (Viridans section), *Streptococcus faecalis*, *Streptococcus bovis*, *Streptococcus* (anaerobic species), *Streptococcus pneumoniae*, *Pathogenic Campylobacter* spp., *Enterococcus* spp., *Haemophilus influenzae*, *Bacillus anthracis*, *Corynebacterium diphtheriae*, *Corynebacterium* spp.The following bacteria are listed: *Erysipelothrix rhusiopathiae*, *Clostridium perfringens*, *Clostridium tetani*, *Enterobacter aerogenes*, *Klebsiella pneumoniae*, *Pasteurella multocida*, *Bacteroides spp.*, *Fusobacterium nucleatum*, *Streptobacillus moniliformis*, *Treponema pertenue*, *Leptospira*, *Actinomyces israelii*, *Burkholderia pseudomallei*, *Burkholderia mallei*, and *Pseudomonas aeruginosa*.

[0131] Preferably, the bacterial antigen is a spirochetal or mycobacterial antigen.

[0132] In some implementations, the disease-associated antigen is a fungal antigen. Examples of fungi from which disease-associated antigens can be derived include: Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Chlamydia trachomatis, and Candida albicans.

[0133] In some implementations, the disease-associated antigen is a parasitic antigen. Parasitic antigens can be derived from protozoa, worms, or ectoparasites. Examples of parasites from which disease-associated antigens can be derived include: *Plasmodium falciparum*, *Plasmodium vivax*, *Plasmodium ovale*, *Plasmodium malariae*, species of *Leishmania*, species of *Trypanosome* (Africa and America), *Cryptosporidium*, species of *isospora*, *Naegleria fowleri*, species of *Acanthamoeba*, *Balamuthia mandrillaris*, *Toxoplasma gondii*, or *Pneumocystis carinii*.

[0134] II.A.2 Tumors / Cancer

[0135] In some implementations, the disease-associated antigen is a tumor or cancer antigen, such as a tumor-associated antigen (TAA) or a tumor-specific antigen (TSA). For the purposes of this disclosure, unless otherwise stated, TAA includes the group of TSA. Tumor or cancer antigens can be found on tumors or cancer cells. Tumor or cancer antigens can be derived from solid tumors or blood cancers.

[0136] Examples of tumors or cancers that can generate disease-associated antigens include leukemia, melanoma, lymphoma, endometrial cancer, kidney cancer, brain cancer, cervical cancer, liver cancer, head and neck cancer, gastrointestinal cancer, lymph node cancer, pancreatic cancer, ear, nose and throat (ENT) cancer, breast cancer, prostate cancer, ovarian cancer, or lung cancer.

[0137] In some implementations, the disease-associated antigen is a tumor-associated antigen. Tumor-associated antigens include Her2, prostate stem cell antigen (PSCA), PSMA (prostate-specific membrane antigen), B-cell maturation antigen (BCMA), ERK5, alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), cancer antigen-125 (CA-125), CA19-9, calretin, MUC-1, epithelial membrane protein (EMA), epithelial tumor antigen (ETA), tyrosinase, melanoma-associated antigen (MAGE), CD34, CD45, CD99, CD117, chromogranin, cytokeratin, myodermin, glial fibrillary acidic protein (GFAP), giant cystic disease fluid protein (GCDFP-15), HMB-45 antigen, and protein melan-A. (Melanoma antigen recognized by T lymphocytes; MART-1), myo-D1, muscle-specific actin (MSA), neurofilaments, neuron-specific enolase (NSE), placental alkaline phosphatase, synaptophysin, thyroglobulin, thyroid transcription factor-1, the dimer form of pyruvate kinase isoenzyme M2 (tumor M2-PK), aberrant ras protein, or aberrant p53 protein. In some embodiments, tumor-associated antigens are CD19, CD22, CD27, CD30, CD70, GD2 (ganglioside G2), EGFRvIII (epidermal growth factor variant III), spermin 17 (Sp17), mesothelin, PAP (prostatic acid phosphatase), prostein, TARP (T cell receptor γ-alternating reading frame protein), Trp-p8, or STEAP1 (prostatic six-transmembrane epithelial antigen 1). In some embodiments, the TAA is a cancer / testis (CT) antigen, such as BAGE, CAGE, CTAGE, FATE, GAGE, HCA661, HOM-TES-85, MAGEA, MAGEB, MAGEC, NA88, NY-ESO-1, NY-SAR-35, OY-TES-1, SPANXB1, SPA17, SSX, SYCP1, or TPTE. In some other embodiments, the TAA is a carbohydrate or ganglioside, such as fuc-GMI, GM2 (Cancer-Fetal Antigen Immunogenicity-1; OFA-I-1); GD2 (OFA-I-2), GM3, GD3, etc. In some other embodiments, the TAA is α-actin-4, Bage-1, BCR-ABL, Bcr-Abl fusion protein, β-catenin, CA 125, CA 15-3 (CA27).29\BCAA), CA 195, CA 242, CA-50, CAM43, Casp-8, cdc27, cdk4, cdkn2a, CEA, coa-1, dek-can fusion protein, EBNA, EF2, Epstein-Barr virus antigen, ETV6-AML1 fusion protein, HLA-A2, HLA-A11, hsp70-2, KIAAO205, Mart2, Mum-1, Mum-2 and Mum-3, neo-PAP, myosin class I, OS-9, pml-RARa fusion protein, PTPRK, K-ras, N-ras, triosephosphate isomerase, Gage 3, Gage 4, Gage 5, Gage 6, Gage 7. GnTV, Herv-K-mel, Lage-1, NA-88, NY-Eso-1 / Lage-2, SP17, SSX-2, TRP2-Int2, gpi00 (Pmel 17), tyrosinase, TRP-1, TRP 2. MAGE-1, MAGE-3, RAGE, GAGE-1, GAGE-2, p15(58), RAGE, SCP-i, Hom / Mel-40, PRAME, p53, H-Ras, HER-2 / neu, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, Human papillomavirus (HPV) antigens E6 and E7, TSP-180, MAGE-4, MAGE-5, MAGE-6, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72-4, CA 19-9, CA 72-4, CAM17.1, NuMa, 13-catenin, Mum-1, p16, TAGE, PSMA (Prostate-specific membrane antigen), B-cell maturation antigen (BCMA), CT7, telomerase, 43-9F, 5T4, 791Tgp72, 13HCG, BCA225, BTAA, CD68 / KPi, CO-O29, FGF-5, G250, Ga733 (EpCAM), HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB / 70K, NY-CO-1, RCAS1, SDCCAG16, TA-90, TAAL6, TAG72, TLP, TPS, integrin cv3 (CD61), prolactin, K Ras (V-Ki-ras2 Kirsten rat sarcoma virus oncogene) or Ral-B.

[0138] Other tumor-associated antigens are known to those skilled in the art and can be combined with the phDCs provided herein to produce corresponding antigen-specific phDCs, or may be present in the compositions disclosed herein.

[0139] II.B Means of providing disease-related antigens

[0140] II.B.1 Antigens derived from pathogenic particles

[0141] Disease-associated antigens can be derived from pathogenic particles, such as cells, biopsies, tissue lysates, or other particles containing pathogenic particles. In one embodiment, the disease-associated antigen can be derived from a tumor or cancer cell to be treated. For example, a sample of tumor cells can be taken from a subject with cancer, such as for a biopsy, or the sample can be taken from a subject with an infectious disease. The sample can be further treated with an agent suitable for releasing the antigen, such as a photoactivated agent combined with light (especially UV light), such as a combination of 8-MOP and UVA. The treated sample can then be combined with phDC.

[0142] In another example, the pathogen can be cultured in a culture medium. The relevant antigen can detach from the pathogen into the culture medium and then be collected for use in combination with phDC. Those skilled in the art are aware of the various methods and further methods known in the art for providing antigens derived from pathogenic particles.

[0143] II.B.2 Antigens expressed from nucleic acids

[0144] The phDC can be loaded with nucleic acid (including its functional fragment) encoding at least one disease-related antigen. In one embodiment of the invention, the phDC is loaded with RNA (including its functional fragment) encoding at least one disease-related antigen. In this embodiment, the at least one disease-related antigen corresponds to at least one antigenic protein. Therefore, the phDC is directly combined with RNA (e.g., mRNA). The RNA (e.g., mRNA) can be translated into the corresponding disease-related antigen (i.e., antigenic protein) after entering the phDC. The RNA can be single-stranded RNA, mRNA, self-amplifying RNA, circular RNA, and / or synthetic RNA. In a preferred embodiment, the RNA is mRNA. Optionally, the mRNA is self-amplifying mRNA. The basic components of an mRNA molecule typically include at least one coding region, a 5' untranslated region (UTR), a 3' UTR, a 5' cap, and a multi-A tail.

[0145] Therefore, in some embodiments, at least one disease-associated antigen (i.e., an antigenic protein) is encoded by mRNA, which is present in the composition with phDC (first aspect). In other words, this document provides a vaccine composition comprising phDC and at least one mRNA (containing a coding sequence) encoding at least one antigenic protein.

[0146] If a multivalent composition is desired, phDC can be in a composition having multiple mRNAs encoding more than one antigenic protein from the same or different pathogens or tumors, such as 2, 3, 4, 5, 6, 7, 8, 9, 10 or more antigenic proteins. Alternatively or additionally, polycistronic mRNAs can be engineered that can be translated into more than one antigenic protein.

[0147] Encoding area

[0148] In one embodiment, the mRNA encodes one or more antigenic proteins of the pathogenic particles listed above, or the mRNA encodes one or more antigenic proteins of the tumor or infectious disease-associated antigens listed above. In one embodiment, the mRNA encodes one or more antigenic proteins of the pathogens listed above. In one embodiment, the mRNA encodes one or more antigenic proteins of the viruses, bacteria, fungi, prions, or parasites listed above. In one embodiment, the mRNA encodes one or more antigenic proteins of the viruses (optionally coronaviruses) listed above. In one embodiment, the mRNA encodes one or more antigenic proteins of the pathogenic particles listed above. In one embodiment, the mRNA encodes one or more antigenic proteins of the tumors or cancers listed above. In one embodiment, the mRNA encodes one or more antigenic proteins of the TAAs listed above. In one embodiment, the at least one mRNA encodes one antigenic protein.

[0149] SARS-CoV-2 sequence

[0150] In one embodiment, the at least one mRNA encodes one or more antigenic proteins derived from SARS-CoV (optionally SARS-CoV-2). Antigenic proteins derived from SARS-CoV or SARS-CoV-2 include spike proteins, envelope proteins, nucleocapsid proteins, membrane proteins, and / or Orf1ab polyproteins. Therefore, in one embodiment, the antigenic protein is a fragment of a spike protein, envelope protein, nucleocapsid protein, membrane protein, and / or Orf1ab polyprotein, or each of the foregoing. In one embodiment, the fragment comprises at least 10, at least 50, at least 100, at least 200, at least 400, or at least 800 amino acid residues. RNA polynucleotides encoding structural proteins are considered to have preferred immunogenic activity due to their surface expression characteristics, and therefore may be most suitable for the compositions of the present invention. In a preferred embodiment, the mRNA encodes one or more antigenic proteins derived from the spike protein or nucleocapsid protein. Even more preferably, the mRNA encodes one or more antigenic proteins derived from the spike protein. The mRNA can encode antigenic proteins derived from the S1 or S2 subunits of the spike protein. Variants of the aforementioned SARS-CoV-2 protein are also explicitly included. For example, a first-generation spike protein variant is referred to as “S-2P” (Pallesen et al. 2017) and contains two proline substitutions at positions 986 and 987 (see, for example, Polack et al. 2020; Bos et al. 2020; Corbett et al. 2020; Wrapp et al. 2020). A second-generation spike construct, referred to as “HexaPro”, contains four additional proline residues at positions 817, 892, 899, and 942. HexaPro is expressed at higher levels than the wild-type spike protein or S-2P and exhibits improved stability relative to S-2P under cryogenic storage and multiple freeze-thaw cycles (Edwards et al. 2020).

[0151] In some embodiments, the mRNA comprises a sequence selected from the group consisting of or a portion thereof: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and / or SEQ ID NO: 5.

[0152] In some embodiments, the mRNA comprises a sequence selected from or a portion of a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and / or SEQ ID NO: 5, or a sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity with the selected sequence. In one embodiment, the mRNA comprises a sequence selected from or a portion of a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and / or SEQ ID NO: 5, or a sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity with the selected sequence, wherein the selected RNA sequence is modified with one or more (e.g., all) naturally occurring modified nucleosides as described in the "RNA Modification" section. In one embodiment, the naturally occurring modified nucleoside is 1-methylpseuuridine.

[0153] The above sequences are based on the reference genome from NCBI (accession number NC_045512.2) and are also shown in Table 1 below. The corresponding amino acid sequences are given in Table 2 below.

[0154] In some implementations, the mRNA comprises a sequence or a portion thereof corresponding to SEQ ID NO: 19 (Table 3) or a sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity with SEQ ID NO: 19.

[0155] Those skilled in the art who read this disclosure will further understand that this describes various mRNA constructs comprising a sequence encoding the full-length SARS-CoV-2 spike protein (e.g., embodiments comprising where such encoded SARS-CoV-2 spike protein may contain at least one or more amino acid substitutions (e.g., proline substitutions as described herein), and / or embodiments wherein the mRNA sequence is optimized, for example, for mammalian (e.g., human) subjects, and / or embodiments wherein the mRNA contains one or more chemical modifications).

[0156] HIV sequence

[0157] In one embodiment, the mRNA encodes one or more HIV-derived antigenic proteins. HIV-derived antigenic proteins include envelope proteins (env), group antigen polyproteins (gag), reverse transcriptases (pol), and / or negative factor proteins (nef). RNA polynucleotides encoding structural proteins are considered to have preferred immunogenic activity due to their surface expression characteristics, and therefore may be best suited for the compositions of the present invention. Variants of the aforementioned HIV proteins are also explicitly included.

[0158] In some embodiments, the mRNA comprises a sequence selected from the group consisting of or a portion thereof: SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 and / or SEQ ID NO: 14.

[0159] In some embodiments, the mRNA comprises a sequence selected from the group consisting of or a portion thereof: SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 and / or SEQ ID NO: 14, or a sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity with the selected sequence.

[0160] The above sequences are based on the reference genome from NCBI (accession number NC_AF033819, HIV-1) and are also shown in Table 1 below. The corresponding amino acid sequences are given in Table 2 below.

[0161] Those skilled in the art who read this disclosure will further understand that this describes various mRNA constructs comprising sequences encoding full-length HIV structural proteins (e.g., embodiments in which such encoded HIV structural proteins may contain at least one or more amino acid substitutions, and / or embodiments in which the mRNA sequence is optimized, for example, for mammalian (e.g., human) subjects, and / or embodiments in which the mRNA contains one or more chemical modifications).

[0162] Table 1

[0163]

[0164]

[0165]

[0166]

[0167]

[0168]

[0169]

[0170]

[0171]

[0172]

[0173]

[0174]

[0175]

[0176]

[0177]

[0178]

[0179] Table 2

[0180]

[0181]

[0182]

[0183]

[0184]

[0185] Table 3

[0186]

[0187]

[0188]

[0189] Tumor-associated antigen sequences

[0190] In one embodiment, at least one mRNA encodes one or more antigen proteins derived from tumors or cancer. In one embodiment, the mRNA encodes a TAA polypeptide, such as the amino acid sequence of TAA. TAAs include Her2, prostate stem cell antigen (PSCA), PSMA (prostate-specific membrane antigen), B cell maturation antigen (BCMA), ERK5, alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), cancer antigen-125 (CA-125), CA19-9, calretinin, MUC-1, epithelial membrane protein (EMA), epithelial tumor antigen (ETA), tyrosinase, melanoma-associated antigen (MAGE), CD34, CD45, CD99, CD117, chromogranin, cytokeratin, desmin, glial fibrillary acidic protein (GFAP), giant cystic disease fluid protein (GCDFP-15), HMB-45 antigen, and protein melan-A. (Melanoma antigen recognized by T lymphocytes; MART-1), myo-D1, muscle-specific actin (MSA), neurofilaments, neuron-specific enolase (NSE), placental alkaline phosphatase, synaptophysin, thyroglobulin, thyroid transcription factor-1, a dimer form of pyruvate kinase isoenzyme M2 (tumor M2-PK), aberrant ras protein, or aberrant p53 protein. In some embodiments, the TAA is CD19, CD22, CD27, CD30, CD70, GD2 (ganglioside G2), EGFRvIII (epidermal growth factor variant III), spermin 17 (Sp17), mesothelin, PAP (prostatic acid phosphatase), prostein, TARP (T cell receptor γ-alternating reading frame protein), Trp-p8, or STEAP1 (prostatic six-transmembrane epithelial antigen 1). In some embodiments, the TAA is a cancer / testis (CT) antigen, such as BAGE, CAGE, CTAGE, FATE, GAGE, HCA661, HOM-TES-85, MAGEA, MAGEB, MAGEC, NA88, NY-ESO-1, NY-SAR-35, OY-TES-1, SPANXB1, SPA17, SSX, SYCP1, or TPTE. In some other embodiments, the TAA or TSA is α-actin-4, Bage-1, BCR-ABL, BCR-ABL fusion protein, β-catenin, CA 125, CA 15-3 (CA 27).29\BCAA), CA 195, CA 242, CA-50, CAM43, Casp-8, cdc27, cdk4, cdkn2a, CEA, coa-1, dek-can fusion protein, EBNA, EF2, Epstein-Barr virus antigen, ETV6-AML1 fusion protein, HLA-A2, HLA-A11, hsp70-2, KIAAO205, Mart2, Mum-1, Mum-2 and Mum-3, neo-PAP, myosin class I, OS-9, pml-RARa fusion protein, PTPRK, K-ras, N-ras, triosephosphate isomerase, Gage 3, Gage 4, Gage 5, Gage 6, Gage 7. GnTV, Herv-K-mel, Lage-1, NA-88, NY-Eso-1 / Lage-2, SP17, SSX-2, TRP2-Int2, gpi00 (Pmel 17), tyrosinase, TRP-1, TRP 2. MAGE-1, MAGE-3, RAGE, GAGE-1, GAGE-2, p15(58), RAGE, SCP-i, Hom / Mel-40, PRAME, p53, H-Ras, HER-2 / neu, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, Human papillomavirus (HPV) antigens E6 and E7, TSP-180, MAGE-4, MAGE-5, MAGE-6, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72-4, CA 19-9, CA 72-4, CAM 17.1, NuMa, 13-catenin, Mum-1, p16, TAGE, PSMA (prostate-specific membrane antigen), B-cell maturation antigen (BCMA), CT7, telomerase, 43-9F, 5T4, 791Tgp72, 13HCG, BCA225, BTAA, CD68 / KPi, CO-O29, FGF-5, G250, Ga733 (EpCAM), HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB / 70K, NY-CO-1, RCAS1, SDCCAG16, TA-90, TAAL6, TAG72, TLP, TPS, integrin cv3 (CD61), prolactin, KRas (V-Ki-ras2 Kirsten rat sarcoma virus oncogene) or Ral-B.

[0191] The mRNA encoding the amino acid sequence of TAA can be a sequence that has at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity with the RNA sequence of the selected TAA.

[0192] RNA modification

[0193] In one implementation, the RNA (e.g., mRNA) is modified RNA, particularly stable mRNA. In some implementations, RNA (e.g., mRNA) may be modified to achieve maximum efficacy, such as improved lifespan in the cell, transcriptional efficiency, non-immunogenic properties (e.g., lack of Toll receptor induction), and / or structural mRNA stability.

[0194] In one embodiment, the RNA (e.g., mRNA) independently comprises at least one chemical modification. The chemical modification may be, for example, a modified nucleoside. In one embodiment, the chemical modification comprises a naturally occurring modified nucleoside. Naturally occurring modified nucleosides include 1-methyladenosine (mRNA). 1 A), N 6 -Methyladenosine (m 6 A), 2'-O-methyladenosine (Am), 5-methylcytidine (m) 5 C), 2'-O-methylcytidine (Cm), 2-thiocytidine (s) 2 C), N 4 - Acetylcytidine (ac) 4 C), 5-formylcytidine (f) 5 C), 2'-O-methylguanosine (Gm), inosine (I), pseudouridine (Ψ), 5-methyluridine (m) 5U), 2'-O-methyluridine (Um). 1-Methylpseudouridine (M1Ψ), 2-thiouridine (s2U), 4-thiouridine (s4U), 5-methoxyuridine (mo5U), 3-methyluridine (m3U). In one embodiment, the RNA (e.g., mRNA) comprises a modified nucleoside replacing at least one uridine. In one embodiment, the RNA comprises a modified nucleoside replacing each uridine. In one embodiment, the modified nucleoside is independently selected from pseudouridine, N1-methyl-pseudouridine, 5-methyl-uridine, and N1-ethylpseudouridine. In one embodiment, the modified nucleoside is N1-methylpseudouridine or N1-ethylpseudouridine. In one embodiment, the modified nucleoside is N1-methylpseudouridine. For example, N1-methylpseudouridine has been found to be superior to several other nucleoside modifications and combinations thereof in terms of translational ability. In some implementations, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the uridine nucleoside in the mRNA is chemically modified.

[0195] Alternatively or additionally to chemical modifications, RNA, particularly mRNA, sequences can be optimized. RNA sequence optimization includes, in particular, codon optimization, G / C content optimization, and optimization of structural elements (e.g., 5' cap, 5' UTR, 3' UTR, and multiple (A) tails).

[0196] In some embodiments, the amino acid sequence of at least one disease-associated antigen (or antigenic protein) is encoded by a coding sequence with codon-optimized and / or increased G / C content compared to the wild-type coding sequence. This also includes embodiments in which one or more regions of the coding sequence are codon-optimized and / or have increased G / C content compared to the corresponding region of the wild-type coding sequence. In one embodiment, the codon optimization and / or increased G / C content preferably does not alter the sequence of the encoded amino acid sequence. In some embodiments, the G / C content of the coding region of the RNA is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 55%, or even more compared to the G / C content of the coding region of the wild-type RNA.

[0197] In some embodiments, the RNA (e.g., mRNA) may contain one or more optimized structural elements. These structural elements include a 5' cap, a 5' UTR, a 3' UTR, and a multiple (A) tail. Thus, in one embodiment, the RNA (e.g., mRNA) contains a 5' untranslated region (UTR), a 3' UTR, a 5' cap, and / or a multiple (A) tail. In one embodiment, the RNA (e.g., mRNA) contains all of these elements. In some embodiments, the RNA contains a 5'-UTR and / or a 3'-UTR. In one embodiment, the RNA (e.g., mRNA) contains a cap. In some embodiments, the RNA (e.g., mRNA) contains a 3'-multiple (A) sequence. In one embodiment, the cap is a Cap1 structure or an m7GpppG cap. In one embodiment, the sequence of the 5' UTR and / or the 3' UTR is optimized. In some embodiments, the mRNA contains a 5' UTR or a 3' UTR derived from a gene whose sequence is different from that encoding the at least one disease-associated antigen (or antigenic protein), i.e., the UTR is a heterologous UTR. In some implementations, the 5' UTR and / or 3' UTR sequences can be derived from stable mRNAs (e.g., globin, actin, GAPDH, tubulin, histones, or citrate cycling enzymes) to increase mRNA stability. For example, as a 5'-UTR sequence, the 5'-UTR sequence of human α-globin mRNA can be used, optionally having an optimized "Kozak sequence" to increase translation efficiency. Alternatively, the 5' UTR sequence of human cytochrome mRNA, such as human cytochrome b-245α mRNA or cytochrome p4502E1 mRNA, can be used. As a 3'-UTR sequence, a combination of two sequence elements (FI element) derived from a "split N-terminal enhancer" (AES) mRNA (referred to as F) and mitochondrial-encoded 12S ribosomal RNA (referred to as I) can be used, placed between the coding sequence and the multiple (A) tail to ensure higher maximum protein levels and extended durability of the mRNA. Alternatively, the 3'-UTR can be the 3'-UTR of two duplicated human β-globin mRNAs.

[0198] In one embodiment, the multi-A sequence comprises at least 100 nucleotides. In another embodiment, the multi-A sequence comprises at least 150 nucleotides. In yet another embodiment, the multi-A sequence comprises at least 250 nucleotides. Furthermore, a multi-(A) tail of 110 nucleotides in length can be used, consisting of a 30-nucleotide extension followed by a 10-nucleotide linker sequence (of random nucleotides) and an additional 70 adenosine residues. This multi-(A) tail sequence enhances RNA stability and translation efficiency. In one embodiment, the multi-(A) tail is 300 to 800 nucleotides in length.

[0199] Furthermore, the secretory signal peptide (sec) can preferably be fused to the antigen coding region in a manner that translates sec into an N-terminal tag. A sequence encoding a short linker peptide, typically used in fusion proteins and primarily composed of the amino acids glycine (G) and serine (S), can be used as a GS linker (glycine-serine linker). In other embodiments, the RNA (e.g., mRNA) can have one or more AU-rich sequences removed. These sequences, sometimes called AURES, are unstable sequences found in the 3' UTR. AURES can be removed from the RNA (e.g., mRNA). Alternatively, AURES can be retained in the RNA (e.g., mRNA).

[0200] lipid nanoparticles

[0201] At least one disease-associated antigen (e.g., at least one mRNA encoding at least one antigenic protein) can be encapsulated in lipid nanoparticles (LNPs). For example, if the at least one disease-associated antigen is an antigenic protein encoded by RNA (e.g., mRNA), then the RNA can be encapsulated in lipid nanoparticles for delivery to the phDC of the present invention. The encapsulated RNA (e.g., mRNA) is combined with the phDC.

[0202] LNPs can contain 3, 4, or 5 types of lipids. LNPs with three types of lipids include: (1) ionizable lipids, (2) PEGylated lipids, and (3) cholesterol-based lipids. LNPs with four types of lipids include: (1) ionizable lipids, (2) PEGylated lipids, (3) cholesterol-based lipids, and (4) accessory lipids. LNPs with five types of lipids include: (1) ionizable lipids, (2) PEGylated lipids, (3) cholesterol-based lipids, (4) accessory lipids, and (5) DSPE-PEG-maleimide or DSPN-PEG-azide. DSPE-PEG-maleimide or DSPN-PEG-azide allows for the addition of ligands for targeted delivery.

[0203] (1) Ionizable lipids

[0204] Ionizable lipids facilitate mRNA encapsulation and can be cationic lipids. Cationic lipids provide a positively charged environment at low pH to facilitate the efficient encapsulation of negatively charged mRNA. In one embodiment, the cationic lipid is cKK-E12 ((3,6-bis(4-(bis(2-hydroxydodecyl)amino)butyl)piperazine-2,5-dione); see Dong et al., PNAS (2014) 111(11):3955-60). Other cationic lipids that can be used include those described in Dong et al., 2014.

[0205] (2) PEG-modified lipids

[0206] PEG-modified lipid components provide control over the particle size and stability of nanoparticles. The addition of such components prevents complex aggregation and provides a means to increase lifetime and enhance LNP delivery to phDC.

[0207] The anticipated PEGylated lipids include, but are not limited to, polyethylene glycol (PEG) chains with a length of up to 5 kDa covalently attached to lipids having an alkyl chain having a length of C6-C20 (e.g., C8, C10, C12, C14, C16, or C18), such as derivatized ceramides (e.g., N-octanoyl-sphingosine-1-[succinyl(methoxypolyethylene glycol)] (C8 PEG ceramide)). In some embodiments, the PEGylated lipid is 1,2-dimyristoyl-rac-glycero-3-methoxy polyethylene glycol (DMG-PEG); 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol (DSPE-PEG); 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol (DLPE-PEG); or 1,2-distearayl-rac-glycero-polyethylene glycol (DSG-PEG). In particularly exemplary embodiments, the PEG has a high molecular weight, for example, 2000-2400 g / mol. In some embodiments, the PEG is PEG2000, also known as PEG-2K. In some implementations, the PEG-modified lipids are DMG-PEG2000, DSPE-PEG2000, DLPE-PEG2000, DSG-PEG2000, or C8 PEG2000.

[0208] (3) Cholesterol-based lipids

[0209] The cholesterol component provides stability to the lipid bilayer structure within the nanoparticles. In some embodiments, the LNP comprises one or more cholesterol-based lipids. Suitable cholesterol-based lipids include, for example, N,N-dimethyl-N-ethylformamido-cholesterol, 1,4-bis(3-N-oleoylaminopropyl)piperazine, imidazole cholesterol ester, β-sitosterol, fucosterol, stigmasterol, and other modified forms of cholesterol. In some embodiments, the cholesterol-based lipid used in the LNP is cholesterol.

[0210] (4) Lipid-assisted

[0211] The assisting lipid enhances the structural stability of the LNP and facilitates endosome escape. It improves the uptake and release of RNA (e.g., mRNA) loaders. The cross-presentation capacity of DCs may be limited by non-specific degradation during endosome maturation. Therefore, in some embodiments, LNPs that provide better endosome escape may be useful (e.g., if the antigen form is, for example, a protein, peptide, or mRNA). In one embodiment, the assisting lipid is a non-cationic lipid. In some embodiments, the assisting lipid is a zwitterionic lipid with fusion-promoting properties for enhancing load uptake and release. Examples of accessory lipids are 1,2-dioleoyl-SN-glycero-3-phosphate ethanolamine (DOPE); 1,2-distearyl-sn-glycero-3-phosphate choline (DSPC); 1,2-dioleoyl-sn-glycero-3-phosphate-L-serine (DOPS); 1,2-dielaidoyl-sn-glycero-3-phosphate choline (DPOC), dipalmitoylphosphatidylcholine (DPPC), 1,2-dilauroyl-sn-glycero-3-phosphate choline (DLPC), 1,2-distearoylphosphatidylethanolamine (DSPE), and 1,2-dilauroyl-sn-glycero-3-phosphate ethanolamine (DLPE).

[0212] In one embodiment, the lipid nanoparticles comprise cKK-E12, cholesterol, C14-PEG 2000-PE, and DOPE. In some embodiments, phDCs can be loaded with different antigens to generate multivalent antigen-specific phDCs. For example, if the antigenic protein is encoded by mRNA encapsulated in an LNP, then the LNP can carry mRNAs encoding more than one antigenic protein (such as 2, 3, 4, 5, 6, 7, 8, 9, 10, or more antigenic proteins) from the same or different pathogens or from the same or different tumors. For example, the LNP can carry multiple mRNA molecules, each encoding a different antigenic protein. The LNP can also carry polycistronic mRNAs that can be translated into more than one antigenic protein. If the LNP carries different mRNA molecules, then typically multiple copies of each mRNA molecule will be present.

[0213] Molar ratio of lipid components

[0214] The specific molar ratios of the above components may be important for the effectiveness of the LNP. The molar ratio of cationic lipids, PEGylated lipids, cholesterol-based lipids, and cofactor lipids is A:B:C:D, where A+B+C+D=100%. In some embodiments, the molar ratio of cationic lipids to total lipids in the LNP (i.e., A) is 35-50%, optionally 35-45%. In some embodiments, the molar ratio of the PEGylated lipid component to total lipids (i.e., B) is 0.25-2.75%, optionally about 1.5%. In some embodiments, the molar ratio of cholesterol-based lipids to total lipids (i.e., C) is 20-46.5%. In some embodiments, the molar ratio of cholesterol-based lipids to total lipids (i.e., C) is about 46.5%. In another embodiment, the molar ratio of cholesterol-based lipids to total lipids (i.e., C) is about 38.5%. In some embodiments, the molar ratio (i.e., D) of the coenzyme lipids relative to the total lipids is about 10-35%, such as 10-25%, or 16-35% (e.g., 16-32%, such as 16%). In some embodiments, the molar ratio (i.e., D) of the coenzyme lipids relative to the total lipids is about 10%. In another embodiment, the molar ratio (i.e., D) of the coenzyme lipids relative to the total lipids is about 16%. In one embodiment, the component ratio is 35: 2.5: 46: 16 (A: B: C: D). In another embodiment, the component ratio is 50:38.5:1.5:10 (A: B: C: D).

[0215] In one embodiment, the lipid nanoparticles comprise cKK-E12, cholesterol, C14-PEG 2000-PE, and DOPE. In another embodiment, the lipid nanoparticles comprise cKK-E12, cholesterol, C14-PEG 2000-PE, and DOPE in a ratio of 35:46.5:2.5:16.

[0216] In some embodiments, the lipid nanoparticles comprise SM102, cholesterol, DMG-PEG-2K, and DSPC. In one embodiment, the lipid nanoparticles comprise SM102, cholesterol, DMG-PEG-2K, and DSPC in a ratio of 50:38.5:1.5:10.

[0217] In some embodiments, the (PEGylated lipid + cholesterol) component has the same molar amount as the cofactor lipid. In some embodiments, the LNP contains a molar ratio of cationic lipid to cofactor lipid greater than 1. To calculate the actual amount of each lipid to be incorporated into the LNP formulation, the molar amount of the cationic lipid is first determined based on the desired N / P ratio, where N is the number of nitrogen atoms in the cationic lipid and P is the number of phosphate groups in the RNA (e.g., mRNA) to be transported by the LNP. Then, the molar amount of each other lipid is calculated based on the molar amount of the cationic lipid and the selected molar ratio. These molar amounts are then converted to weight using the molecular weight of each lipid.

[0218] Lipid nanoparticles containing antigens (e.g., in the form of RNA, particularly mRNA encoding at least one antigenic protein) can be supplied frozen. For example, it could be useful if phDC containing antigens (e.g., in the form of RNA, particularly mRNA encoding at least one antigenic protein) and lipid nanoparticles were supplied as a kit.

[0219] Size and amount of lipid nanoparticles

[0220] Suitable LNPs can be prepared in a variety of sizes. In some embodiments, most purified LNPs, i.e., LNPs larger than about 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 98%, or 99%, have a size of about 50 to 200 nm (e.g., about 145 nm, about 140 nm, about 135 nm, about 130 nm, about 125 nm, about 120 nm, about 115 nm, about 110 nm, about 105 nm, about 100 nm, about 95 nm, about 90 nm, about 85 nm, or about 80 nm). In some embodiments, substantially all (e.g., greater than 80% or 90%) of the purified lipid nanoparticles have a size of about 70 nm to 200 nm (e.g., about 145 nm, about 140 nm, about 135 nm, about 130 nm, about 125 nm, about 120 nm, about 115 nm, about 110 nm, about 105 nm, about 100 nm, about 95 nm, about 90 nm, about 85 nm, or about 80 nm). In some embodiments, greater than 70%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% of the LNPs in the compositions of the present invention have a size ranging from about 85 to 100 nm (e.g., about 45-85 nm, about 50-80 nm, about 55-75 nm, about 60-70 nm) or about 50-70 nm (e.g., 55-65 nm). Smaller sizes are particularly suitable for inhalation delivery via nebulization. In some embodiments, phDC is combined with at least about 1 μg, 2 μg, 3 μg, 4 μg, 5 μg, 10 μg, or 20 μg of encapsulated RNA, particularly mRNA. In some embodiments, phDC is combined with about 10 μg of encapsulated RNA, particularly mRNA. In some embodiments, phDC is combined with about 20 μg of encapsulated RNA, particularly mRNA.

[0221] lipoplex

[0222] At least one disease-associated antigen (e.g., at least one mRNA encoding at least one antigenic protein) may be contained in a lipoplex. Mixing RNA (e.g., mRNA) with positively charged liposomes results in the formation of lipoplex particles through spontaneous self-assembly. Liposomes typically contain at least two components: cationic lipids and neutral lipids. Lipoplexes have been extensively described in the art, see, for example, Nanomedicine: Nanotechnology, Biology and Medicine, 2009. In one embodiment, the neutral lipid is an accessory lipid as defined above. In an exemplary embodiment, the cationic lipid is DOTMA, and the neutral lipid is DOPE.

[0223] In some embodiments, the molar ratio of at least one cationic lipid to at least one neutral lipid is about 10:0 to about 1:9, about 4:1 to about 1:2, or about 3:1 to about 1:1. In specific embodiments, this molar ratio may be about 3:1, about 2.75:1, about 2.5:1, about 2.25:1, about 2:1, about 1.75:1, about 1.5:1, about 1.25:1, or about 1:1. In an exemplary embodiment, the molar ratio of at least one cationic lipid to at least one additional lipid is about 2:1. The RNA lipoplex particles described herein have an average diameter ranging from about 200 nm to about 1000 nm, about 200 nm to about 800 nm, about 250 nm to about 700 nm, about 400 nm to about 600 nm, about 300 nm to about 500 nm, or about 350 nm to about 400 nm in one embodiment. In specific embodiments, the RNA lipoplex particles have an average diameter of about 200 nm, about 225 nm, about 250 nm, about 275 nm, about 300 nm, about 325 nm, about 350 nm, about 375 nm, about 400 nm, about 425 nm, about 450 nm, about 475 nm, about 500 nm, about 525 nm, about 550 nm, about 575 nm, about 600 nm, about 625 nm, about 650 nm, or about 700 nm. In one embodiment, the RNA lipoplex particles have an average diameter ranging from about 250 nm to about 700 nm. In another embodiment, the RNA lipoplex particles have an average diameter ranging from about 300 nm to about 500 nm. In an exemplary embodiment, the RNA lipoplex particles have an average diameter of about 400 nm.

[0224] In one embodiment, the (mRNA) lipoplex particle comprises at least one cationic lipid and at least one neutral lipid. In one embodiment, the at least one cationic lipid comprises 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA) and / or 1,2-dioleoyl-3-trimethylammonium propane (DOTAP). In one embodiment, the at least one neutral lipid comprises 1,2-di-(9Z-octadecenyl)-sn-glycerol-3-phosphate ethanolamine (DOPE), cholesterol (Chol), and / or 1,2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC). In one embodiment, the at least one cationic lipid comprises 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), and the at least one neutral lipid comprises 1,2-di-(9Z-octadecenyl)-sn-glycerol-3-phosphate ethanolamine (DOPE). In one embodiment, the lipoplex particle comprises 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA) and 1,2-di-(9Z-octadecenyl)-sn-glycerol-3-phosphate ethanolamine (DOPE). Targeted spleen RNA lipoplex particles are described in WO 2013 / 143683, which is incorporated herein by reference. It has been found that lipoplex particles with a net negative charge can be used to preferentially target spleen tissue or spleen cells, such as antigen-presenting cells, particularly dendritic cells.

[0225] oligomers, polymers, or lipids containing an oligomeric (alkyleneamine) moiety

[0226] At least one antigen (e.g., at least one mRNA encoding at least one antigen) may be contained in an oligomer, polymer, or lipid comprising an oligomeric (alkyleneamine) moiety (e.g., a characteristic oligomeric (alkyleneamine) moiety as described in PCT / EP2014 / 063756). Specifically, at least one antigen (e.g., at least one mRNA encoding at least one antigen) may be contained in an oligomer, polymer, or lipid as described in PCT / EP2014 / 063756. A key characteristic of oligomers, polymers, or lipids comprising an oligomeric (alkyleneamine) moiety is that they contain a common structural entity of formula (I):

[0227]

[0228] Such oligomers, polymers, or lipids containing an oligomeric (alkyleneamine) moiety can be selected from:

[0229] a) Oligomers or polymers containing multiple groups of formula (II) as side chains and / or as terminal groups:

[0230]

[0231] The variables are a, b, p, m, n, and R. 2 To R 6 Each of the multiple such groups in formula (II) is defined independently as follows:

[0232] a is 1 and b is an integer from 2 to 4; or a is an integer from 2 to 4 and b is 1.

[0233] p is 1 or 2

[0234] m is 1 or 2; n is 0 or 1 and m + n ≥ 2; and

[0235] R 2 To R 5 They are independently selected from hydrogen; the group –CH2-CH(OH)-R 7 -CH(R) 7 -CH2-OH, -CH2-CH2-(C=O)-OR 7 - CH2-CH2-(C=O)-NH-R 7 or -CH2-R 7 , where R 7 Selected from C3-C18 alkyl or C3-C18 alkenyl with one C-C double bond; protecting group of amino group; -C(NH)-NH2; poly(ethylene glycol) chain;

[0236] R 6 Selected from hydrogen; group –CH2-CH(OH)-R 7 -CH(R) 7 -CH2-OH, -CH2-CH2-(C=O)-OR 7 -CH2-CH2-(C=O)-NH-R 7 or -CH2-R 7 , where R 7 Selected from C3-C18 alkyl or C3-C18 alkenyl having a C-C double bond; a protecting group of an amino group; -C(NH)-NH2; a poly(ethylene glycol) chain; and an acceptor ligand, wherein one or more of the nitrogen atoms shown in formula (II) may be protonated to provide a cationic group of formula (II);

[0237] b) Oligomers or polymers containing multiple groups of formula (III) as repeating units:

[0238]

[0239] The variables are a, b, p, m, n, and R. 2 To R 5 Each of the multiple such groups, specifically the group of formula (III), is defined independently as follows:

[0240] a is 1 and b is an integer from 2 to 4; or a is an integer from 2 to 4 and b is 1.

[0241] p is 1 or 2

[0242] m is 1 or 2; n is 0 or 1 and m + n ≥ 2; and

[0243] R 2 To R 5 They are independently selected from hydrogen; the group –CH2-CH(OH)-R 7 -CH(R) 7 -CH2-OH, -CH2-CH2-(C=O)-OR 7 - CH2-CH2-(C=O)-NH-R 7 or -CH2-R 7 , where R 7 Selected from C3-C18 alkyl or C3-C18 alkenyl with one C-C double bond; protecting group of amino group; -C(NH)-NH2; and poly(ethylene glycol) chain;

[0244] Furthermore, one or more of the nitrogen atoms shown in formula (III) may be protonated to provide a cationic group of formula (III); and

[0245] c) Lipids having the structure of formula (IV):

[0246]

[0247] The variables are a, b, p, m, n, and R. 2 To R 6 The definition is as follows:

[0248] a is 1 and b is an integer from 2 to 4; or a is an integer from 2 to 4 and b is 1.

[0249] p is 1 or 2

[0250] m is 1 or 2; n is 0 or 1 and m + n ≥ 2; and

[0251] R 2 To R 6 They are independently selected from hydrogen; the group –CH2-CH(OH)-R 7 -CH(R) 7 -CH2-OH, -CH2-CH2-(C=O)-OR 7 - CH2-CH2-(C=O)-NH-R 7 or -CH2-R 7 , where R 7Selected from C3-C18 alkyl or C3-C18 alkenyl groups having one C-C double bond; protecting groups of amino groups; -C(NH)-NH2; poly(ethylene glycol) chains; and acceptor ligands.

[0252] The condition is R 1 To R 6 At least two residues in it are the group –CH2-CH(OH)-R 7 -CH(R) 7 -CH2-OH, -CH2-CH2-(C=O)-OR 7 - CH2-CH2-(C=O)-NH-R 7 or -CH2-R 7 , where R 7 Selected from C3-C18 alkyl or C3-C18 alkenyl having a C-C double bond; and wherein one or more nitrogen atoms shown in formula (IV) may be protonated to provide a cationic lipid of formula (IV).

[0253] Preferably, the oligomer, polymer, or lipid comprising the oligomeric (alkyleneamine) moiety is selected from a) and b), wherein

[0254] a) is an oligomer or polymer containing groups of multiple formulas (IIa) as side chains and / or as terminal groups:

[0255]

[0256] Where a, b, m, n and R 2 To R 6 As defined above, and wherein one or more of the nitrogen atoms shown in formula (IIa) can be protonated to provide a cationic oligomer or polymer structure; and

[0257] b) Oligomers or polymers containing multiple groups of formula (IIIa) as repeating units:

[0258]

[0259] Where a, b, m, n and R 2 To R 5 As defined above, and wherein one or more of the nitrogen atoms shown in formula (IIIa) may be protonated to provide a cationic oligomer or polymer structure.

[0260] Furthermore, lipids containing oligomeric (alkyleneamine) moieties can be selected from lipids having the structure of formula (IVa):

[0261]

[0262] Where a, b, m, n and R1 To R 6 As defined above, and in which one or more of the nitrogen atoms shown in formula (IVa) can be protonated to provide cationic lipids.

[0263] For such oligomers, polymers, or lipids containing an oligomeric (alkyleneamine) moiety, n may be 1 in formula (II), (IIa), (III), (IIIa), (IV), or (IVa); or m may be 1 and n may be 1.

[0264] Furthermore, for such oligomers, polymers, or lipids containing an oligomeric (alkyleneamine) moiety, in formulas (II), (IIa), (III), (IIIa), (IV), or (IVa), a may be 1 and b may be 2; or a may be 2 and b may be 1.

[0265] A non-limiting example of such oligomers, polymers, or lipids containing an oligomeric (alkyleneamine) moiety is a cationic lipid prepared by mixing 100 mg of N,N'-bis(2-aminoethyl)-1,3-propanediamine (0.623 mmol) with 575.07 mg of 1,2-epoxydodecane (3.12 mmol, (N-1) equivalents, where N is 2x the amount of primary amine plus 1x the amount of secondary amine per oligomeric (alkyleneamine) moiety) and mixing at 80°C with constant shaking for 96 h. Such oligomers, polymers, or lipids are also referred to as lipids "C12-(2-3-2)". Oligomers, polymers, or lipids containing an oligomeric (alkyleneamine) moiety, especially polymers, can be copolymers, particularly statistical copolymers. Such copolymers can be copolymers containing a statistical / random arrangement of repeating alkylene units of alternating lengths (e.g., in contrast to less preferred polymers containing non-alternating lengths of repeating alkylene units in a similar arrangement). The copolymer can be a cationic (e.g., protonated) copolymer. Copolymers to be used are known in the art and are described, for example, in EP 14 19 9439.2, WO 01 / 00708, EP-A11 198 489, and CA-A1 2,377,207.

[0266] In particular, the copolymer can be a statistical copolymer comprising a plurality of repeating units (a) independently selected from repeating units of formulas (a1) and (a2):

[0267]

[0268] And multiple repeating units (b) independently selected from repeating units of the following formulas (b1) to (b4):

[0269]

[0270]

[0271]

[0272]

[0273] The molar ratio of the total number of repeating units (a) to the total number of repeating units (b) is in the range of 0.7 / 1.0 to 1.0 / 0.7, and one or more nitrogen atoms of the repeating units (a) and / or (b) contained in the copolymer can be protonated to provide a cationic copolymer.

[0274] The copolymer can be a statistical copolymer, wherein any repeating unit (a) and any repeating unit (b) are statistically distributed in the copolymer macromolecule. It is typically obtained by copolymerization of a mixture of monomers that produce repeating units (a) during polymerization and monomers that produce repeating units (b) during polymerization. Preferably, the copolymer is a random copolymer, wherein any repeating units (a) and any repeating units (b) are randomly distributed in the polymer macromolecule. Such copolymers can be linear, branched, or dendritic copolymers. As those skilled in the art will understand, repeating units of formula (a1), (b1), or (b3) having two valences (i.e., open bonds with adjacent units) result in a linear propagation of the copolymer structure. Thus, a linear copolymer may contain repeating units of formula (a1) and one or more types of repeating units of formulas (b1) and (b3), but not repeating units of formulas (a2), (b2), or (b4). As will be further understood, the presence of repeating units of formula (a2), (b2), or (b4) having three valences provides branching points in the copolymer structure. Therefore, branched copolymers comprise one or more types of repeating units of formula (a2), (b2), and (b4), and may further comprise one or more types of repeating units of formula (a1), (b1), and (b3). Such copolymers may comprise a plurality of repeating units (a) independently selected from repeating units of formula (a1) and (a2) as defined above, and a plurality of repeating units (b) independently selected from repeating units of formulas (b1) to (b4) as defined above. Preferably, copolymers comprise a plurality of repeating units (a) and a plurality of repeating units (b), wherein the repeating unit (a) is independently selected from repeating units of formula (a1) and (a2) as defined above, and the repeating unit (b) is independently selected from repeating units of formula (b1) and (b2) as defined above. Preferably, such copolymers are branched copolymers comprising one or more types of repeating units selected from repeating units (a2), (b2), and (b4), and optionally further comprising one or more types of repeating units of formula (a1), (b1), and (b3), and particularly comprising repeating units of formula (a2) and one or more types of repeating units of formula (b2) and (b4), and optionally further comprising one or more types of repeating units of formula (a1), (b1), and (b3). Therefore, according to the above, a more preferred copolymer is a branched copolymer comprising repeating units of formula (a2) and repeating units of formula (b2), and optionally further comprising one or more types of repeating units of formula (a1) and (b1). In the copolymer, the total number of repeating units (a) and repeating units (b) is typically 20 or more, preferably 50 or more, and more preferably 100 or more. Typically, the total number of repeating units (a) and repeating units (b) is 10,000 or less, preferably 5,000 or less, and more preferably 1,000 or less.Furthermore, for copolymers, it is preferred that repeating units (a) and (b) constitute 80 mol% or more of all repeating units in the copolymer, more preferably 90 mol% or more. Further preferred are copolymers in which repeating units (a) selected from (a1) and (a2) and repeating units (b) selected from (b1) and (b2) constitute 80 mol% or more of all repeating units in the copolymer, more preferably 90 mol% or more. Most preferably, all repeating units in the copolymer are repeating units (a) or (b), particularly all repeating units in the copolymer are repeating units (a) selected from (a1) and (a2) or repeating units (b) selected from (b1) and (b2). The weight-average molecular weight of the copolymer (e.g., measured relative to a linear poly(ethylene oxide) standard via size exclusion chromatography) typically ranges from 1,000 to 500,000 Da, preferably from 2,500 to 250,000 Da, and more preferably from 5,000 to below 50,000 Da. The terminal groups of such copolymers typically contain one or more types of groups (c), which are independently selected from groups of formulas (c1) to (c3), preferably groups of formulas (c1) and (c2):

[0275]

[0276] Preferably, the terminal groups in the copolymer consist of one or more types of groups (c), which are independently selected from groups of formulas (c1) to (c3), preferably groups of formulas (c1) and (c2). As those skilled in the art will understand, the number of terminal groups depends on the structure of the copolymer. While linear copolymers have only two ends, branched copolymers, particularly dendritic copolymers, contain a greater number of terminal groups. As will be further understood, one or more nitrogen atoms of the terminal groups (c) contained in the copolymer may also be protonated to provide a cationic copolymer. In the copolymer, the molar ratio of the total number of repeating units (a) to the total number of repeating units (b) is in the range of 0.7 / 1.0 to 1.0 / 0.7, and preferably in the range of 0.8 / 1.0 to 1.0 / 0.8. This molar ratio can be determined, for example, by NMR. Therefore, it should be understood that this ratio is generally determined for multiple macromolecules of the copolymer and generally represents the total ratio of the total number of repeating units (a) to the total number of repeating units (b) in multiple macromolecules. As described above, one or more nitrogen atoms of the copolymer can be protonated to produce a copolymer in cationic form (typically oligocationic or polycationic). It should be understood that primary, secondary, or tertiary amine groups in repeating unit (a) or (b) or in the terminal group (c) can act as proton acceptors, particularly in water and aqueous solutions (including physiological fluids). Therefore, such copolymers typically have a total positive charge in aqueous solutions with a pH below 7.5. As described herein, an aqueous solution is one in which the solvent comprises 50% (vol. / vol.) or more, preferably 80% or 90% or more, and most preferably 100% water. Furthermore, if the composition is in contact with physiological fluids (including, for example, blood and pulmonary fluids) having a pH below 7.5, these typically contain repeating units (a) and (b) in which the nitrogen atoms are protonated. The pK value of the copolymer used in the composition can be determined by acid-base titration using an automated pK titrator. The net charge at a given pH can then be calculated, for example, from the Henderson-Hasselbach equation. Any charge can be shared by several basic centers and is not necessarily attributed to a single point. Typically, in solutions at physiological pH, copolymers used in compositions contain repeating units with protonated amino groups and repeating units with unprotonated amino groups. However, as those skilled in the art will understand, copolymers can also be provided as dry salts containing a cationic form of the copolymer. It will be further understood that the counterion (anion) for the positive charge of the protonated amino groups in compositions containing copolymers and nucleic acids (particularly mRNA) is typically provided by the anionic portion contained in the nucleic acid. If the positively charged groups are in excess compared to the anionic portion in the nucleic acid, the positive charge can be balanced by other anions (particularly those commonly encountered in physiological fluids, such as Cl or HCO3-).Consistent with the above, the preferred copolymer is a random copolymer, wherein 80 mol% or more of all repeating units, more preferably all repeating units are formed from a plurality of repeating units (a) independently selected from repeating units of formulas (a1) and (a2).

[0277]

[0278]

[0279] And multiple repeating units (b) independently selected from repeating units of the following formulas (b1) and (b2):

[0280]

[0281]

[0282] The molar ratio of the total number of repeating units (a) to the total number of repeating units (b) is in the range of 0.7 / 1.0 to 1.0 / 0.7, more preferably in the range of 0.8 / 1.0 to 1.0 / 0.8; 35 wherein the terminal groups of the copolymer are formed by groups (c) independently selected from formulas (c1) and (c2):

[0283] Furthermore, one or more nitrogen atoms of the repeating units (a) and / or (b) and / or terminal groups (c) contained in the copolymer can be protonated to provide a cationic copolymer. More preferably, the copolymer is a branched copolymer comprising units (a2) and (b2), optionally together with units (a1) and / or (b1). The preparation of the copolymer is described in EP 4 223 306 A2, which is incorporated herein by reference in its entirety. In principle, lipids are preferred nanoparticles, particularly compared to oligomers, and even more particularly compared to polymers.

[0284] At least one disease-associated antigen may be an antigenic protein encoded by RNA (e.g., mRNA, modified or unmodified), wherein the RNA may be encapsulated in lipid nanoparticles (LNPs) for delivery to the phDC of the present invention. The encapsulated RNA (e.g., mRNA) is combined with the phDC. The uptake of RNA (e.g., mRNA, optionally modified mRNA) by the phDC can be monitored or evaluated by means known in the art. These means include, but are not limited to, fluorescent LNPs, luminescent LNPs, radioactive LNPs, or cascade reactions or detection of expressed antigenic proteins encoded by RNA encapsulated in LNPs. In one embodiment, the transfection efficacy of phDCs (e.g., human phDCs) is evaluated by co-incubating phDCs generated from isolated PBMCs with mRNA encoding GPI-anchored nanoluciferase (NLuc) (LNP[NLuc]) and observing the luminescence signal. In some embodiments, the spatiotemporal distribution of the LNPs is monitored.

[0285] III. Pharmaceutical compositions or vaccine compositions

[0286] This invention provides pharmaceutical or vaccine compositions comprising phDCs and at least one RNA (e.g., mRNA) containing a coding sequence encoding at least one antigenic protein. The terms pharmaceutical composition, vaccine, and vaccine composition are used synonymously herein. The at least one mRNA can be any form described in the first aspect and detailed description of the invention. In one embodiment, the pharmaceutical composition of the invention contains at least 1, at least 10, at least 100, at least 1000, or at least 1 x 10^6 molecules. 4 At least 1 x 10 5 One or at least 1 x 10 6 The number of phDCs can be estimated, for example, from the volume of whole blood, which is converted to apheresed blood components and passed through a flow chamber (e.g., a plate, bag, or mixture) or directly through a flow chamber (e.g., a plate, bag, or mixture). At least one mRNA encapsulated in nanoparticles (e.g., LNPs) can be provided, wherein the pharmaceutical composition contains at least about 0.1 μg, 0.5 μg, 5 μg, 10 μg, 100 μg, 500 μg, or 1000 μg of encapsulated RNA, particularly mRNA. The vaccine composition provides a pharmaceutically effective amount of antigen-specific phDCs. In some embodiments, the vaccine composition according to the invention contains at least 1, at least 10, at least 100, at least 1000, or at least 1 x 10^10 phDCs. 4 At least 1 x 10 5 One or at least 1 x 10 6Each antigen-specific phDC. In terms of dose savings, the dose of RNA-containing, particularly mRNA-containing nanoparticles (e.g., LNPs) can be reduced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% compared to the dose used for mRNA vaccines that do not contain dendritic cells (e.g., LNPs). Typical doses of mRNA vaccines can be from 0.1 to 100 μg, for example, 1 μg, 5 μg, 10 μg, 30 μg, or 50 μg. In one particular embodiment, the phDC of the present invention is combined with up to 4.5 μg of RNA-containing, particularly mRNA-containing nanoparticles (e.g., LNPs).

[0287] The pharmaceutical or vaccine compositions of the present invention optionally comprise a pharmaceutically acceptable carrier and / or diluent. Additionally, the pharmaceutical or vaccine compositions may comprise adjuvants and / or immunomodulators to enhance the activity of the pharmaceutical or vaccine composition and the subject's response. Such adjuvants and / or immunomodulators are understood by those skilled in the art and are readily described in available publications. The pharmaceutical compositions of the present invention may contain one or more T-cell activators. As contemplated herein, and depending on the type of vaccine produced, vaccine production can be scaled up, if desired, by culturing cells in a bioreactor or fermenter or other such container or apparatus suitable for large-scale cell growth. Other steps in vaccine preparation can be personalized to meet the requirements of a specific vaccine. Such additional steps will be understood by those skilled in the art.

[0288] In all embodiments of the present invention, the pharmaceutical or vaccine compositions are used to prevent diseases such as cancer and / or infectious diseases. The pharmaceutical or vaccine compositions of the present invention can induce antigen-specific T cell and / or high-titer antibody responses, thereby triggering an immune response against or responsive to a disease (e.g., cancer or infectious disease) associated with the expression of the corresponding antigen. In some embodiments, the induced or triggered immune response can be a cellular immune response, a humoral immune response, or both. In some embodiments, the induced or triggered cellular immune response includes inducing or secreting interferon-γ (IFN-γ) and / or tumor necrosis factor α (TNF-α). In certain embodiments, the pharmaceutical composition or vaccine composition acts by one or more of the following: (i) inducing humoral immunity to generate antibodies via a B-cell response; (ii) increasing cytotoxic T lymphocytes such as CD8+ (CTL) to attack and kill pathogenic granules expressing corresponding antigens; (iii) increasing T helper cell responses; (iv) increasing inflammatory responses via IFN-γ and / or TNF-α; (v) increasing natural killer cell responses; and (vi) increasing central memory T cells and stem cell-like T cell subsets. In one embodiment, the pharmaceutical composition or vaccine composition acts by all of the foregoing. In one embodiment, the pharmaceutical composition or vaccine composition acts by at least (ii), (v), and (vi). In one embodiment, the pharmaceutical composition or vaccine composition acts by at least (vi).

[0289] In other embodiments, the induced or induced immune response may reduce or suppress one or more immunosuppressive factors that promote disease onset. In the case of cancer, the induced or induced immune response may reduce or suppress one or more immunosuppressive factors that promote the growth of tumors or cancers expressing antigens, such as, but not limited to, downregulating MHC-presenting factors, upregulating antigen-specific regulatory T cells, PD-L1, FasL, cytokines such as IL-10 and TFG-β, tumor-associated macrophages, tumor-associated fibroblasts, soluble factors produced by immunosuppressive cells, CTLA-4, PD-1, MDSC, MCP-1, and immune checkpoint molecules. The pharmaceutical or vaccine compositions may be combined with other pharmaceutical products or compounds. The pharmaceutical or vaccine compositions of the present invention may be used to prevent diseases such as cancer or infectious diseases in an individual, or may be used in individuals who may be at risk of developing a specific disease such as cancer or acquiring an infection. The pharmaceutical or vaccine compositions of the present invention may be particularly useful for subjects with compromised immune systems.

[0290] In some embodiments, subjects vaccinated with the vaccine composition of the present invention experience fewer adverse effects after vaccination compared to subjects vaccinated with a vaccine (particularly an mRNA vaccine) that does not contain dendritic cells (control subjects). Adverse events can be graded, for example, according to FDA recommendations (US Department of Health and Human Services, Administration, F. and D. & Research, C. for BE and. Toxicity grading scale for healthy adult and adolescent volunteers enrolled in preventive vaccine clinical trials. (2007)). In one embodiment, subjects exhibited less fatigue, pain, headache, and / or fever than control subjects. For example, grade 3 fatigue, pain, headache, or fever, as recommended by the FDA, was observed in less than 20%, less than 10%, less than 5%, or 0% of subjects after vaccination with the pharmaceutical or vaccine composition of the present invention (e.g., 7 or 14 days after vaccination). Comparisons can be made with a control group of subjects vaccinated with a vaccine that does not contain DCs or phDCs, particularly an mRNA vaccine.

[0291] Depending on the vaccine target, vaccines can be administered via various routes, such as subcutaneous, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intrasheath, intrahepatic, intralesional, intracranial, transdermal, intradermal, intrapulmonary, intraperitoneal, intracardiac, intra-arterial, or sublingual. Preferred routes of administration are intradermal or intramuscular. The composition can also be administered by inhalation. In one example, the vaccine composition of the present invention is administered intradermally, subcutaneously, or intramuscularly to the limbs, arms, and legs of a subject being treated. In another example, the vaccine composition of the present invention is administered intravenously. The vaccine composition of the present invention can be administered as a primary and / or booster dose. In one embodiment, the vaccine composition of the present invention is administered as a primary dose. In one embodiment, the vaccine composition of the present invention is administered as a booster dose. In one embodiment, the vaccine composition of the present invention is administered as both a primary and booster dose.

[0292] This invention provides articles of manufacture, such as kits, which provide phDC in one container and at least one disease-associated antigen (e.g., at least one mRNA encoding at least one antigenic protein, optionally encapsulated in nanoparticles) in another container. The containers may be pretreated glass or plastic vials or ampoules. The articles of manufacture may include instructions for use.

[0293] IV. Definition

[0294] When the term "comprising" is used in this specification and claims, it does not exclude other elements. For the purposes of this invention, the term "composes" is considered to be the preferred embodiment of the term "comprising". If a group is defined below as comprising at least a certain number of embodiments, this should also be understood to disclose a group preferably consisting only of these embodiments.

[0295] For the purposes of this invention, the term "obtained" is considered to be a preferred embodiment of the term "available". If phDC is defined below, for example, as obtainable by a particular method, it should also be understood that this discloses phDC obtained by that method.

[0296] When an indefinite or definite article, such as “a,” “an,” or “the,” is used to refer to a singular noun, this includes the plural form of that noun, unless otherwise specified. Unless the context clearly indicates otherwise, the term “or” means “and / or” and is used interchangeably with the term “and / or.” In the context of this invention, the terms “about” or “approximately” represent a range of accuracy that, as those skilled in the art will understand, still ensures the technical effect of the features under discussion. This term typically indicates a deviation from the indicated value of ±20%, preferably ±15%, more preferably ±10%, and even more preferably ±5%.

[0297] Furthermore, the terms “first,” “second,” “third,” or “(a),” “(b),” “(c),” “(d),” or “(i),” “(ii),” “(iii),” “(iv)” in the specification and claims are used to distinguish similar elements and are not necessarily used to describe an order or chronological order. It should be understood that such terms are interchangeable where appropriate, and embodiments of the invention described herein can be operated in orders other than those described or illustrated herein. Where the terms “first,” “second,” “third,” or “(a),” “(b),” “(c),” “(d),” or “(i),” “(ii),” “(iii),” “(iv)” relate to steps of a method, use, or measurement, unless otherwise stated, there is no temporal or time interval consistency between the steps; that is, the steps may be performed simultaneously or there may be time intervals of seconds, minutes, hours, days, weeks, months, or even years between these steps, unless otherwise stated in the application set forth above or below.

[0298] The term "about" means a number, level, value, quantity, frequency, percentage, dimension, size, amount, weight, or length that varies by as much as 30%, 25%, 20%, 15%, 30%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% relative to a reference number, level, value, quantity, frequency, percentage, dimension, size, amount, weight, or length. When the term "about" is used in conjunction with a numerical range, it modifies the range by extending the boundaries above and below the stated value. In the context of this invention, the term "about" indicates a range of accuracy that, as those skilled in the art will understand, still ensures the technical effect of the feature in question. Typically, the term "about" is intended to modify numerical values ​​above and below the stated value by a variation range of ±10%.

[0299] Technical terms are used according to their common meanings. If a particular meaning is conveyed to certain terms, then the definition of the term will be given below in the context in which the term is used.

[0300] Nucleic acids and proteins

[0301] As used herein, the terms "polynucleotide" or "nucleic acid" are intended to include DNA and RNA, such as genomic DNA, cDNA, mRNA, recombinant-derived, and chemically synthesized molecules. Nucleic acids can be single-stranded or double-stranded. RNA includes in vitro transcribed RNA or synthetic RNA. In one embodiment of all aspects of the invention, RNA encoding an antigenic protein is expressed in phDCs (phnuclear cells) obtained from a donor or a subject to be inoculated. The loaded phDCs, i.e., phDCs containing RNA encoding the antigenic protein, present the antigenic protein to the subject's immune system.

[0302] The nucleic acids described herein can be recombinant and / or isolated molecules. In this disclosure, the term "RNA" refers to nucleic acid molecules comprising ribonucleotide residues. In a preferred embodiment, the RNA contains all or most of the ribonucleotide residues. As used herein, "ribonucleotide" refers to a nucleotide having a hydroxyl group at the 2' position of the β-D-furanonitrosourea group. RNA encompasses, but is not limited to, double-stranded RNA, single-stranded RNA, isolated RNA such as partially purified RNA, substantially pure RNA, mRNA, circular RNA, synthetic RNA, recombinant RNA, synthetic RNA, self-amplifying RNA, and modified RNA (e.g., modified mRNA) that differs from naturally occurring RNA by the addition, deletion, substitution, and / or alteration of one or more nucleotides. The aforementioned RNA types, such as "RNA," "mRNA," "self-amplifying RNA," "modified mRNA," or "synthetic RNA," always include a segment of it that remains functional. A "segment" can refer to a portion of a nucleotide sequence, i.e., a sequence shortened at the 5' or 3' end. The fragment of the RNA (e.g., mRNA) sequence contains, for example, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the residues from the RNA sequence. The fragment of the RNA (e.g., mRNA) sequence preferably contains at least 18, particularly at least 24, at least 36, at least 45, at least 60, at least 90, at least 150, or at least 300 consecutive residues from the RNA sequence.

[0303] The alterations can target internal RNA nucleotides or the addition of non-nucleotide substances to one or more ends of the RNA. This document also considers that the nucleotides in RNA (e.g., mRNA) can be non-standard nucleotides, such as chemically synthesized nucleotides, naturally occurring modified nucleotides, or deoxynucleotides. For the purposes of this disclosure, these altered RNAs are considered analogs of naturally occurring RNA.

[0304] In some embodiments of the invention, the RNA is messenger RNA (mRNA) associated with an RNA transcript encoding a peptide or protein. As known in the art, mRNA typically comprises a 5' untranslated region (5'-UTR), a coding region, and a 3' untranslated region (3'-UTR). In some embodiments, the RNA (e.g., mRNA) is produced by in vitro transcription or chemical synthesis. In one embodiment, mRNA is produced by in vitro transcription using a DNA template, wherein the DNA refers to a nucleic acid containing deoxyribonucleotides. In one embodiment, the RNA (e.g., mRNA) is in vitro transcribed RNA (IVT-RNA) and can be obtained by in vitro transcription of a suitable DNA template. The promoter used to control transcription can be any promoter of any RNA polymerase.

[0305] DNA templates for in vitro transcription can be obtained by cloning nucleic acids, particularly cDNA, and introducing them into a suitable vector for in vitro transcription. cDNA can be obtained by reverse transcription of RNA.

[0306] In one embodiment, the RNA, particularly the mRNA, described herein may have a modified nucleoside. In some embodiments, the RNA, particularly the mRNA, contains a modified nucleoside that replaces at least one (e.g., each) uridine.

[0307] "Pseudouridine" is an example of a modified nucleoside that is an isomer of uridine, wherein uracil is attached to a pentose ring via a carbon-carbon bond rather than a nitrogen-carbon glycosidic bond. Another exemplary modified nucleoside is N1-methyl-pseudouridine. Another exemplary modified nucleoside is 5-methyl-uridine. In some embodiments, one or more uridine residues in the RNA (e.g., mRNA) described herein are replaced by modified uridine residues (e.g., N1-methyl-pseudouridine or 5-methyl-uridine or combinations thereof). In one embodiment, the RNA comprises other modified nucleosides or comprises further modified nucleosides, such as modified cytidine. For example, in one embodiment, cytidine is partially or completely substituted in the RNA, preferably completely substituted with 5-methyl-cytidine. In one embodiment, the RNA comprises 5-methylcytidine and one or more selected from pseudouridine, N1-methyl-pseudouridine, and 5-methyl-uridine. In one embodiment, the RNA comprises 5-methylcytidine and N1-methyl-pseudouridine. In some embodiments, the RNA comprises 5-methylcytidine in place of each cytidine and N1-methyl-pseudouridine in place of each uridine. In some embodiments, the RNA (e.g., mRNA) may comprise more than one type of modified nucleoside. In some embodiments, only a subset of uridine residues is modified. In some embodiments, all uridine residues are modified.

[0308] In some embodiments, the modified nucleoside is a modified uridine. Exemplary nucleobases and nucleosides having modified uracil include pseudouridine (ψ), pyridin-4-ketoribonucleoside, 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s2U), 4-thio-uridine (s4U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho5U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), 3-methyl-uridine (m3U), and 5-methoxy-uridine (mo5U). Uridine 5-hydroxyacetic acid (cmo5U), methyl uridine 5-hydroxyacetate (mcmo5U), 5-carboxymethyl-uridine (cm5U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm5U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm5U), 5-methoxycarbonylmethyl-uridine (mcm5U), 5-methoxycarbonylmethyl-2-thio-uridine (mcm5s2U), 5-aminomethyl-2-thio-uridine (nm5s2U), 5-methylaminomethyl-uridine (mnm5U), 5-methyl 5-Methylaminomethyl-2-thiouridine (mnm5s2U), 5-methylaminomethyl-2-selenouridine (mnm5se2U), 5-carbamoylmethyluridine (ncm5U), 5-carboxymethylaminomethyluridine (cmnm5U), 5-carboxymethylaminomethyl-2-thiouridine (cmnm5s2U), 5-propynyluridine, 1-propynyl-pseudouridine, 5-tauronic acid methyluridine (τm5U), 1-tauronic acid methyl-pseudouridine, 5-tauronic acid methyl-2-thiouridine (τm5s2U) 1-Taurine methyl-4-thio-pseudouridine, 5-methyluridine (m5U, i.e., with nucleobase deoxythymidine), 1-methyl-pseudouridine (M1ψ), 5-methyl-2-thio-uridine (m5s2U), 1-methyl-4-thio-pseudouridine (m1s4ψ), 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m3ψ), 2-thio-1-methyl-pseudouridine, 1-methyl-1-deazo-pseudouridine, 2-thio-1-methyl-1-deazo-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-Dihydrouridine, 5-Methyl-Dihydrouridine (m5D), 2-Thio-Dihydrouridine, 2-Thio-Dihydropseudouridine, 2-Methoxy-uridine, 2-Methoxy-4-Thio-uridine, 4-Methoxy-pseudouridine, 4-Methoxy-2-Thio-pseudouridine, N1-Methyl-pseudouridine, 3-(3-amino-3-carboxypropyl)uridine (acp3U), 1-Methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp3Ψ), 5-(isopentenylaminomethyl)uridine (inm5U), 5-(isopentenylaminomethyl)-2-thiouridine (inm5S2U), α-Thio-uridine, 2'-O-Methyl-uridine (Um), 5,2'-O-Dimethyl-uridine (m5Um), 2'-O 1-Methyl-pseudouridine (ψm), 2-thio-2'-O-methyluridine (s2Um), 5-methoxycarbonylmethyl-2'-O-methyluridine (mcm5Um), 5-carbamoylmethyl-2'-O-methyluridine (ncm5Um), 5-carboxymethylaminomethyl-2'-O-methyluridine (cmnm5Um), 3,2'-O-dimethyluridine (m3Um) and 5-(isopentenylaminomethyl)-2'-O-methyluridine (inm5Um), 1-thio-uridine, deoxythymidine, 2'-F-ara-uridine, 2'-F-uridine, 2'-OH-ara-uridine, 5-(2-methoxycarbonylvinyl)uridine and 5-[3-(1-E-propenylamino)]uridine.

[0309] In some embodiments, the modified nucleoside is a modified cytidine. Exemplary nucleobases and nucleosides having modified cytosine include 5-aza-cytidine, 6-aza-cytidine, pseudo-isocytidine, 3-methyl-cytidine (m3C), N4-acetyl-cytidine (ac4C), 5-formyl-cytidine (f5C), N4-methyl-cytidine (m4C), 5-methyl-cytidine (m5C), 5-halo-cytidine (e.g., 5-iodo-cytidine), and 5-hydroxymethyl-cytidine (hm5C). ), 1-methyl-pseudocytidine, pyrrolo-cytidine, 2-thio-cytidine (s2C), 2-thio-5-methyl-cytidine, 4-thio-pseudocytidine, 4-thio-1-methyl-pseudocytidine, 4-thio-1-methyl-1-deazo-pseudocytidine, 1-methyl-1-deazo-pseudocytidine, zebulin rine), 5-aza-za-zabralin, 5-methyl-zabralin, 5-aza-2-thio-zabralin, 2-thio-zabralin, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudo-cytidine, 4-methoxy-1-methyl-pseudo-cytidine, lysine (k2C), α-thio-cytidine, 2'-O-methyl-cytidine (Cm), 5,2'-O-dimethyl -Cytidine (m5Cm), N4-acetyl-2'-O-methyl-cytidine (ac4Cm), N4,2'-O-dimethyl-cytidine (m4Cm), 5-formyl-2'-O-methyl-cytidine (f5Cm), N4,N4,2'-O-trimethyl-cytidine (m42Cm), 1-thio-cytidine, 2'-F-afa-cytidine, 2'-F-cytidine and 2'-OH-afa-cytidine.

[0310] In some implementations, the modified nucleoside is a modified adenosine. Exemplary nucleobases and nucleosides having modified adenine include 2-amino-purine, 2,6-diamino-purine, 2-amino-6-halo-purine (e.g., 2-amino-6-chloro-purine), 6-halo-purine (e.g., 6-chloro-purine), 2-amino-6-methyl-purine, 8-azido-adenosine, 7-deadenine, 7-deadenine-8-aza-adenosine, 7-deadenine-2-amino-purine, 7-deadenine-2,6-diamino-purine, 7-deadenine-8-aza-2,6-diamino-purine, 1-methyl-adenosine (m1A), 2-methyl-adenosine (m2A), N6-methyl-adenosine (m6A), 2-methylthio-N6-methyl-adenosine (ms2m6A), and N6-isopentanyl-adenosine. Alkenyl-adenosine (i6A), 2-methylthio-N6-isopentenyl-adenosine (ms2i6A), N6-(cis-hydroxyisopentenyl)adenosine (io6A), 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine (ms2io6A), N6-glycylcarbamoyl-adenosine (g6A), N6-threonylcarbamoyl-adenosine (t6A), N6-methyl-N6-threonylcarbamoyl-adenosine (m6t6A), 2-methylthio-N6-threonylcarbamoyl-adenosine (ms2g6A), N6,N6-dimethyl-adenosine (m62A), N6-hydroxynor-valylcarbamoyl-adenosine (N6-hydroxynor-valylcarbamoyl-adenosine) (hn6A), 2-methylthio-N6-hydroxydevaline-carbamoyl-adenosine (ms2hn6A), N6-acetyl-adenosine (ac6A), 7-methyl-adenosine, 2-methylthio-adenosine, 2-methoxy-adenosine, α-thio-adenosine, 2'-O-methyl-adenosine (Am), N6,2'-O-dimethyl-adenosine (m6Am), N6,N6,2'-O- Trimethyl-adenosine (n62Am), 1,2'-O-dimethyl-adenosine (m1Am), 2'-O-ribosyl-adenosine (phosphate) (Ar(p)), 2-amino-N6-methyl-purine, 1-thio-adenosine, 8-azido-adenosine, 2'-F-aa-adenosine, 2'-F-adenosine, 2'-OH-ara-adenosine, and N6-(19-amino-pentaoxane-nonadecanyl)-adenosine.

[0311] In some embodiments, the modified nucleoside is a modified guanidine. Exemplary nucleobases and nucleosides having modified guanine include inosine (I), 1-methyl-inosine (m1l), wyoside (imG), methylwyosine (mimG), 4-demethyl-wyoside (imG-14), isowyoside (imG2), wyoside (yW), peroxywyoside (O2yW), hydroxywyoside (OhyW), under-modified hydroxywyoside (OhyW*), 7-deazo-guanosine, guanosine (Q), epoxy-guanosine (oQ), galactosyl-guanosine (galQ), mannosyl-guanosine (manQ), 7-cyano-7-deazo-guanosine (... preQo), 7-aminomethyl-7-deazo-guanosine (preQ1), archaenoside (G+), 7-deazo-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deazo-guanosine, 6-thio-7-deazo-8-aza-guanosine, 7-methyl-guanosine (m7G), 6-thio-7-methyl-guanosine, 7-methyl-inosine, 6-methoxy-guanosine, 1-methyl-guanosine (m1G), N2-methyl-guanosine (m2G), N2,N2-dimethyl -Guanin (m22G), N2,7-dimethyl-guanin (m2,7G), N2,N2,7-dimethyl-guanin (m2,2,7G), 8-oxo-guanin, 7-methyl-8-oxo-guanin, 1-methyl-6-thio-guanin, N2-methyl-6-thio-guanin, N2,N2-dimethyl-6-thio-guanin, α-thio-guanin, 2'-O-methyl-guanin (Gm), N2-methyl-2'-O-methyl-guanin (m2Gm), N2, N2-Dimethyl-2'-O-methyl-guanosine (m22Gm), 1-Methyl-2'-O-methyl-guanosine (m1Gm), N2,7-Dimethyl-2'-O-methyl-guanosine (m2,7Gm), 2'-O-methyl-inosine (Im), 1,2'-O-dimethyl-inosine (m1lm), 2'-O-ribosylguanosine (phosphate) (Gr(p)), 1-thio-guanosine, O6-methyl-guanosine, 2'-F-ara-guanosine and 2'-F-guanosine.

[0312] In some embodiments, the mRNA contains a 5'-cap. In one embodiment, the mRNA does not have an uncapped 5'-triphosphate. In one embodiment, the mRNA can be modified with a 5'-cap analogue. The term "5'-cap" refers to a structure found at the 5' end of the mRNA molecule and is typically composed of a guanosine nucleotide linked to the mRNA via a 5'-to-5'-triphosphate bond. In one embodiment, the guanosine is methylated at the 7-position. Providing mRNA with a 5'-cap or a 5'-cap analogue can be achieved through in vitro transcription, wherein the 5'-cap is co-transcribed into the mRNA strand, or it can be attached to the mRNA post-transcriptionally using a capping enzyme.

[0313] The term "untranslated region" or "UTR" refers to a region in a DNA molecule that is transcribed but not translated into an amino acid sequence, or to a corresponding region in an RNA molecule such as mRNA. An untranslated region (UTR) can be located at the 5' (upstream) end of an open reading frame (5'-UTR) and / or the 3' (downstream) end of an open reading frame (3'-UTR). The 5'-UTR (if present) is located at the 5' end upstream of the start codon in a protein-coding region. The 5'-UTR is downstream of the 5'-cap (if present), for example, directly adjacent to the 5'-cap. The 3'-UTR (if present) is located at the 3' end downstream of the stop codon in a protein-coding region, but the term "3'-UTR" preferably does not include the poly(A) sequence. Therefore, the 3'-UTR is upstream of the poly(A) sequence (if present), for example, directly adjacent to the poly(A) sequence.

[0314] As used herein, the term "multiple (A) sequence" or "multiple A tail" refers to a continuous or discontinuous sequence of adenosine residues, typically located at the 3' end of an RNA molecule (e.g., an mRNA molecule). Multiple (A) sequences are known to those skilled in the art and can occur after the 3'-UTR in the RNA (e.g., mRNA) described herein. A continuous multiple (A) sequence is characterized by a series of adenosine residues. Essentially, a continuous multiple (A) sequence is typical. The RNA (e.g., mRNA) according to the invention can have a free 3' end multiple (A) sequence that is attached to the RNA (e.g., mRNA) post-transcriptionally by a template-independent RNA polymerase, or a multiple (A) sequence encoded by DNA and transcribed by a template-dependent RNA polymerase. A poly(A) sequence of approximately 120 A nucleotides has a beneficial effect on RNA levels in transfected eukaryotic cells and on protein levels, wherein the protein is translated from an open reading frame located upstream (5') of the poly(A) sequence (Holtkamp et al., 2006, Blood, vol.108, pp. 4009-4017). The poly(A) sequence can be of any length. In some embodiments, the poly(A) sequence comprises, is substantially composed of, or consists of at least 20, at least 30, at least 40, at least 80, or at least 100 and at most 800, at most 400, at most 300, at most 200, or at most 150 A nucleotides. In one embodiment, the poly(A) sequence comprises, is substantially composed of, or consists of 300 nucleotides. In this context, "consistently composed of" means that the majority of nucleotides in the poly(A) sequence are typically at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of the nucleotides in the poly(A) sequence, but the remaining nucleotides are allowed to be nucleotides other than A nucleotides, such as U nucleotides (uridine monophosphate), G nucleotides (guanosine monophosphate), or C nucleotides (cytidine monophosphate). In this context, "consisting of" means that all nucleotides in the poly(A) sequence are 100% A nucleotides by nucleotide number. The term "A nucleotide" or "A" refers to adenosine monophosphate. In some embodiments, the poly(A) sequence is attached during RNA transcription, such as during the preparation of RNA (e.g., mRNA) transcribed in vitro, based on a DNA template containing repeating dT nucleotides (deoxythymidine monophosphate) in a strand complementary to the coding strand. The DNA sequence encoding the poly(A) sequence (coding strand) is called a poly(A) cassette.

[0315] The term "codon-optimized" refers to altering the codons in the coding region of a nucleic acid molecule to reflect the typical codon selection of the host organism, preferably without changing the amino acid sequence encoded by the nucleic acid molecule. In the context of this invention, the coding region is preferably codon-optimized for optimal expression in subjects to be treated with the RNA (e.g., mRNA) molecules described herein. Codon optimization is based on the finding that translation efficiency is also determined by the varying frequencies of tRNA occurrence in cells. Therefore, the sequence of RNA (e.g., mRNA) can be modified such that the readily available codon insertion of frequently occurring tRNA replaces "rare codons."

[0316] In some embodiments of the invention, the guanosine / cytosine (G / C) content of the coding region of the RNA (e.g., mRNA) described herein is increased compared to the G / C content of the corresponding coding sequence of wild-type RNA, wherein the amino acid sequence encoded by the RNA (e.g., mRNA) is preferably not modified compared to the amino acid sequence encoded by wild-type RNA. This modification of the RNA (e.g., mRNA) sequence is based on the fact that the sequence of any RNA region to be translated is important for the efficient translation of that RNA (e.g., mRNA). Sequences with increased G / C content are more stable than sequences with increased A (adenosine) / U (uracil) content. Regarding the fact that several codons encode the same amino acid (so-called genetic code degeneracy), the most favorable codons for stability can be determined (so-called alternative codon selection). Depending on the amino acid to be encoded by the RNA, there are multiple possibilities for modification of the RNA (e.g., mRNA) sequence compared to its wild-type sequence. In particular, codons containing A and / or U nucleotides can be modified by replacing these codons with other codons encoding the same amino acid but not containing A and / or U, or containing a lower content of A and / or U nucleotides.

[0317] In the context of this invention, the term "transcription" refers to the process of transcribing the genetic code in a DNA sequence into RNA (e.g., mRNA). Subsequently, the RNA (e.g., mRNA) can be translated into peptides or proteins.

[0318] According to the invention, the term "transcription" includes "in vitro transcription," wherein the term "in vitro transcription" refers to a process in which RNA, particularly mRNA, is synthesized in vitro in a cell-free system, preferably using suitable cell extracts. Preferably, cloning vectors are used to generate transcripts. These cloning vectors are generally designated as transcription vectors and are covered by the term "vector" according to the invention.

[0319] With regard to RNA (e.g., mRNA), the terms "expression" or "translation" refer to the process in cellular ribosomes through which the mRNA chain directs the assembly of an amino acid sequence to prepare a peptide or protein. In one embodiment, at least a portion of the RNA (e.g., mRNA) (e.g., formulated as RNA (e.g., mRNA) lipid particles) described herein is delivered to the phDC after being combined with it. In one embodiment, the RNA (e.g., mRNA) is translated by the phDC to produce the peptide or protein it encodes. RNA (e.g., mRNA) particles, such as the RNA (e.g., mRNA) lipid particle antigen described herein, are used to deliver the RNA to the phDC.

[0320] "Encoding" refers to the inherent characteristic of a specific nucleotide sequence in a polynucleotide (such as a gene, cDNA, RNA, or mRNA) that serves as a template for the synthesis of other polymers and macromolecules with defined nucleotide sequences (i.e., rRNA, tRNA, and mRNA) or defined amino acid sequences and the resulting biological properties during biological processes. Therefore, if the translation of RNA (e.g., mRNA) occurs in a cell, then the RNA (e.g., mRNA) sequence can encode a protein (e.g., an antigen).

[0321] As used herein, terms such as “reduce,” “effectively reduce,” “lower,” “inhibit,” or “damage” refer to the ability to reduce or cause an overall reduction, preferably at a level of at least 5%, at least 10%, at least 20%, at least 50%, at least 75%, or even more. These terms include complete or substantially complete inhibition, i.e., reduction to zero or substantially reduction to zero. Terms such as “increase,” “enhance,” or “exceed” preferably refer to an increase or enhancement of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 80%, at least 100%, at least 200%, at least 500%, or even more.

[0322] According to this disclosure, the term "peptide" includes oligopeptides and polypeptides, and refers to a substance comprising about two or more, about three or more, about four or more, about six or more, about eight or more, about ten or more, about thirteen or more, about sixteen or more, about twenty or more, and at most about 50, about 100 or about 150 consecutive amino acids linked together by peptide bonds. The terms "protein" or "polypeptide" refer to macropeptides, particularly peptides having at least about 150 amino acids, but unless otherwise specified, the terms "peptide," "protein," and "polypeptide" are used synonymously herein.

[0323] "Fragment" can refer to a part of an antigen, such as disease-associated antigens, amino acid sequences, antigenic proteins, RNA (including the types of RNA listed above), especially mRNA.

[0324] If the fragment involves an amino acid sequence (e.g., an antigenic protein), the term refers to a portion of the amino acid sequence, specifically a sequence representing a shortened amino acid sequence (e.g., of an antigenic protein) at the N-terminus and / or C-terminus. A C-terminal shortened fragment (N-terminal fragment) can be obtained, for example, by translating a truncated open reading frame lacking the 3' end. An N-terminal shortened fragment (C-terminal fragment) can be obtained, for example, by translating a truncated open reading frame lacking the 5' end, provided that the truncated open reading frame contains a start codon for initiating translation. The amino acid sequence fragment contains, for example, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the amino acid residues from the amino acid sequence. The amino acid sequence fragment preferably contains at least 6, particularly at least 8, at least 12, at least 15, at least 20, at least 30, at least 50, or at least 100 consecutive amino acids from the amino acid sequence.

[0325] The term "variant" as used herein refers to an amino acid sequence that differs from the parent amino acid sequence due to at least one amino acid modification. The parent amino acid sequence may be a naturally occurring or wild-type (WT) amino acid sequence, or a modified version of the wild-type amino acid sequence. Preferably, the variant amino acid sequence has at least one amino acid modification compared to the parent amino acid sequence, for example, 1 to about 20 amino acid modifications, 1 to about 10 amino acid modifications, or 1 to about 5 amino acid modifications compared to the parent.

[0326] The terms "wild-type," "WT," or "natural" in this article refer to amino acid sequences found in nature, including allelic variations. Wild-type amino acid sequences, peptides, or proteins have amino acid sequences that have not been intentionally modified.

[0327] A “variant” of an amino acid sequence (peptide, protein, or polypeptide) includes amino acid insertion variants, amino acid addition variants, amino acid deletion variants, and / or amino acid substitution variants. The term “variant” includes all mutants, splicing variants, post-translational modification variants, conformations, isoforms, allelic variants, species variants, and species homologs, particularly those that are naturally occurring. The term “variant” specifically includes fragments of amino acid sequences. Preferably, the degree of similarity (or identity) between a given amino acid sequence and an amino acid sequence that is a variant of said given amino acid sequence will be at least about 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. Alignment for determining sequence similarity, preferably sequence identity, can be performed using tools known in the art, preferably using optimal sequence alignment, such as Align, using standard settings, preferably EM-BOSS::needle, Matrix: Blosum62, etc. "Sequence similarity" indicates the percentage of identical or conserved amino acid substitutions. "Sequence identity" between two amino acid sequences indicates the percentage of identical amino acids between the sequences. "Sequence identity" between two nucleic acid sequences indicates the percentage of identical nucleotides between the sequences. Percentage identity is obtained by determining the number of identical positions corresponding to the sequences to be compared, dividing that number by the number of positions being compared (e.g., the number of positions in the reference sequence), and multiplying the result by 100.

[0328] In one embodiment, the fragment or “variant” is preferably a “functional fragment” or “functional variant.” The terms “functional fragment” or “functional variant” refer to any fragment or variant that exhibits one or more functional properties identical or similar to those of the unfragmented agent from which it is derived (e.g., an unfragmented amino acid sequence, an unfragmented RNA sequence, an unfragmented mRNA sequence, etc.), i.e., they are functionally equivalent. With respect to an antigen or antigenic protein, a particular function is one or more immunogenic (“antigenic” activities, synonymous herein) activities exhibited by the amino acid sequence from which the fragment or variant is derived. As used herein, the terms “functional fragment” or “functional variant” specifically refer to variant molecules or sequences that contain a sequence with one or more altered amino acid or nucleotide sequences compared to a parent molecule or sequence, and are still capable of achieving one or more functions of the parent molecule or sequence, such as inducing an immune response (i.e., antigenicity). In one embodiment, modifications to the amino acid sequence of the parent molecule or sequence do not significantly affect or alter the characteristics of the molecule or sequence. In different embodiments, the function of the functional fragment or variant may be reduced but still significantly present; for example, the immunogenicity of the functional fragment or variant may be at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of that of the parent agent, molecule, or sequence. However, in other embodiments, the immunogenicity of the functional fragment or variant may be enhanced compared to the parent molecule or sequence.

[0329] Composition and application

[0330] As used herein, the term "vaccine" refers to a composition that induces an immune response when administered to a subject. In some embodiments, the induced immune response provides protective immunity. Different types of vaccines include, in particular, live vaccines, attenuated vaccines, inactivated vaccines, toxoid vaccines or subunit and conjugate vaccines (also known as conventional vaccines), and mRNA vaccines. Conventional vaccines such as mRNA vaccines, as understood herein, do not include dendritic cells (DCs), particularly phDCs.

[0331] For the vaccine compositions of the present invention to be used as vaccines, an immune response against disease-associated antigens (e.g., antigenic proteins) must be induced in cells, tissues, or subjects (e.g., humans). In some embodiments, the vaccine compositions of the present invention induce an immune response against at least one disease-associated antigen (e.g., antigenic protein) in cells, tissues, or subjects (e.g., humans). In some cases, the vaccine compositions of the present invention induce a protective immune response in mammals. Prophylactic administration of the vaccine compositions of the present invention (i.e., prevention of disease or condition or mitigation of the course of disease or condition). However, vaccination occurs before the onset of a pathological event or exposure to an infectious agent, such as before infection with an infectious disease or before the onset of a tumor. The vaccine compositions of the present invention can be administered to subjects at risk (or susceptible to) developing a disease or condition. Such subjects can be identified using standard clinical methods. In the context of the present invention, prophylactic administration occurs before the onset of a pathological event or exposure to an infectious agent, such that the disease or condition is prevented or alternatively delayed in its progression or its symptoms are mitigated. In the context of the medical field, the term "prevention" encompasses any activity that reduces the burden of mortality or morbidity from disease. In some embodiments, administration of the vaccine compositions of the present invention can be performed by a single dose or by booster doses. If the application is referred to as “initial” or “primary immunization” in this document, this corresponds to the first application. Any subsequent application is referred to as a “booster”. Boosters are typically administered at least 14, 21, 28, 35, 42, 49, or 56 days or more after the primary immunization.

[0332] This invention contemplates immune responses that can be protective, preventive, and / or prophylactic. As used herein, “induce / stimulate (or induce / stimulate) an immune response” can indicate the absence of an immune response against a specific antigen prior to induction / stimulation, or it can indicate a baseline level of immune response against a specific antigen present prior to induction, which is enhanced after induction. Therefore, “induce / stimulate (or induce / stimulate) an immune response” includes “enhance (or enhance) an immune response.” The terms “immunity” or “vaccination” describe the process of administering an antigen to an individual for the purpose of stimulating an immune response for preventive reasons.

[0333] In contrast to vaccination, the term "treatment" or "therapeutic treatment" involves treating a subject (e.g., a mammal, such as a human) or cells to alter the current course of the disease. Therefore, (therapeutic) treatment refers to a subject or cell diagnosed with a disease or condition, previously diagnosed with a disease or condition, or identified as having a disease or condition (as opposed to prevention). (Therapeutic) treatment is administered after the onset of a pathological event or exposure to an infectious agent.

[0334] immunity

[0335] As used herein, the term "antigen" or "antigenic protein" refers to a compound, composition, or substance that can stimulate antibody production or a T-cell response in an animal, including compositions injected or absorbed into an animal. Antigens or antigenic proteins react with products of specific humoral or cellular immunity, including those induced by heterologous immunogens. The term antigen may be used interchangeably with the term "immunogen." The term "antigen" or "antigenic protein" includes all relevant antigenic epitopes. The terms "antigen," "antigen molecule / protein," or "immunogen" include fragments that can still function as antigens. Throughout this application, the term "antigenic protein" may be used interchangeably with "immunogen" or "disease-associated antigen." An "antigenic protein" comprises a number of amino acids ranging from small peptides to large proteins, such as 4 to 2000 or more, 4 to 1800, 4 to 1600, or 4 to 1400 amino acids. In one embodiment, the antigenic protein encoded by RNA (e.g., mRNA) comprises at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1200, at least 2000, or at least 3000 amino acids. In one embodiment, the antigenic protein encoded by RNA (e.g., mRNA) comprises at least 50 amino acids. In one embodiment, the antigenic protein encoded by RNA (e.g., mRNA) comprises at least 500 amino acids. In one embodiment, the antigenic protein encoded by RNA (e.g., mRNA) comprises at least 1000 amino acids. In one embodiment, the antigenic protein comprises 100 to 1500 amino acids. In one embodiment, the antigenic protein comprises 200 to 1300 amino acids. In one embodiment, the antigenic protein comprises 400 to 1300 amino acids.

[0336] An epitope is a site on an antigen that triggers a B-cell response and / or a T-cell response.

[0337] As used in this article, the term “immunogenicity” refers to the ability of a substance, cell, or part thereof, such as an antigen or antigenic protein, to elicit an immune response in a human or animal.

[0338] "Cell-mediated immunity," "cellular immunity," "cellular immune response," or similar terms refer to cellular responses that include the expression of antigens or antigenic proteins, particularly cells that present class I or II MHC antigens. Cellular responses involve immune effector cells, particularly cells called T cells or T lymphocytes, which act as "helpers" or "killers." Helper T cells (also known as CD4+ T cells) play a central role in regulating the immune response, while killer cells (also known as cytotoxic T cells, cytolytic T cells, CD8+ T cells, or CTLs) kill diseased cells, such as virus-infected cells, preventing the generation of more diseased cells.

[0339] Humoral immunity, or the humoral immune response, is an aspect of immunity mediated by macromolecules found in the extracellular fluid, such as secreted antibodies, complement proteins, and certain antimicrobial peptides. It contrasts with cell-mediated immunity. The aspect involving antibodies is often referred to as antibody-mediated immunity. Humoral immunity refers to antibody production and its accompanying accessory processes, including: Th2 activation and cytokine production, germinal center formation and allotype switching, affinity maturation, and memory cell generation.

[0340] Dendritic cells, also referred to as "DCs" in this document, are antigen-presenting immune cells that process antigenic material and present it to other cells of the immune system, most notably T cells. The function of DCs is to capture and process antigens. When DCs endocytose antigens, they process them into smaller fragments (usually peptides) that are displayed on their surface. On this surface, these fragments are presented to, for example, antigen-specific T cells via MHC molecules. After antigen uptake, DCs migrate to lymph nodes. During maturation, DCs can be promoted by various signals, including signaling via Toll-like receptors (TLRs), to express co-stimulatory signals that induce the activation and proliferation of homologous effector T cells (Teffs), thereby initiating a T cell-mediated immune response against the antigen. Alternatively, DCs may present antigens to antigen-specific T cells without providing co-stimulatory signals (or simultaneously provide co-inhibitory signals), preventing proper Teff activation. Such presentation can cause, for example, the death or anergy of antigen-recognizing T cells, or can induce the generation and / or expansion of regulatory T cells (Tregs). The term "dendritic cell" includes differentiated dendritic cells, immature and mature dendritic cells. These cells can be characterized by the expression of certain cell surface markers (e.g., CD11c, MHC class II, and at least low levels of CD80 and CD86), CD11b, CD304 (BDCA4)). In some embodiments, dendritic cells express CD8, CD103, CD1d, etc. Other dendritic cells can be identified by the absence of lineage markers such as CD3, CD14, CD19, CD56, etc. Furthermore, dendritic cells can be functionally characterized by their ability to stimulate allogeneic responses and mixed lymphocyte responses (MLRs). In one instance, phDCs combined with SARS-CoV-2-derived spike protein antigens (antigenic proteins) or the mRNA encoding them will process and present spike protein-associated antigens; that is, phDCs will be antigen-specific to the spike protein.

[0341] The term "AB serum" or "human AB serum" refers to cell culture reagents known in the art for use with certain human cell types, which provide growth factors, vitamins, nutrients, as well as trace elements and transport factors. Human AB serum is collected from healthy male donor volunteers with the AB serotype.

[0342] The term "FBS" refers to a widely used growth supplement for cell culture media. It typically contains high levels of embryonic growth-promoting factors.

[0343] disease

[0344] The term "disease" or "symptom" refers to any disease or symptom involving an antigen or antigenic protein, such as a disease characterized by the presence of an antigen or antigenic protein. A disease can be, for example, an infectious disease or a neoplastic disease (cancer). As mentioned above, an antigen can be a disease-associated antigen, such as a viral antigen or a tumor antigen. In one embodiment, a disease involving an antigen is a disease involving cells expressing an antigen (preferably on the cell surface). In the context of this invention, a disease is caused by pathogenic particles. The concentration of pathogenic particles in a subject can be determined by any suitable method known to those skilled in the art. For example, viral load can be determined by quantitative PCR and expressed as copies per ng of tissue. If appropriate, expression markers can also be used to quantify pathogenic particles.

[0345] The term "hyperproliferative disorder" includes tumors and cancer. The term "tumor" refers to a disease in which some cells in the body grow uncontrollably and spread to other parts of the body. Tumors can be cancerous or non-cancerous (benign). In the context of this invention, the terms tumor and cancer are used interchangeably. Cancerous tumors spread to or invade nearby tissues and can move to distant parts of the body to form new tumors (also known as metastases). Cancerous tumors are also called malignant tumors. Many cancers form solid tumors, but blood cancers, such as leukemia, do not form solid tumors. Benign tumors do not spread to or invade nearby tissues. Blood cancers include leukemia, lymphoma, myelodysplastic syndromes (MDS), myeloproliferative disorders (MPD), multiple myeloma, and all their subtypes.

[0346] The term "infectious disease" refers to any illness (e.g., the common cold) that can be transmitted from person to person or from organism to organism and is caused by a microbial pathogen. Infectious diseases are those known in the art and include, for example, viral diseases, bacterial diseases, fungal diseases, prion diseases, or parasitic diseases caused by viruses, bacteria, fungi, prions, and parasites, respectively. In this regard, infectious diseases can be, for example, hepatitis, sexually transmitted diseases (e.g., chlamydia or gonorrhea), tuberculosis, HIV / Acquired Immunodeficiency Syndrome (AIDS), diphtheria, hepatitis B, hepatitis C, cholera, severe acute respiratory syndrome (SARS), Covid-19, avian influenza, and influenza.

[0347] "Coronavirus disease 2019," or COVID-19, is an infectious disease caused by Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2). The outbreak of SARS-CoV-2, which causes atypical pneumonia, has raged since mid-December 2019 and has developed into a Public Health Emergency of International Concern. SARS-CoV-2 (MN908947.3) belongs to the β-coronavirus lineage B. It shares at least 70% sequence similarity with SARS-CoV. Specifically, COVID-19 refers to the disease as defined in the current International Classification of Diseases (ICD-11, World Health Organization, version: 09 / 2020). More specifically, COVID-19 is used to indicate a disease diagnosed clinically, epidemiologically, or otherwise, regardless of whether laboratory tests are definitive, indeterminate, or unavailable. Generally, coronaviruses have four structural proteins: envelope (E), membrane (M), nucleocapsid (N), and spike (S). The E and M proteins play important functions in viral assembly, and the N protein is essential for viral RNA synthesis. The key glycoprotein S is responsible for viral binding and entry into target cells. The S protein is synthesized as a single-stranded, inactive precursor, which is cleaved in the producing cell by a furin-like host protease into two non-covalently associated subunits, S1 and S2. The S1 subunit contains a receptor-binding domain (RBD) that recognizes the host cell receptor. The S2 subunit contains a fusion peptide, two heptapeptide repeats, and a transmembrane domain, all of which are essential for mediating viral-host cell membrane fusion through a large conformational rearrangement. The S1 and S2 subunits trimerize to form a large pre-fusion spike. The SARS-CoV-2 S precursor protein can be proteasically cleaved into S1 (685 aa) and S2 (588 aa) subunits. The S1 subunit, composed of the receptor-binding domain (RBD), mediates viral entry into susceptible cells via the host angiotensin-converting enzyme 2 (ACE2) receptor. COVID-19 prevention may include preventing at least one of the following in subjects with COVID-19: pulmonary fibrosis, interstitial pneumonia, acute lung injury (ALI), acute respiratory distress syndrome (ARDS), alveolar injury, kidney injury, vascular disease, cardiac injury, acute myocardial injury, chronic cardiovascular injury, thrombosis, and venous thromboembolism. In certain implementations, pulmonary fibrosis, interstitial pneumonia, acute lung injury (ALI), acute respiratory distress syndrome (ARDS), kidney injury (such as proteinuria and acute kidney injury), and vascular disease are triggered by COVID-19.SARS-CoV-2 is used to denote all variants of the virus, which, according to the ICTV, belong to the domain Riboviria, kingdom Orthornavirae, phylum Pisuviricota, class Pisoniviricetes, order Nidovirales, family Coronaviridae, genus Betacoronavirus, subgenus Sarbecovirus, species Severe Acute Respiratory Syndrome-associated Coronavirus, and strain 2 of Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV-2).

[0348] The term "HIV" refers to the Human Immunodeficiency Virus. HIV is a member of the genus Lentivirus (part of the family Retroviridae). Two types of HIV have been characterized: HIV-1 and HIV-2. HIV-1 is the virus that was first discovered and is known as lymph node-associated virus (LAV) and human T-lymphotropic virus 3 (HTLV-III). HIV-1 is more virulent and more infectious than HIV-2 and is responsible for the majority of HIV infections worldwide. HIV infects people and, over time, leads to acquired immunodeficiency syndrome (AIDS). AIDS manifests as progressive failure of the immune system, which allows life-threatening opportunistic infections and cancers to develop.

[0349] As used herein, the term "animal" or "mammal" covers all mammals, including humans. Preferably, the mammal of the present invention is a human subject. In one embodiment, the subject is a human.

[0350] The terms “individual” and “subject” are used interchangeably herein. They refer to a person or another mammal (e.g., a mouse, rat, rabbit, dog, cat, cow, pig, sheep, horse, or primate) that may have or is susceptible to a disease or condition. In many embodiments, an individual is a human being. Unless otherwise stated, the terms “individual” and “subject” do not indicate a specific age and therefore encompass adults, the elderly, children, and newborns. In some embodiments, the term “subject” includes a person who is at least 50 years old, at least 55 years old, at least 60 years old, at least 65 years old, at least 70 years old, or older. In some embodiments, the term “subject” includes a person who is at least 65 years old, such as 65 to 80 years old, 65 to 75 years old, or 65 to 70 years old. In some embodiments of the invention, “individual” or “subject” is a “patient.”

[0351] The term "patient" refers to an individual or subject receiving treatment or preventative treatment. This invention relates to preventative treatment, namely vaccination.

[0352] The invention will now be described with respect to specific examples, however, these examples are for illustrative purposes and not intended to be limiting.

[0353] The present invention further relates to the following embodiments:

[0354] 1. A vaccine composition comprising a pharmaceutically effective amount of physiological DC (phDC) and at least one mRNA, said mRNA comprising a coding sequence encoding at least one antigenic protein.

[0355] 2. The vaccine composition according to 1, further comprising a pharmaceutically acceptable carrier or diluent.

[0356] 3. The vaccine composition according to 1 or 2, wherein the at least one mRNA is contained in lipid nanoparticles.

[0357] 4. The vaccine composition according to any one of 1 to 3, wherein the at least one mRNA comprises at least one modified nucleoside.

[0358] 5. The vaccine composition according to 4, wherein the at least one modified nucleoside is a modified uridine, optionally wherein all uridine residues are replaced with modified uridine.

[0359] 6. The vaccine composition according to 5, wherein the modified uridine is N1-methyl-pseudouridine.

[0360] 7. The vaccine composition according to any one of 1 to 6, wherein the at least one mRNA is partially modified with N1-methyl-pseudouridine.

[0361] 8. The vaccine composition according to any one of 1 to 6, wherein the at least one mRNA is completely modified with N1-methyl-pseuuridine.

[0362] 9. The vaccine composition according to any one of 1 to 8, wherein the at least one mRNA comprises a 5' UTR, a 3' UTR, a 5' cap and / or multiple (A) tails.

[0363] 10. A vaccine composition according to any one of 1 to 9, wherein the at least one mRNA comprises an optimized sequence; optionally, wherein the sequences of the 5' UTR, 3' UTR and / or the coding sequence of the antigenic protein are optimized.

[0364] 11. The vaccine composition according to any one of 1 to 10, wherein the lipid nanoparticles comprise cationic lipids, polyethylene glycol (PEG) modified lipids, cholesterol-based lipids, and / or non-cationic lipids.

[0365] 12. The vaccine composition according to 11, wherein the cationic lipids are present at a molar ratio between 30% and 40%, the PEG-modified lipids are present at a molar ratio between 1.5% and 4.0%, the cholesterol-based lipids are present at a molar ratio between 40% and 52%, and the non-cationic lipids are present at a molar ratio between 11% and 21%, wherein all molar ratios are relative to the total lipid content of LNP.

[0366] 13. The vaccine composition according to 11 or 12, wherein the cationic lipid is cKK-E12 lipid.

[0367] 14. The vaccine composition according to any one of 1 to 13, wherein the at least one antigenic protein is associated with a disease.

[0368] 15. The vaccine composition according to 14, wherein the disease-associated antigenic protein is an infectious disease-associated antigenic protein.

[0369] 16. The vaccine composition according to 15, wherein the infectious disease-associated antigenic protein is a viral antigenic protein, a bacterial antigenic protein, a fungal antigenic protein, a prion antigenic protein, or a parasitic antigenic protein.

[0370] 17. The vaccine composition according to 16, wherein the viral antigenic protein is a coronavirus antigenic protein or a human immunodeficiency virus (HIV) antigenic protein.

[0371] 18. The vaccine composition according to 17, wherein the coronavirus antigenic protein is derived from the spike protein, envelope protein, nucleocapsid protein, membrane protein and / or Orf1ab polymer.

[0372] 19. The vaccine composition according to 17 or 18, wherein the coronavirus antigenic protein is derived from the spike protein.

[0373] 20. The vaccine composition according to any one of 17 to 19, wherein the coronavirus antigenic protein is a β-coronavirus antigenic protein.

[0374] 21. The vaccine composition according to 20, wherein the β-coronavirus antigenic protein is the SARS-CoV-2 antigenic protein.

[0375] 22. The vaccine composition according to any one of 1 to 21, wherein the at least one mRNA comprises at least 75%, 85%, 90%, 95%, 98%, 99%, or 100% of the same nucleotide sequence as SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5.

[0376] 23. The vaccine composition according to any one of 1 to 22, wherein the at least one mRNA comprises at least 75%, 85%, 90%, 95%, 98%, 99%, or 100% of the same nucleotide sequence as SEQ ID NO:1.

[0377] 24. The vaccine composition according to any one of 1 to 21, wherein the at least one mRNA comprises at least 75%, 85%, 90%, 95%, 98%, 99%, or 100% of the same nucleotide sequence as SEQ ID NO:19.

[0378] 25. The vaccine composition according to 16, wherein the bacterial antigenic protein is an antigenic protein of a species of *Treponema* or *Mycobacterium*.

[0379] 26. The vaccine composition according to 16, wherein the fungal antigenic protein is an antigenic protein of Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Chlamydia trachomatis, or Candida albicans.

[0380] 27. The vaccine composition according to 16, wherein the parasite antigenic protein is an antigenic protein of Plasmodium malariae.

[0381] 28. The vaccine composition according to 14, wherein the disease-associated antigenic protein is a tumor-associated antigenic protein.

[0382] 29. The vaccine composition according to 28, wherein the tumor-associated antigenic protein is a blood cancer antigenic protein.

[0383] 30. The vaccine composition according to 29, wherein the blood cancer antigenic protein is a leukemia antigenic protein, a lymphoma antigenic protein, or a myeloma antigenic protein.

[0384] 31. The vaccine composition according to 28, wherein the tumor-associated antigenic protein is a solid tumor antigenic protein.

[0385] 32. The vaccine composition according to 31, wherein the solid tumor antigenic protein is a melanoma antigenic protein, an endometrial cancer antigenic protein, a renal cancer antigenic protein, a brain cancer antigenic protein, a cervical cancer antigenic protein, a liver cancer antigenic protein, a head and neck cancer antigenic protein, a gastrointestinal cancer antigenic protein, a lymph node cancer antigenic protein, a pancreatic cancer antigenic protein, an ear, nose and throat (ENT) cancer antigenic protein, a breast cancer antigenic protein, a prostate cancer antigenic protein, an ovarian cancer antigenic protein, or a lung cancer antigenic protein.

[0386] 33. The vaccine composition according to any one of 1 to 32, wherein phDC can be obtained by subjecting monocytes to physical forces.

[0387] 34. The vaccine composition according to 33, wherein the physical force is applied by passing the mononuclear cells through the flow chamber.

[0388] 35. The vaccine composition according to 34, wherein the flow chamber is a plate or a flexible bag, optionally a flexible plastic bag.

[0389] 36. The vaccine composition according to 33 to 35, wherein the mononuclear cells are autologous.

[0390] The vaccine composition of any one of 37.1 to 36, in a preventive method for preventing or reducing disease in a subject, the method comprising administering the vaccine composition to the subject.

[0391] The vaccine composition of any one of 38.15 to 27, in a prophylactic method for preventing or reducing an infectious disease in a subject, the method comprising administering the vaccine composition to the subject.

[0392] The vaccine composition of any one of 39.16 to 24, in a prophylactic method for preventing or reducing viral diseases in a subject, the method comprising administering the vaccine composition to the subject.

[0393] The vaccine composition of any one of 40.17 to 24, used in a preventive or reduction method for coronavirus disease in a subject, preferably a preventive method for coronavirus disease 2019 (Covid-19), the method comprising administering the vaccine composition to the subject.

[0394] The vaccine composition of any one of 41.28 to 32, in a prophylactic method for preventing or reducing tumors or cancer in a subject, the method comprising administering the vaccine composition to the subject.

[0395] The vaccine composition of any one of 42.29 or 30, in a prophylactic method for preventing or reducing blood cancers in a subject, the method comprising administering the vaccine composition to the subject.

[0396] 43.31 or 32, a vaccine composition for use in a prophylactic method of preventing or reducing solid tumors in a subject, the method comprising administering the vaccine composition to the subject.

[0397] 44. The vaccine composition used in any one of 37 to 43, wherein the administration of the vaccine composition is intradermal, intravenous or intramuscular.

[0398] 45. The vaccine composition used in any one of 37 to 43, wherein the administration of the vaccine composition is by inhalation.

[0399] 46. ​​The vaccine composition used in any one of 37 to 45, wherein the vaccine composition is administered once or multiple times.

[0400] 47. The vaccine composition used in any one of 37 to 46, wherein at least two doses are administered to the subject.

[0401] 48. The vaccine composition used in any one of 37 to 47, wherein the at least two doses are administered to the subject at intervals of 2 to 6 weeks between each dose, optionally at intervals of 2, 3, 4, 5 or 6 weeks between each dose.

[0402] 49. A method for preparing antigen-specific physiological dendritic cells (phDCs), the method comprising combining phDCs obtained from a donor with at least one mRNA encoding at least one antigenic protein.

[0403] 50. The method according to embodiment 49, wherein the phDC is generated by subjecting mononuclear cells obtained from the donor to physical forces.

[0404] 51. The method according to embodiment 49 or 50, wherein the physical force is applied by passing the mononuclear cell obtained from the donor through the flow chamber.

[0405] 52. The method according to any one of embodiments 49 to 51, wherein the method includes the further step of incubating the at least one mRNA with the phDC.

[0406] 53. The method according to implementation plan 52, wherein the incubation step is carried out for at least 0.5 h, 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 8 h, 12 h or 24 h.

[0407] 54. The method of any one of claims 49 to 53, wherein the mononuclear cells obtained from the donor are derived from an in vitro blood sample or PBMC from the donor.

[0408] 55. The method according to any one of embodiments 49 to 54, wherein the flow chamber is a plate.

[0409] 56. The method according to any one of embodiments 49 to 55, wherein the flow chamber is a bag, optionally a flexible bag or a plastic bag.

[0410] 57. The method according to embodiment 56, wherein the plastic bag is made of a material comprising: polyolefin, polyethylene, fluoropolymer, polyvinyl chloride, ethylene-vinyl acetate copolymer, ethylene vinyl alcohol, polyvinylidene fluoride, and / or other plastics comprising materials approved for medical use.

[0411] 58. The method according to any one of embodiments 49 to 57, wherein the at least one mRNA comprises at least one modified nucleoside.

[0412] 59. The method according to embodiment 58, wherein the at least one modified nucleoside is a uridine that replaces some or all of the uridine residues with at least one modified uridine, optionally some or all of the uridine residues being replaced with N1-methyl-pseudouridine.

[0413] 60. The method according to embodiment 59, wherein some or all of the modified uridine residues comprise one or more uniquely modified uridine residues.

[0414] 61. The method according to embodiment 60, wherein the one or more uniquely modified uridine residues comprise N1-methyl-pseudouridine.

[0415] 62. The method according to any one of embodiments 49 to 61, wherein the at least one mRNA is partially modified with N1-methyl-pseudouridine.

[0416] 63. The method according to any one of embodiments 49 to 61, wherein the at least one mRNA is completely modified with N1-methyl-pseudouridine.

[0417] 64. The method according to any one of embodiments 49 to 63, wherein the at least one mRNA comprises a 5' UTR, a 3' UTR, a 5' cap and / or a multiple (A) tail.

[0418] 65. The method according to embodiment 64, wherein the 5' cap is a Cap1 structure or an m7GpppG cap.

[0419] 66. The method according to any one of embodiments 49 to 65, wherein the mRNA comprises a coding sequence encoding an antigenic protein.

[0420] 67. The method according to any one of embodiments 49 to 66, wherein the at least one mRNA comprises an optimized sequence; preferably, the sequences of the 5' UTR, 3' UTR and / or the coding sequence of the antigenic protein are optimized.

[0421] 68. The method according to any one of embodiments 49 to 67, wherein the at least one mRNA encoding at least one antigenic protein is provided in the lipid nanoparticles.

[0422] 69. The method according to embodiment 68, wherein the lipid nanoparticles comprise cationic lipids, PEG-modified lipids, cholesterol and / or non-cationic lipids.

[0423] 70. The method according to any one of embodiments 49 to 69, wherein the antigenic protein is associated with a disease, optionally an infectious disease-associated antigenic protein or a tumor-associated antigenic protein.

[0424] 71. The method according to embodiment 70, wherein the infectious disease-associated antigenic protein is a viral antigenic protein, a bacterial antigenic protein, a fungal antigenic protein, a prion antigenic protein, or a parasitic antigenic protein.

[0425] 72. The method according to embodiment 71, wherein the viral antigenic protein is a coronavirus or an HIV antigenic protein.

[0426] 73. The method according to embodiment 72, wherein the coronavirus antigenic protein is a SARS-CoV antigenic protein, optionally a SARS-CoV-2 antigenic protein.

[0427] 74. The method according to embodiment 73, wherein the SARS-CoV or SARS-CoV-2 antigenic protein is derived from the spike protein, envelope protein, nucleocapsid protein, membrane protein and / or Orf1ab polymer.

[0428] 75. The method according to embodiment 71, wherein the bacterial antigenic protein is an antigenic protein of a species of *Treponema* or *Mycobacterium*.

[0429] 76. The method according to embodiment 71, wherein the fungal antigenic protein is an antigenic protein of Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Chlamydia trachomatis, or Candida albicans.

[0430] 77. The method according to embodiment 71, wherein the parasite antigenic protein is an antigenic protein of Plasmodium malariae.

[0431] 78. The method according to embodiment 70, wherein the tumor-associated antigenic protein is a leukemia antigenic protein, melanoma antigenic protein, lymphoma antigenic protein, endometrial cancer antigenic protein, renal cancer antigenic protein, brain cancer antigenic protein, cervical cancer antigenic protein, liver cancer antigenic protein, head and neck cancer antigenic protein, gastrointestinal cancer antigenic protein, lymph node cancer antigenic protein, pancreatic cancer antigenic protein, ear, nose and throat (ENT) cancer antigenic protein, breast cancer antigenic protein, prostate cancer antigenic protein, ovarian cancer antigenic protein, or lung cancer antigenic protein.

[0432] 79. The method according to any one of embodiments 49 to 74, wherein the mRNA comprises at least 75%, 85%, 90%, 95%, 98%, 99%, or 100% identical nucleotide sequences to or a portion thereof selected from the following sequences: SEQ ID NO: 1 (spike protein transcript), SEQ ID NO: 2 (enveloping protein transcript), SEQ ID NO: 3 (nucleocapsid protein transcript), SEQ ID NO: 4 (membrane protein transcript) and / or SEQ ID NO: 5 (Orf1ab polyprotein transcript).

[0433] 80. An antigen-specific phDC that can be obtained by any one of the methods in embodiments 49 to 79.

[0434] 81. A vaccine composition comprising a pharmaceutically effective amount of antigen-specific phDC and at least one mRNA encoding at least one antigenic protein.

[0435] 82. The vaccine composition according to embodiment 81 further comprises a pharmaceutically acceptable carrier or diluent.

[0436] 83. The vaccine composition according to embodiment 81 or 82, wherein the mRNA is contained in lipid nanoparticles.

[0437] 84. A vaccine composition according to any one of embodiments 81 to 83, wherein the at least one mRNA comprises at least one modified nucleoside.

[0438] 85. The vaccine composition according to embodiment 84, wherein the at least one modified nucleoside is a substitution of some or all of the uridine residues.

[0439] 86. The vaccine composition according to embodiment 85, wherein some or all of the modified uridine residues comprise one or more uniquely modified uridine residues.

[0440] 87. The vaccine composition according to embodiment 85 or 86, wherein one or more modified uridine residues comprise N1-methyl-pseudouridine.

[0441] 88. A vaccine composition according to any one of embodiments 81 to 87, wherein the at least one mRNA is partially modified with N1-methyl-pseudouridine.

[0442] 89. A vaccine composition according to any one of embodiments 81 to 87, wherein the at least one mRNA is completely modified with N1-methyl-pseudouridine.

[0443] 90. A vaccine composition according to any one of embodiments 81 to 89, wherein the at least one mRNA comprises a 5'UTR, a 3'UTR, a 5' cap, and / or multiple (A) tails.

[0444] 91. The vaccine composition according to embodiment 90, wherein the 5' cap is a Cap1 structure or an m7GpppG cap.

[0445] 92. A vaccine composition according to any one of embodiments 81 to 91, wherein the mRNA comprises a coding sequence encoding at least one antigenic protein.

[0446] 93. A vaccine composition according to any one of embodiments 81 to 92, wherein the at least one mRNA comprises an optimized sequence; optionally, wherein the sequences of the 5' UTR, 3' UTR and / or the coding sequence of the antigenic protein are optimized.

[0447] 94. A vaccine composition according to any one of embodiments 83 to 93, wherein the lipid nanoparticles comprise cationic lipids, PEG-modified lipids, cholesterol and / or non-cationic lipids.

[0448] 95. A vaccine composition according to any one of embodiments 81 to 94, wherein the antigenic protein is associated with a disease, optionally an infectious disease-associated antigenic protein or a tumor-associated antigenic protein.

[0449] 96. The vaccine composition according to embodiment 95, wherein the infectious disease-associated antigenic protein is a viral antigenic protein, a bacterial antigenic protein, a fungal antigenic protein, a prion antigenic protein, or a parasitic antigenic protein.

[0450] 97. The vaccine composition according to embodiment 96, wherein the viral antigenic protein is a coronavirus or HIV antigenic protein, preferably a SARS-CoV-2 antigenic protein.

[0451] 98. The vaccine composition according to embodiment 97, wherein the SARS-CoV-2 antigenic protein is derived from the spike protein, envelope protein, nucleocapsid protein, membrane protein and / or Orf1ab polymer.

[0452] 99. The vaccine composition according to embodiment 96, wherein the bacterial antigenic protein is an antigenic protein of a species of *Treponema* or *Mycobacterium*.

[0453] 100. The vaccine composition according to embodiment 96, wherein the fungal antigenic protein is an antigenic protein of Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Chlamydia trachomatis, or Candida albicans.

[0454] 101. The vaccine composition according to embodiment 96, wherein the parasite antigenic protein is an antigenic protein of Plasmodium malariae.

[0455] 102. The vaccine composition according to embodiment 96, wherein the tumor-associated antigenic protein is a leukemia antigenic protein, melanoma antigenic protein, lymphoma antigenic protein, endometrial cancer antigenic protein, renal cancer antigenic protein, brain cancer antigenic protein, cervical cancer antigenic protein, liver cancer antigenic protein, head and neck cancer antigenic protein, gastrointestinal cancer antigenic protein, lymph node cancer antigenic protein, pancreatic cancer antigenic protein, ear, nose and throat (ENT) cancer antigenic protein, breast cancer antigenic protein, prostate cancer antigenic protein, ovarian cancer antigenic protein, or lung cancer antigenic protein.

[0456] 103. A vaccine composition according to any one of embodiments 81 to 102, wherein the at least one mRNA comprises at least 75%, 85%, 90%, 95%, 98%, 99%, or 100% identical nucleotide sequences to sequences selected from or a portion of sequences selected from the group consisting of: SEQ ID NO: 1 (spike protein transcript), SEQ ID NO: 2 (enveloping protein transcript), SEQ ID NO: 3 (nucleocapsid protein transcript), SEQ ID NO: 4 (membrane protein transcript) and / or SEQ ID NO: 5 (Orf1ab polyprotein transcript).

[0457] 104. A vaccine composition according to any one of embodiments 81 to 103, wherein the phDC is generated by subjecting mononuclear cells obtained from a donor to physical forces prior to combining the phDC with the at least one mRNA.

[0458] 105. The vaccine composition according to embodiment 104, wherein the physical force is applied by passing the mononuclear cells obtained from the donor through a flow chamber.

[0459] 106. A vaccine composition according to embodiment 104 or 105, wherein the mononuclear cells obtained from the donor are derived from an in vitro blood sample or PBMC from the donor.

[0460] 107. A vaccine composition according to any one of embodiments 104 to 106, wherein the flow chamber is a plate.

[0461] 108. A vaccine composition according to any one of embodiments 104 to 106, wherein the flow chamber is a bag, optionally a flexible bag or a plastic bag.

[0462] 109. A vaccine composition according to any one of embodiments 104 to 108, wherein the method includes the further step of incubating the at least one mRNA with the phDC.

[0463] 110. The vaccine composition according to embodiment 109, wherein the incubation step is performed for at least 0.5 h, 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 8 h, 12 h or 24 h.

[0464] 111. An antigen-specific phDC according to embodiment 80 or a vaccine composition according to any one of claims 81 to 110, in a prophylactic method for preventing or reducing disease in a subject, said method comprising administering the antigen-specific phDC or vaccine composition to said subject.

[0465] 112. The antigen-specific phDC or vaccine composition used according to embodiment 111, wherein the vaccine composition is administered subcutaneously, intravenously, intramuscularly, intra-articularly, intrasynovially, intrasternally, intrasheathically, intrahepatically, intralesionally, intracranially, transdermally, intradermally, intrapulmonaryly, intraperitoneally, intracardiacly, intraarterially, intranasally, or sublingually; preferably intradermally, intravenously, or intramuscularly.

[0466] 113. An antigen-specific phDC or vaccine composition used according to embodiment 111 or 112, wherein the disease is caused by pathogenic particles, optionally wherein the pathogenic particles are viruses, bacteria, fungi, parasites and / or tumor cells.

[0467] 114. The antigen-specific phDC or vaccine composition used according to embodiment 113, wherein the concentration of said pathogenic particles is lower in the subject at the time of infection compared with the concentration of pathogenic particles in a second infected subject who has not been administered the vaccine composition.

[0468] 115. The antigen-specific phDC or vaccine composition used according to embodiment 113, wherein the concentration of the pathogenic particle is lower in the subject at the time of infection compared with the concentration of the pathogenic particle in a second infected subject who has been vaccinated with a different vaccine against the same pathogenic particle.

[0469] 116. The antigen-specific phDC or vaccine composition used according to embodiment 115, wherein the different vaccines do not contain dendritic cells.

[0470] 117. The antigen-specific phDC or vaccine composition used according to embodiment 115 or 116, wherein the different vaccines are RNA vaccines.

[0471] 118. The antigen-specific phDC or vaccine composition used according to embodiment 115 or 116, wherein the concentration of the pathogenic particles in the subject is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% compared to the concentration of the pathogenic particles in the second subject.

[0472] 119. The antigen-specific phDC or vaccine composition used according to embodiment 113, wherein the concentration of the pathogenic particles is low in the body in the subject after infection.

[0473] 120. The antigen-specific phDC or vaccine composition used according to embodiment 113, wherein the concentration of the pathogenic particles is locally low, optionally wherein the concentration of the pathogenic particles is low in the brain.

[0474] 121. The antigen-specific phDC or vaccine composition used according to any one of embodiments 111 to 120, wherein the disease in the subject is an infectious disease.

[0475] 122. The antigen-specific phDC or vaccine composition used according to any one of embodiments 111 to 120, wherein the disease in the subject is a viral disease.

[0476] 123. An antigen-specific phDC or vaccine composition used according to any one of embodiments 111 to 120, wherein the disease in the subject is Covid-19.

[0477] 124. An antigen-specific phDC or vaccine composition used according to any one of embodiments 111 to 113, wherein the disease in the subject is a tumor.

[0478] 125. An antigen-specific phDC or vaccine composition used according to any one of embodiments 111 to 113, wherein the disease in the subject is a blood cancer.

[0479] 126. Use of a vaccine composition according to any one of embodiments 113 to 125 for vaccination.

[0480] 127. A kit containing physiological dendritic cells and at least one mRNA, wherein the at least one mRNA encodes at least one antigenic protein.

[0481] 128. The kit according to embodiment 127, wherein the phDC is obtained by passing mononuclear cells obtained from a donor through a flow chamber.

[0482] 129. The kit according to embodiment 127 or 128, wherein the at least one mRNA encodes at least one viral antigenic protein.

[0483] 130. The kit according to embodiment 127 or 128, wherein the at least one mRNA encodes at least one tumor-associated antigenic protein.

[0484] 131. The kit according to embodiment 129, wherein the viral antigenic protein is a coronavirus antigenic protein, preferably a SARS-CoV-2 antigenic protein.

[0485] 132. The kit according to embodiment 131, wherein the SARS-CoV-2 antigenic protein is derived from the spike protein, envelope protein, nucleocapsid protein, membrane protein and / or Orf1ab polymer.

[0486] 133. A kit according to any one of embodiments 127 to 132, wherein the at least one mRNA is formulated in lipid nanoparticles.

[0487] 134. A method for preventing or reducing disease in a subject, the method comprising administering to the subject an antigen-specific phDC of embodiment 80 or a vaccine composition according to any one of embodiments 81 to 110.

[0488] 135. The method according to embodiment 134, wherein the vaccine composition is administered subcutaneously, intravenously, intramuscularly, intra-articularly, intra-synovially, intrasternally, intrasheathically, intrahepatically, intralesionally, intracranially, transdermally, intradermally, intrapulmonaryly, intraperitoneally, intracardiacly, intra-arterially, or sublingually; preferably intradermally, intravenously, or intramuscularly.

[0489] 136. The method according to implementation scheme 134 or 135, wherein the disease is caused by pathogenic particles, optionally wherein the pathogenic particles are viruses, bacteria, fungi, parasites and / or tumor cells.

[0490] 137. The method according to embodiment 136, wherein the concentration of pathogenic particles in a subject at the time of infection is lower than the concentration of pathogenic particles in a second infected subject who has not been administered the vaccine composition.

[0491] 138. The method according to implementation plan 137, wherein the concentration of pathogenic particles in the subject at the time of infection is lower than the concentration of pathogenic particles in a second infected subject who has been vaccinated with a different vaccine against the same pathogenic particles.

[0492] 139. The method according to implementation scheme 137 or 138, wherein the concentration of pathogenic particles in the subject is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% compared to the concentration of pathogenic particles in the second subject.

[0493] 140. The method according to implementation plan 136, wherein the concentration of pathogenic particles is low in the systemic level of the subjects after infection.

[0494] 141. The method according to embodiment 136, wherein the concentration of pathogenic particles is locally low, optionally wherein the concentration of pathogenic particles is low in the brain.

[0495] 142. The method according to any one of implementation schemes 134 to 141, wherein the disease in the subject is an infectious disease.

[0496] 143. The method according to any one of implementation schemes 134 to 141, wherein the disease in the subject is a viral disease.

[0497] 144. The method according to any one of implementation schemes 134 to 141, wherein the disease in the subject is Covid-19.

[0498] 145. The method according to any one of implementation schemes 134 to 136, wherein the disease in the subject is a tumor.

[0499] 146. The method according to any one of implementation schemes 134 to 136, wherein the disease in the subject is blood cancer. Example

[0500] Example 1. Preparation of phDC transduced by LNP particles

[0501] Isolation of mouse peripheral blood mononuclear cells (PBMCs)

[0502] Peripheral blood (100–200 μL / mouse) is collected from experimental and control mice (and any additional bleeding mice if needed) as required for the experiment; for example, on days 1 and 7 for prophylactic vaccination and booster studies, or twice weekly during treatment for therapeutic tumor vaccination studies. Whole blood is collected in 1:100 5,000 U / mL heparin (McKesson Packaging Services). Platelet-containing peripheral blood mononuclear cells (PBMCs) are isolated from peripheral whole blood via Lympholyte M gradient separation (Cedarlane Labs). Autologous serum is collected from a separate cohort of syngeneic donor mice and reserved for subsequent steps (overnight culture).

[0503] Transimmunization room

[0504] A miniaturized ECP device suitable for use in animal models is called the Transimmune (TI) chamber, designed and manufactured by Transimmune AG in collaboration with the Fraunhofer Institute for Biomedical Engineering, Saarland, Germany, for Dr. Edelson's laboratory. The sterile polystyrene TI chamber has external dimensions of 25 * 75 mm, a flow path of 18 * 66 mm, and a flow channel height of 290 + / - 15 μm.

[0505] PBMC TI processing solution

[0506] The isolated PBMCs containing mouse platelets were resuspended in fetal bovine serum (FBS). The cells were then incubated in a TI chamber at 37°C for 1 hour. This step allowed platelet-activated plasma proteins to deposit in the chamber, and platelets to adhere to the coated chamber surface, as confirmed by optical microscopy. The cells were then passed through the TI chamber using an infusion pump at a rate of 0.09 mL / min. After plate passage, the cells were collected, and the TI chamber was washed with 100% FBS at 0.49 mL / min, while simultaneously physically agitating the chamber surface by tapping or flicking to help detach any adhered cells. The collected cells were washed and cultured overnight under standard conditions in phenol red-free (Gibco)-free RPMI supplemented with 15% autologous mouse serum and 1% penicillin / streptomycin / L-glutamine (Invitrogen).

[0507] LNP containing mRNA transfected phDC

[0508] When setting up the culture, the desired amount of LNPs (cKK-E12, cholesterol, C14-PEG2000-PE, and DOPE in a ratio of 35:46.5:2.5:16) containing mRNA targeting the antigen of interest (such as the SARS-CoV-2 spike protein or sample tumor antigen) was directly added to the overnight phDC culture. Based on our experience, the amount of LNP can range from 20 μg to 10 ng, depending on the LNPs and antigens used.

[0509] reintroducing phDC into laboratory animals

[0510] The next day, LNP-transfected cells were harvested by scraping, washed, resuspended in sterile PBS (Gibco), and administered intravenously at 100 μL / animal via the retro-orbital plexus.

[0511] Example 2. Internalization of phDCs containing viral antigens in LNPs

[0512] method

[0513] phDCs were prepared from blood of healthy C57BL / 6 donor mice using the standard protocol described in Example 1. The desired amount of LNPs containing mRNA targeting the viral antigen of interest (such as SARS-CoV-2 spike protein mRNA) was added to the PBMCs passed through the plate. In this example, 20 μg / mL of cKK-E12-based LNPs containing SARS-CoV-2 spike protein mRNA (LNPs prepared and supplied by the Santangelo Laboratory, Emory University, mRNA sequence corresponding to SEQ ID NO: 19) was used. The PBMCs passed through the plate were incubated with the LNPs overnight to allow for LNP uptake and antigen expression.

[0514] Cells of interest were identified using an appropriate fluorescently labeled detection antibody (anti-human SARS-CoV-2 spike protein antibody, kindly provided by the Santangelo Laboratory, Emory University) and any necessary antibodies (e.g., CD11b of mouse phDCs within PBMCs passing through a plate). + Ly6G - ;CD11b Biolegend clone M1 / 70; Ly6GBiolegend clone 1A8), antigen expression was monitored after overnight incubation.

[0515] The binding of antibodies to phDCs can be detected by flow cytometry (using a Cytoflex cytometer and FlowJo v10 software) or by confocal microscopy.

[0516] result

[0517] Among all other immune cell subsets contained in PBMCs, mouse phDCs specifically internalized cKK-E12 LNPs and efficiently expressed the spike protein encoded by the mRNA contained in these LNPs. Compared to cultures without LNP transfection, FACS analysis showed significant differences in CD11b... + phDCs specifically showed positivity for the spike protein (Figure 1 A, B). This was confirmed by confocal microscopy analysis, which showed positivity for CD11b. + Ly6G - phDC specifically shows spike protein expression on the cell surface and in the cytoplasm (projected Z-stack images, Fig. 1 C, D; Z-plane slice images, Fig. 1 E).

[0518] Example 3. Therapeutic phDC administration in an EG7-OVA lymphoma mouse model using LNP (ovalbumin (OVA) antigen) containing tumor antigen mRNA.

[0519] Experimental design in Figure 2A The diagram is illustrated in the text.

[0520] For tumor induction, 3x10 6 One EG7-OVA tumor cell was subcutaneously injected into the right abdomen of wild-type C57BL / 6J mice at a dose of 100 mL. Therapeutic treatment was initiated on day 2 post-tumor implantation. For each treatment, phDC was prepared from the blood of each experimental group (200 μL of blood collected from each animal) using the standard protocol described in Example 1. Blood from groups not treated with phDC was discarded.

[0521] 10 ng (0.5 ug / kg LNP, equivalent to the dose of the Pfizer mRNA LNP vaccine for COVID-19 in the current human vaccine scenario) of cKK-E12-based LNPs containing mRNA targeting a tumor antigen of interest was added to PBMCs via plate. Here, the tumor antigen of interest was the model antigen ovalbumin (OVA), which had been introduced into EL4 lymphoma cells to generate the EG7-OVA tumor model (SEQ ID NO: 20, also used in all subsequent experiments related to LNPs containing OVA mRNA). All mRNAs and LNPs were prepared and supplied by the Santangelo Laboratory, Emory University.

[0522] After overnight incubation, cells co-cultured with LNPs containing OVA mRNA were collected and intravenously injected into the retroorbital plexus of animals in the "phDC"-treated experimental group in 100 μL of sterile PBS. Simultaneously, an equal dose of LNPs containing OVA mRNA in 10–50 μL of sterile PBS was injected intramuscularly ("IM") into the thigh of each animal in the "untreated" ("no Tx") group. Animals in the "no treatment" ("no Tx") group received no treatment.

[0523] Treatment was repeated twice a week until the end of the experiment, determined by the maximum tumor growth rate allowed by the animal care facility. Mice were typically bled on Mondays and Thursdays for therapeutic production of phDCs, with phDC or IM treatment performed on Tuesdays and Fridays. Typically, 5-6 therapeutic immunotherapy treatments could be administered every two weeks.

[0524] Tumor volume was monitored by measuring the vertical diameter and height of the tumor every two weeks using calipers, and the tumor volume was calculated as (tumor length x width x height) / 2.

[0525] At the end of the experiment (day 30 post-tumor inoculation), spleen cells were harvested from all treatment and control groups for further analysis to characterize the resulting anti-tumor immune response. Elispot analysis: CD8+ spleen T cells from treated mice (negatively selected using the Miltenyi CD8 T cell isolation kit) were immediately introduced at 1*10-1 per well, with or without 10 μg / mL SIINFEKL peptide. 5 Cells were placed in an 18-hour IFN-γ Elispot assay. Evaluation of SIINFEKL-specific CD8 T cells: CD8 (BioRad clone KT1.5) and specific H2d-SIINFEKL dextramer staining (Immudex) were performed, followed by flow cytometry (Cytoflex). Phenotypic analysis of Tem (effective) / Tcm (central memory) type: expression of CD44 (Biolegend clone IM7) and CD62L (Biolegend clone MEL-14) on antigen-specific T cells. Staining was performed, followed by flow cytometry (Cytoflex). Stem cell-like T cell analysis: IL7Ra (Biolegend clone A7R34) and SCA-1 (Biolegend clone E13-161.7) expression on antigen-specific T cells were stained, followed by flow cytometry (Cytoflex). Evaluation of T cell exhaustion markers: PD-1 (Biolegend clone 29F.1A12) expression on antigen-specific T cells was assessed, followed by flow cytometry (Cytoflex).

[0526] result

[0527] Compared to the untreated control group (“No Tx”), therapeutic phDC treatment with a clinically relevant dose of 10 ng of LNP containing OVA mRNA (0.5 μg / kg LNP, equivalent to the dose of the current human vaccine setting Pfizer mRNA LNP for COVID-19) successfully controlled the growth of EG7-OVA tumors, while direct intramuscular administration of the same amount of LNP containing OVA mRNA (“IM” group) did not significantly alter tumor growth kinetics. Figure 2B ).

[0528] To better understand the therapeutic effect of phDC relative to the IM vaccination method, spleen cells were collected at the end of the experiment (day 30 post-tumor inoculation) for further analysis. Figure 3 AC, experimental schematic). Overall responsiveness to the OVA-dominant immune peptide SIINFEKL, measured by IFNg Elispot analysis of spleen cells from different groups, showed: some responses in the untreated control group, as expected in tumor-bearing mice expressing OVA; stronger responses in the phDC-treated group; and interestingly, the strongest responses in the IM vaccine group. Figure 3 DE), although such responses are clinically unproductive. Figure 2B ).

[0529] Therefore, it is clear that the total amount of model tumor antigen OVA-specific T-cell responses does not control tumor growth. Thus, to explain the strong therapeutic effect of phDC treatment, it must be the quality of the generated T-cell responses. The quality of T-cell responses on day 30 was further investigated in spleen cells of tumor-bearing treated mice. This analysis showed that antigen-specific T cells in phDC-treated mice exhibited a phenotype characterized by the presence of central memory T cells and stem cell-like T-cell subsets (Fig. 4A), which have been associated with productive anti-cancer immune responses in human and mouse studies. In contrast, T cells from the IM vaccination group showed a strong effector T-cell-dominated phenotype (Fig. 4A), consistent with the strong responses observed in the IFNg Elispot assay. Figure 3 DE).

[0530] The effector-like response generated by IM vaccination appears to be clinically ineffective in a tumor context; this may be due to the potential exhaustion of such effector T cells, as evidenced by their high PD1 expression compared to antigen-specific T cells from phDC-treated animals. Figure 4B ).

[0531] Example 4. Analysis of spontaneous immune response in prophylactic phDC vaccination using LNPs containing ovalbumin (OVA) sample antigen mRNA.

[0532] method

[0533] For each treatment, phDC was prepared from the blood of healthy C57BL / 6 mice from each experimental group (200 μL of blood collected from each animal) using the standard protocol described in Example 1. Blood from groups not treated with phDC was discarded.

[0534] Six μg of cKK-E12-based LNPs containing mRNA targeting the tumor antigen of interest (in this case, the model antigen ovalbumin (OVA)) were added to the PBMCs of the culture via plate. All LNPs were prepared and supplied by the Santangelo Laboratory, Emory University.

[0535] PBMCs were incubated with LNPs overnight via the plate to allow for LNP uptake and antigen expression. The following day, PBMCs were collected and prepared as described in Example 1, and administered intravenously at 100 μL / animal via the retroorbital plexus to the experimental group inoculated with “phDC [ova]”. In parallel, an equal dose of LNPs containing OVA mRNA was injected intramuscularly (“IM [ova]”) into the thigh of each animal in the experimental group inoculated with IM [ova]”. Animals in the “untreated” group received no treatment.

[0536] Mice were exsanguinated on days -8 and -1, and prophylactically vaccinated with phDC or intramuscular LNP on days -7 and 0. Intact undifferentiated spleen cells were collected on days 0, 5, and 13 post-vaccination for analysis of spontaneous immune responses using IFNgElispot assay. Elispot assay: In the absence of any additional stimulation, intact undifferentiated spleen cells from all experimental groups were immediately collected at 1*10-1 saturates per well. 5 The cells were placed in an 18-hour IFN-g Elispot assay. To further identify the cells responsible for IFN-g production, some day 13 samples were further fractionated into CD8 T cells (negative selection using the Miltenyi CD8 T cell isolation kit) or NK cells (positive selection using the Miltenyi NK1.1 cell isolation kit).

[0537] result

[0538] Interestingly, animals that were prophylactically vaccinated and boosted with phDC (instead of intramuscular LNP injection) exhibited spontaneous IFNg production in the absence of any additional stimulation. Figure 5 (AC). This spontaneous response is detectable for at least 13 days after vaccination. Figure 5 C).

[0539] Further investigation into the source of this spontaneous IFNg signaling by isolating CD8 T cells or NK cells revealed that NK cells were identified as an IFNg-secreting cell subset. This indicates that phDC vaccination not only initiates antigen-specific T cell and B cell responses (Figures 4 and 5), but also extensively participates in the innate immune system, such as NK cells.

[0540] Example 5. Prophylactic phDC vaccination in an EG7-OVA lymphoma mouse model using LNP (ovalbumin (OVA) antigen) containing tumor antigen mRNA.

[0541] Experimental design in Figure 6 A is described schematically.

[0542] For each vaccination treatment, phDC was prepared from the blood of each experimental group (200 μL of blood collected from each animal) using the standard protocol described in Example 1. Blood from groups not treated with phDC was discarded.

[0543] For initial vaccination (day -14), 1 μg of cKK-E12-based LNPs containing mRNA targeting the tumor antigen of interest (in this case, ovalbumin (OVA), a model antigen that has been introduced into EL4 lymphoma cells to generate the EG7-OVA tumor model) were added to the PBMCs in the culture via plate. All LNPs were prepared and supplied by the Santangelo Laboratory, Emory University. After overnight incubation, cells co-cultured with LNPs containing OVA mRNA were collected and intravenously injected into the retro-orbital plexus of animals in the “phDC”-treated group in 100 μL of sterile PBS. Simultaneously, 50 μg of soluble OVA protein in 100 μL of sterile PBS was injected into the retro-orbital plexus of animals in the “soluble OVA”-treated group.

[0544] A booster vaccination is given one week after the initial vaccination treatment (day -7), in the same manner as described above.

[0545] For tumor attack on day 0, 1x10 in 100 mL 6 EG7-OVA tumor cells were subcutaneously injected into the right abdomen of mice from all treatment groups. Tumor volume was monitored by measuring the vertical tumor diameter and height every two weeks using calipers, and the tumor volume was calculated as (tumor length x width x height) / 2.

[0546] result

[0547] Prophylactic phDC vaccination with LNPs containing OVA mRNA successfully inhibited the growth of EG7-OVA tumors, while vaccination with soluble OVA (“soluble Ova” group) did not protect against EG7-OVA tumor attack. Figure 6 B).

[0548] Experiment 6. Detection of OVA protein in phDC transduced with OVA mRNA and LNP.

[0549] method

[0550] PhDCs were prepared from blood of healthy C57BL / 6 donor mice using the standard protocol described in Experiment 1. The desired amount of LNPs containing mRNA targeting the antigen of interest, such as 1 μg / mL of cKK-E12-based LNPs containing OVA or SARS-CoV-2 spike protein mRNA, was added to the PBMCs passed through the plate to the culture. The PBMCs were incubated overnight with the LNPs to allow for LNP uptake and antigen expression.

[0551] After overnight incubation, antigen expression was monitored by intracellular staining of cells with fluorescently labeled anti-OVA antibodies (Rockland Immunochemicals) and any antibodies necessary for identifying cells of interest (e.g., CD11b+ for mouse phDCs in PBMCs passing through the plate; CD11b Biolegend clone M1 / 70).

[0552] The binding of antibodies to phDCs can be detected by flow cytometry (using a Cytoflex cytometer and FlowJo v10 software).

[0553] result

[0554] Among all other immune cell subsets containing PBMCs, mouse phDCs specifically express the OVA protein encoded by mRNA contained in cKK-E12 LNPs. FACS analysis specifically showed OVA protein positivity in CD11b+ phDCs compared to cultures transfected with mimic (spike protein) LNPs. Figure 7 ).

[0555] Experiment 7. Detection of SIINFEKL-bound MHC I (H-2Kb) in mouse phDCs loaded with soluble OVA, tumor cells expressing OVA, or OVA mRNA LNP.

[0556] method

[0557] PhDCs were prepared from blood of healthy C57BL / 6 donor mice using the standard protocol described in Experiment 1. Antigen sources, such as soluble OVA protein (10 μg / mL; 50 μg / mL); EG7-OVA tumor cells treated with 8-MOP / UVA (200 ng / mL 8-MOP, UVADEX, Therakos; 2 or 4 J / cm² UVA); or LNPs containing OVA protein mRNA (1 μg / mL or 5 μg / mL cKK-E12-based LNPs) (LNPs prepared and supplied by the Santangelo Laboratory, Emory University), were incubated overnight with the corresponding antigen source to allow antigen uptake and / or expression and processing.

[0558] After overnight incubation, the presentation of the antigenic OVA peptide SIINFEKL on the phDC H-2Kb MHCI molecule was detected by staining cells with fluorescently labeled 25.D1 TCR-like antibody (Biolegend, clone 25-D1.16) and any necessary antibodies used to identify cells of interest (e.g., by staining cells in PBMCs of mouse phDCs for CD11b+, Ly6G-; CD11b Biolegend clone M1 / 70; Ly6G Biolegend clone 1A8).

[0559] The binding of antibodies to phDCs can be detected by flow cytometry (using a Cytoflex cytometer and FlowJo v10 software).

[0560] result

[0561] Of all the other immune cell subsets contained in PBMCs, mouse phDCs specifically process and present the antigenic OVA SIINFEKL peptide in their MHCI molecules. In contrast to delivery via soluble OVA protein or by dying tumor cells expressing OVA protein, antigenic peptide presentation was significantly higher when OVA protein was expressed via mRNA LNP (Figure 8).

[0562] Experiment 8. Time-kinetics of surface SIINFEKL-MHC I complex expression in phDC transduced with OVA mRNA LNP (via 25.D1 ab staining)

[0563] method

[0564] PhDCs were prepared from blood of healthy C57BL / 6 donor mice using the standard protocol described in Experiment 1. The desired amount of LNPs containing mRNA targeting the antigen of interest, such as 1 μg / mL of cKK-E12-based LNPs containing OVA protein mRNA, was added to the PBMCs passed through the plate. (LNPs were prepared and supplied by the Santangelo Laboratory, Emory University). The PBMCs passed through the plate were incubated with LNPs for 2, 4, 6, 8, 12, or 20 hours, followed by washing and fixation for staining.

[0565] After incubation for 2–20 hours, the expression of the antigenic OVA peptide SIINFEKL on the phDC H-2Kb MHCI molecule was detected by staining cells with fluorescently labeled 25.D1 TCR-like antibody (Biolegend, clone 25-D1.16) and any necessary antibody used to identify cells of interest (e.g., by staining cells with CD11b+ of mouse phDCs within PBMCs of the plate; CD11b Biolegend clone M1 / 70).

[0566] The binding of antibody to phDCs can be detected by flow cytometry and by the percentage of antibody-positive cells / the mean fluorescence intensity (MFI) of the quantitative positive cells (Cytoflex cytometer, analyzed using FlowJo v10 software).

[0567] result

[0568] The percentage of phDCs displaying antigenic OVA SIINFEKL peptides bound to MHCI molecules on their surface, and the amount of SIINFEKL / MHCI per cell, increased with incubation time of phDCs with OVA mRNA LNPs (Fig. 9). The expression level of 25.D1 in CD11b+ cells at different time points is shown (FACS plot, top). The percentage of 25.D1-positive cells in the CD11b+ subset and the MFI level of 25.D1 are shown in the bar plot (bottom).

[0569] Experiment 9. Detection of SIINFEKL-bound MHC I (H-2Kb) on mouse phDCs or BMDCs transduced with OVA mRNA LNP (at reduced LNP concentrations).

[0570] method

[0571] PhDCs were prepared from the blood of healthy C57BL / 6 donor mice using the standard protocol described in Experiment 1. BMDCs were cultured from the bone marrow of healthy C57BL / 6 donor mice according to the standard protocol. Briefly, bone marrow cells were plated in cell culture medium containing GM-CSF (20 ng / ml); after 5 days, non-adherent cells were washed and replated in fresh GM-CSF-supplemented medium, and incubated for another 48 hours; subsequently, non-adherent BMDC cells were removed, washed, and immediately used for experiments.

[0572] Add the desired amount of LNP containing mRNA targeting the antigen of interest, such as 0.1 μg / mL of cKK-E12-based LNP containing OVA protein mRNA (LNP prepared and supplied by the Santangelo Laboratory, Emory University), to the PBMCs or BMDCs passed through the plate. Incubate the PBMCs or cultured BMDCs with the LNPs overnight to allow for LNP uptake and antigen expression.

[0573] After incubation, the expression of the antigenic OVA peptide SIINFEKL on the phDC H-2Kb MHCI molecule was detected by staining cells with fluorescently labeled 25.D1 TCR-like antibody (Biolegend, clone 25-D1.16) and any necessary antibodies used to identify cells of interest (e.g., by staining cells in PBMCs of the plate with CD11b+ of mouse phDCs; CD11b Biolegend clone M1 / 70; CD11c of mouse BMDCs, Biolegend clone N418).

[0574] The binding of antibodies to phDC or BMDC can be detected by flow cytometry (using a Cytoflex cytometer and FlowJov10 software).

[0575] result

[0576] The percentage of phDCs displaying the antigenic OVA SIINFEKL peptide bound to the MHCI molecule on their surface was significantly higher than that of BMDCs displaying the peptide, indicating that phDCs are superior in terms of transfection with OVA mRNA LNP or antigen expression and processing from LNP mRNA antigen sources or all of the above (Figure 10).

[0577] Experiment 10. Surface 25.D1 staining in phDC transduced with SIINFEKL peptide mRNA and OVA protein mRNA.

[0578] method

[0579] PhDCs were prepared from blood of healthy C57BL / 6 donor mice using the standard protocol described in Experiment 1. The desired amount of LNPs containing mRNA targeting the antigen of interest was added to the PBMCs passed through the plate, such as 1 μg / mL of cKK-E12-based LNPs containing OVA protein mRNA, or 0.05, 0.1, 0.5, 1, or 5 μg of LNPs containing immunogenic OVASIINFEKL peptide mRNA (LNPs were prepared and supplied by the Santangelo Laboratory, Emory University, SEQ ID NO: 21). The PBMCs passed through the plate were incubated with the LNPs overnight to allow for LNP uptake and antigen expression.

[0580] After overnight incubation, the expression of the antigenic OVA peptide SIINFEKL on the phDC H-2Kb MHCI molecule was detected by staining cells with a fluorescently labeled 25.D1 TCR-like antibody (Biolegend, clone 25-D1.16) and any necessary antibody used to identify cells of interest (e.g., by staining cells with CD11b+ of mouse phDCs within the PBMCs of the plate; CD11b Biolegend clone M1 / 70).

[0581] The binding of antibodies to phDC or BMDC can be detected by flow cytometry (using a Cytoflex cytometer and FlowJov10 software).

[0582] result

[0583] At the same LNP concentration (1 μg / mL), phDCs transduced with SIINFEKL peptide mRNA LNPs exhibited a higher percentage of antigenic OVA bound to MHCI molecules on their surface compared to phDCs transduced with OVA protein mRNA LNPs (Figure 11). PhDC transduction with SIINFEKL peptide mRNA LNPs was also effective at much lower LNP concentrations, exhibiting surface antigen / MHCI complexes with as little as 0.05 μg / mL of LNPs used. In conclusion, these data demonstrate that loading phDCs with a single immunogenic peptide, rather than with the whole protein, as an antigen can be highly efficient.

[0584] Experiment 11. OT1 proliferation assay: phDCs cultured with OT1 T cells were subjected to a titration of LNP or soluble OVA protein pulses.

[0585] method

[0586] PhDCs were prepared from blood of healthy C57BL / 6 donor mice using the standard protocol described in Experiment 1. Antigen sources, such as soluble OVA protein (50 μg / mL; 200 μg / mL); or LNPs containing OVA protein mRNA (1, 0.1, 0.01, or 0.001 μg / mL cKK-E12-based LNPs) were added to the PBMCs passed through the plate (LNPs were prepared and supplied by the Santangelo Laboratory, Emory University). The PBMCs passed through the plate were incubated overnight with the corresponding antigen source to allow antigen uptake and / or expression and processing.

[0587] After overnight incubation, antigen-loaded cells were harvested and cultured in 96-well plates at a rate of 2 x 10⁻⁶ cells / well under standard conditions. 5 cells / mL and 1*10 5CFSE-labeled OVA-specific OT1 CD8 T cells / mL (isolated from the spleen of C57Bl / 6-Tg(TcraTcrb)1100Mjb / J mice, recognizing OVA peptide residues 257 to 264 against an H-2Kb background, Jackson Laboratory) were co-cultured for 3 days. (Note that the phDCs were unpurified; the cell count is for total PBMCs, of which 3-10% are CD11+ phDCs; therefore, the number of phDCs in PBMCs is at most approximately 2*10^6 cells / mL.) 4 Cells / mL). At the end of co-culture, cells were stained with anti-CD8 antibody to identify antigen-reactive T cells (Biolegend, clone 53-6.7).

[0588] As indicated by CFSE dilution in OT1 CD8+ T cells, antigen-specific CD8 T cell proliferation can be assessed by flow cytometry (Cytoflex cytometer, analyzed using FlowJo v10 software).

[0589] result

[0590] When phDCs transduced with OVA protein mRNA LNPs were cultured together, the percentage of OVA-responsive OT1 CD8 T cells proliferating was very high, even at the lowest LNP concentration (0.001 ug / mL) (Figure 12). This demonstrates that LNP-transduced phDCs are extremely potent in stimulating antigen-specific CD8 T cell responses. Furthermore, phDC transduction with the lowest LNP concentration (0.001 ug / mL) was at least as potent in stimulating CD8 T cell responses as phDCs loaded with the highest concentration of soluble OVA protein (200 ng / mL), indicating that mRNA LNPs are a significantly superior antigen source.

[0591] Experiment 12. OT1 proliferation assay: OT1 T cells cultured with titrated phDC (at 1 μg / mL LNP)

[0592] method

[0593] PhDCs were prepared from blood of healthy C57BL / 6 donor mice using the standard protocol described in Experiment 1. Antigen sources, such as LNPs containing OVA protein mRNA (1 μg / mL cKK-E12-based LNPs) (LNPs prepared and supplied by the Santangelo Laboratory, Emory University), were added to the PBMCs passed through the plate in culture. The PBMCs passed through the plate were incubated overnight with the corresponding antigen source to allow for antigen uptake and / or expression and processing.

[0594] After incubation overnight, cells were harvested and cultured in 96-well plates at a rate of 2 x 10⁻⁶ cells / well under standard conditions. 5 1*10 5 5*10 4 2.5*10 4 Or 1.2*10 4 cells / mL and 1*10 5 CFSE-labeled OVA-specific OT1 CD8 T cells / mL (isolated from the spleen of C57Bl / 6-Tg (TcraTcrb) 1100Mjb / J mice, recognizing OVA peptide residues 257 to 264 against an H-2Kb background, Jackson Laboratory) were co-cultured for 3 days. (Note that the phDCs were unpurified; the cell count is for total PBMCs, of which 3-10% are CD11+ phDCs; therefore, the number of phDCs in PBMCs is at most approximately 2*10^6 cells / mL.) 4 1*10 4 5*10 3 2.5*10 3 Or 1.2*10 3 Cells / mL). At the end of co-culture, cells were stained with anti-CD8 antibody to identify antigen-reactive T cells (Biolegend, clone 53-6.7).

[0595] As indicated by CFSE dilution in CD8+ T cells, CD8 T cell proliferation can be assessed by flow cytometry (Cytoflex cytometer, analyzed using FlowJo v10 software).

[0596] result

[0597] When co-cultured with titrated OVA protein mRNA LNP-transduced phDCs, the percentage of proliferating OVA-responsive OT1CD8 T cells remained exceptionally high, with as low as 1,200 phDCs / mL (120 cells per well in a 96-well plate) retaining the ability to stimulate proliferation in up to 47% of OT1 CD8 T cells (Figure 13). This again demonstrates that mRNA LNP-transduced phDCs are an extremely potent and efficient stimulator of antigen-specific CD8 T cell responses.

[0598] Experiment 13. Therapeutic phDC administration in an EG7-OVA lymphoma mouse model using LNP (ovalbumin (OVA) antigen) containing tumor antigen mRNA.

[0599] method

[0600] The experimental protocol was as described in Experiment 3, with the only difference being that control mice received the same amount (10 ng / mouse) of mimic mRNA LNP (which expresses the SARS-CoV-2 spike protein and is irrelevant in the EG7-OVA tumor model background) intramuscularly every two weeks, instead of remaining untreated.

[0601] Therefore, the experimental group is:

[0602] -Intramuscular vaccination with mimicry (SARS-CoV-2 spike protein) mRNA LNP

[0603] -OVA mRNA LNP intramuscular vaccination

[0604] -OVA mRNA LNP phDC retroorbital vaccination

[0605] Treatment was administered on days 4, 8, 11, 15, 18, and 25 after EG7-OVA tumor implantation. Figure 14A ).

[0606] result

[0607] Compared to the control mimicry treatment group (“mimicry mRNA IM”), therapeutic phDC administration (“OVA mRNA phDC”) at a clinically relevant dose of 10 ng of LNP containing OVA mRNA (0.5 ug / kg LNP, equivalent to the dose of Pfizer’s COVID-19 mRNA LNP vaccine in the current human vaccine context) successfully controlled the growth of EG7-OVA tumors, while direct intramuscular administration of the same amount of LNP containing OVA mRNA (“OVA mRNA IM (without phDC)” group) did not significantly alter tumor growth kinetics. Figure 14B ).

[0608] Experiment 14. SIINFEKL tetramer analysis and adoptive T cell transfer

[0609] method

[0610] At the end of Experiment 13 above, spleen cells were harvested from all treatment and control groups (harvested at the end of the in vivo tumor surveillance period (day 32 post-tumor inoculation)) for further analysis to characterize the obtained anti-tumor immune response.

[0611] Evaluation of SIINFEKL-specific CD8 T cells: The isolated spleen cells were stained with CD8 (BioRad, clone KT1.5) and specific H2d-SIINFEKL dextramer (Immudex), and then flow cytometry was performed for analysis (Cytoflex).

[0612] Adoptive T cell transfer: CD3+ T cells isolated from spleen cells in the three experimental groups of Experiment 13 (Miltenyi T cell isolation kit) were transferred at a rate of 1.5*10-1. 7 T cells / mouse were intravenously transferred to antigen-naive C57BL / 6 mice that had recently been inoculated with EG7-OVA tumors (as described in Experiment 3), and tumor development was monitored over a period of 19 days.

[0613] result

[0614] Both therapeutic OVA protein mRNA LNP-transduced phDC vaccination (“OVA phDC”) and intramuscular OVA protein mRNA LNP vaccination (“OVA IM”) successfully induced OVA antigen-specific T cells, as measured by the percentage of H-2Kb SIINFEKL tetramer-positive CD8 T cells. Figure 15 A). Animals treated with the mimic (“mimic IM”) also showed some tetramer positivity, reflecting the presence of background innate T immunity in mice carrying EG7-OVA tumors.

[0615] However, although tumor-reactive T cells were present in all groups, only T cells isolated from the spleen of mice inoculated with phDCs transduced with OVA protein mRNA LNP (“OVA phDCs”) conferred protective immunity against EG7-OVA tumors in antigen-naïve uninoculated mice. Figure 15 B). This indicates that only phDC therapeutic vaccination provides true anti-tumor immunity.

[0616] Experiment 15: Detection of ELISpot spike protein in SARS-CoV-2 reactive T cells

[0617] LNPs containing spike protein mRNA transduced into human phDC

[0618] The PBMCs (containing neonatal phDCs from healthy human donors) generated according to the method described in Experiment 1 were processed at a 5*10⁻⁶ ppm. 5Cells / well were seeded in human IFNγ ELISpot plates, in triplicate for each experimental group. 62.5–250 ng / well of LNP containing spike protein mRNA (cKK-E12, cholesterol, C14-PEG 2000-PE, and DOPE in a ratio of 35:46.5:2.5:16) (LNP prepared and supplied by the Santangelo Laboratory, Emory University; RNA sequence corresponds to SEQ ID NO: 19) was added to each well in 200 μL of medium supplemented with 15% autologous human plasma and 1% penicillin / streptomycin / L-glutamine (Invitrogen) without phenol red (RPMI, Gibco). Positive control wells were treated with an overlap pool of spike class I and II peptides (Miltenyi Biotec, PepTivator SARS-CoV-2 Prot-SComplete). The negative control wells were treated with LNP containing unrelated (e.g., Nanoluciferase) protein mRNA based on cKK-E12 at a dose of 62.5–250 ng / well.

[0619] Elispot readings

[0620] After overnight incubation, wells were washed, and IFNγ spots were detected using biotinylated anti-human IFNγ mAb, streptavidin-ALP, and BCIP / NBT-plus substrates according to the manufacturer's protocol (MAbTech, 3420-2AST-2). Spot-forming units (SFU) for each experimental condition were quantified using an ELISpot reader and ELISpot 6.0 iSpot software (Autoimmun Diagnostika GmbH, Strasburg, Germany). As a standard practice, an ELISpot signal was considered positive if the ELISpot signal was higher than 50 SFU / million cells, and / or the experimental group signal was at least 2-fold higher than the control (mimic) group signal.

[0621] CD4 and CD8 T cell depletion

[0622] To test the contribution of T cell subsets to cytokine production, CD4 or CD8 T cells can be selectively depleted from PBMCs after plate passage but before overnight incubation with LNPs in Elispot wells. A standard depletion kit (Miltenyi; CellSep) is used to deplete the T cells.

[0623] result

[0624] Figure 16A : An example of phDC ELISpot using PBMCs isolated from a single human donor 4 weeks post-SARS-CoV-2 infection. IFNγ release was significantly increased in the presence of phDCs [spikes] compared to a negative control. This demonstrates the detection of human spike-specific T cells in phDCs transduced via mRNA in a Covid convalescent donor.

[0625] Figure 16B This is an example of phDC ELISpot dose response using PBMCs from a single human donor, 4 weeks post-SARS-CoV-2 infection. When LNP [spike] antigen was added at 62.5 ng / well, IFNγ release increased to levels exceeding 150 SFU / million. At higher doses, T cell activation was further improved, but evidence of a response plateau was shown at 250 ng / well. This demonstrates detectable phDC LNP spike dose response down to 62.5 ng / well.

[0626] Figure 17A Eighteen previously vaccinated human donors were screened from phDC ELISpots targeting the SARS-CoV-2 spike antigen. Donors were divided into two cohorts based on whether they had previously been infected with SARS-CoV-2 (black) or not (blue). Statistical analysis employed an unpaired, two-tailed Mann-Whitney U test. This demonstrates that human phDCs transduced with LNP [spike] can distinguish between T-cell responses associated with innate immunity and responses associated with vaccination alone.

[0627] Figure 17B Eleven previously vaccinated human donors in the convalescent phase were screened from phDC ELISpots targeting the SARS-CoV-2 spike antigen. The dashed line represents the standard threshold cutoff for a positive response (50 SFU / million cells). This indicates that 1) response strength is generally negatively correlated with convalescence; and 2) the phDC-induced IFNγ response is durable and detectable up to one year post-Covid infection.

[0628] Figure 17C Prior to incorporation into a standard 18hr phDC [spike] ELISpot IFNγ assay, PBMCs from vaccinated and convalescent donors were depleted of either CD8 or CD4 T cells via a plate. CD8 T cell depletion eliminated most of the phDC [spike] response, while CD4 depletion had a minimal effect. This suggests that the convalescent phDC [spike] IFNγ response is largely driven by CD8+ T cells.

[0629] Figure 17DExamples of phDC ELISpot assays performed using PBMCs isolated from single-human donors six weeks prior to and following SARS-CoV-2 infection. Post-infection IFNγ release increased above the positive threshold of 50 SFU / million cells. This demonstrates the increased IFNγ T cell response induced by phDCs following natural Covid infection, thus showcasing the feasibility and practicality of longitudinal immune surveillance.

Claims

1. A vaccine composition comprising a pharmaceutically effective amount of physiological DC (phDC) and at least one mRNA, said mRNA comprising a coding sequence encoding at least one antigenic protein.

2. The vaccine composition according to claim 1, wherein the at least one mRNA is contained in lipid nanoparticles.

3. The vaccine composition according to claim 1 or 2, wherein the at least one mRNA is modified with N1-methyl-pseudouridine instead of uridine.

4. The vaccine composition according to any one of claims 1 to 3, wherein the lipid nanoparticles comprise cKK-E12 lipid, SM-102 lipid, or MC3 lipid.

5. The vaccine composition according to any one of claims 1 to 4, wherein the phDC can be obtained by subjecting the mononuclear cells to shear force by passing them through a flow chamber obtained from a donor.

6. The vaccine composition according to any one of claims 1 to 5, wherein the at least one antigenic protein is an infectious disease-associated antigenic protein, optionally a viral antigenic protein, a bacterial antigenic protein, a fungal antigenic protein, a prion antigenic protein, or a parasitic antigenic protein.

7. The vaccine composition according to claim 6, wherein the viral antigenic protein is a coronavirus antigenic protein or a human immunodeficiency virus (HIV) antigenic protein.

8. The vaccine composition according to any one of claims 1 to 7, wherein the at least one antigenic protein is exogenous for the phDC.

9. The vaccine composition according to any one of claims 1 to 6, wherein the at least one mRNA comprises at least 75%, 85%, 90%, 95%, 98%, 99%, or 100% of the same nucleotide sequence as SEQ ID NO:

1.

10. The vaccine composition according to any one of claims 1 to 7, wherein the at least one mRNA comprises at least 75%, 85%, 90%, 95%, 98%, 99%, or 100% of the same nucleotide sequence as SEQ ID NO:

19.

11. The vaccine composition according to any one of claims 1 to 9, wherein the at least one mRNA is a heterologous mRNA or exogenous mRNA relative to the phDC.

12. The vaccine composition according to any one of claims 1 to 10, wherein the mRNA is at least partially contained in the phDC.

13. The vaccine composition of claim 11, wherein substantially all of the mRNA is contained in the phDC.

14. The vaccine composition of claim 12, wherein the composition comprises unincorporated mRNA.

15. The vaccine composition according to any one of claims 1 to 10, wherein the mRNA is at least partially not incorporated into the phDC.

16. The vaccine composition according to any one of claims 14, wherein more than 30%, 40%, 50%, 60%, 70%, 80% or 90% of the mRNA is not incorporated into the phDC.

17. The vaccine composition according to any one of claims 1 to 16, wherein the at least one antigenic protein is a tumor-associated antigenic protein.

18. The vaccine composition of claim 17, wherein the tumor-associated antigenic protein is a blood cancer antigenic protein.

19. The vaccine composition according to any one of claims 6 to 16, for use in preventing or reducing infectious diseases, preferably viral diseases, in a subject, the method comprising administering the vaccine composition to the subject.

20. A method for preventing or reducing coronavirus disease, preferably coronavirus disease 2019 (Covid-19), in a subject using a vaccine composition according to any one of claims 7 to 16, the method comprising administering the vaccine composition to the subject.

21. The vaccine composition according to any one of claims 17 or 18, a prophylactic method for preventing or reducing tumors or cancer in a subject, the method comprising administering the vaccine composition to the subject.

22. The vaccine composition of claim 18, and a prophylactic method for preventing or reducing blood cancers in a subject, the method comprising administering the vaccine composition to the subject.

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