Cell vaccine platform and method of use

Abstract

The present invention provides cell vaccine platforms, such as vaccine-immune virus opsonization platforms, for inducing a host immune response, as well as methods for producing and using such cell vaccine platforms. [Solution] Several embodiments provide genetically engineered human cells comprising (a) genomic disruption in at least one human leukocyte antigen (HLA) gene or at least one transcription regulator of an HLA gene, and (b) an exogenous nucleic acid encoding a cell surface protein that binds to a protein expressed on the surface of phagocytic or cytolytic immune cells, or a functional fragment or functional variant of the cell surface protein, wherein the binding results in activation of the phagocytic or cytolytic activity of the immune cells.

Description

Technical Field

[0001] Cross - reference to Related Applications This international PCT application claims priority and benefit to U.S. Provisional Patent Application No. 63 / 013,387, filed on April 21, 2020, and U.S. Provisional Patent Application No. 63 / 056,460, filed on July 24, 2020, the contents of which are hereby incorporated by reference in their entirety.

[0002] Sequence Listing This application includes a sequence listing submitted electronically in ASCII format, which is hereby incorporated by reference in its entirety into this specification. The ASCII copy created on April 19, 2021, is named 199827 - 757601_SL.txt and is 40,840 bytes in size.

Background Art

[0003] Current vaccine development strategies mainly rely on the delivery of attenuated or inactivated microbial pathogens combined with immunostimulatory adjuvants to induce the host's immune response and the production of persistent antigen - specific antibodies and memory lymphocytes. However, these strategies rely on non - physiological methods of antigen exposure and non - physiological adjuvants and may not be able to fully generate the necessary immune response for vaccines. Therefore, there is a need for new vaccine strategies that more closely mimic and regulate the physiological immune response.

[0004] Incorporation by Reference All publications, patents, and patent applications cited herein are hereby incorporated by reference into this specification to the same extent as if each individual publication, patent, or patent application were specifically and individually indicated to be incorporated by reference. In case of conflict between the terms herein and those in the incorporated references, the terms herein shall prevail.

Summary of the Invention

[0005] Provided herein, among other things, are cell vaccines, allogeneic universal vaccine-producing cells, and methods for producing and using them.

[0006] Some embodiments provide genetically engineered human cells comprising (a) genomic disruption in at least one human leukocyte antigen (HLA) gene or at least one transcription regulator of an HLA gene, and (b) an exogenous nucleic acid encoding a cell surface protein that binds to a protein expressed on the surface of phagocytic or cytolytic immune cells, or a functional fragment or functional variant of the cell surface protein, wherein the binding results in activation of the phagocytic or cytolytic activity of the immune cells.

[0007] In some embodiments, the genome disruption inhibits the expression of the HLA protein encoded by the at least one HLA gene on the surface of the genetically engineered human cell. In some embodiments, the genome disruption results in reduced HLA or MHC-mediated T cell activation and / or proliferation compared to equivalent cells lacking the genome disruption. In some embodiments, the genome disruption reduces HLA or MHC-mediated T cell activation and / or proliferation compared to equivalent cells lacking the genome disruption. In some embodiments, the equivalent cells include human cells lacking the genome disruption. In some embodiments, the equivalent cells include human cells expressing the HLA gene. In some embodiments, the equivalent cells include the genetically engineered human cells lacking the disruption.

[0008] In some embodiments, the genome disruption completely inhibits the expression of the HLA protein encoded by the at least one HLA gene on the surface of the genetically engineered human cell.

[0009] In some embodiments, the genomic disruption in at least one human leukocyte antigen (HLA) gene or at least one transcription factor of the HLA gene results in reduced HLA or MHC-mediated T cell activation or proliferation upon administration of the genetically modified human cells to a subject, compared to administration of equivalent cells without the genomic disruption in at least one human leukocyte antigen (HLA) gene or at least one transcription factor of the HLA gene.

[0010] In some embodiments, the genome disruption is located in an HLA class I gene. In some embodiments, the HLA class I gene is an HLA-A gene, an HLA-B gene, an HLA-C gene, or a β-microglobulin gene. In some embodiments, the HLA class I gene is a β-microglobulin gene.

[0011] In some embodiments, the genome disruption is located in an HLA class II gene. In some embodiments, the HLA class II gene is the HLA-DP gene, HLA-DM gene, HLA-DOA gene, HLA-DOB gene, HLA-DQ gene, or HLA-DR gene.

[0012] In some embodiments, at least one transcription factor of the HLA gene is the CIITA gene, the RFX5 gene, the RFXAP gene, or the RFXANK gene. In some embodiments, the HLA gene is the CIITA gene.

[0013] In some embodiments, the genetically engineered human cells include genomic disruption of at least one HLA class I gene or at least one transcription factor of the HLA class I gene, and genomic disruption of at least one HLA class II gene or at least one transcription factor of the HLA class II gene.

[0014] In some embodiments, the genetically engineered human cells include genomic disruption in at least one HLA class I transcription factor gene and genomic disruption in at least one HLA class II transcription factor.

[0015] In some embodiments, the immune cells are innate immune cells. In some embodiments, the innate immune cells are NK cells, macrophages, dendritic cells, neutrophils, or eosinophils. In some embodiments, the innate immune cells are NK cells.

[0016] In some embodiments, the binding results in the activation of the cytolytic activity of the NK cells.

[0017] In some embodiments, the cell surface protein is a ligand that specifically binds to natural killer (NK) cell activating receptors expressed on the surface of NK cells. In some embodiments, the cell surface protein is selected from the group consisting of MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, CD155, CD112 (nectin-2), B7-H6, Necl-2, and immunoglobulin Fc.

[0018] In some embodiments, the cell surface protein is a natural killer (NK) cell activating ligand. In some embodiments, the natural killer cell activating ligand is selected from the group consisting of MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, CD155, CD112 (nectin-2), B7-H6, and Necl-2.

[0019] In some embodiments, the cells comprise a secretory protein that binds to a receptor expressed on the surface of a phagocytic or cytolytic immune cell, or an exogenous nucleic acid encoding a functional fragment or functional variant of the secretory protein, the protein attracting the immune cell toward the genetically engineered human cell.

[0020] In some embodiments, the genetically engineered human cells include exogenous proteins, antigenic fragments thereof, or nucleic acids encoding suicide genes. In some embodiments, the exogenous proteins include microbial proteins.

[0021] In some embodiments, the microbial protein comprises a nucleocapsidrin protein having at least about 85% sequence identity with respect to SEQ ID NO: 54.

[0022] In some embodiments, the microbial protein is secreted by the genetically modified human cell, expressed on the surface of the genetically modified human cell, or expressed in the cytoplasm of the genetically modified human cell.

[0023] In some embodiments, the microbial protein is a virus, bacterium, parasite, or protist protein. In some embodiments, the microbial protein is a viral protein. In some embodiments, the viral protein is from a virus of the order Nidovirales. In some embodiments, the viral protein is from a virus of the family Coronaviridae. In some embodiments, the viral protein is from the subfamily Orthocoronamiae. In some embodiments, the viral protein is from viruses of the genera Alphacoronavirus, Betacoronavirus, Gammacoronavirus, and Deltacoronavirus. In some embodiments, the viral protein is from a virus of the genus Betacoronavirus. In some embodiments, the viral protein is from a virus of the subgenus Salvecovirus. In some embodiments, the viral protein is from the virus of two Severe Acute Respiratory Syndrome-associated Coronaviruses. In some embodiments, the viral protein is from the virus of two strains of Severe Acute Respiratory Syndrome Coronavirus 2. In some embodiments, the viral protein is the spike protein of Severe Acute Respiratory Syndrome Coronavirus 2. In some embodiments, the viral protein is the spike protein of SEQ ID NO: 1. In some embodiments, the viral protein is the spike protein encoded by SEQ ID NO: 53.

[0024] In some embodiments, the viral protein is of a virus selected from the group consisting of influenza, Epstein - Barr virus (EBV), megavirus, norovirus, coxsackievirus, Middle East respiratory syndrome - related coronavirus, severe acute respiratory syndrome - related coronavirus, SARS - Cov - 2 virus, hepatitis B, varicella - zoster virus, parvovirus, adenovirus, Marburg virus, Ebola virus, rabies, smallpox, HIV, hantavirus, dengue fever, rotavirus, MERS - CoV, mumps virus, cytomegalovirus (CMV), herpes virus, papillomavirus, chikungunya virus, or any combination thereof.

[0025] In some embodiments, the genetically engineered human cells are differentiated from stem cells. In some embodiments, the stem cells are induced pluripotent stem cells (iPSCs), embryonic stem cells (ESCs), adult stem cells (ASCs), pluripotent stem cells (PSCs), or hematopoietic stem and progenitor cells (HSPCs). In some embodiments, the stem cells are induced pluripotent stem cells (iPSCs).

[0026] In some embodiments, the genetically engineered human cells are epithelial cells or endothelial cells. In some embodiments, the genetically engineered human cells are not cancer cells. In some embodiments, the genetically engineered human cells are irradiated with radiation. In some embodiments, the genetically modified cells are stem cells.

[0027] In some embodiments, the stem cells are induced pluripotent stem cells (iPSCs), embryonic stem cells (ESCs), adult stem cells (ASCs), pluripotent stem cells (PSCs), or hematopoietic stem and progenitor cells (HSPCs). In some embodiments, the stem cells are induced pluripotent stem cells (iPSCs). In some embodiments, the genetically engineered human cells cannot proliferate in vitro, in vivo, or both.

[0028] In some embodiments, the genetically modified human cells are intended for use in vaccines.

[0029] In some embodiments, the at least one genome disruption is mediated by an endonuclease. In some embodiments, the endonuclease is a CRISPR endonuclease, a zinc finger nuclease (ZFN), or a transcription activator-like effector nuclease (TALEN).

[0030] In some embodiments, the disruption of at least one genome is mediated by a CRISPR system comprising an endonuclease and a guide RNA (gRNA), wherein the gRNA comprises an RNA sequence complementary to the DNA sequence of the at least one HLA gene or at least one transcription regulator of the HLA gene.

[0031] Some embodiments provide genetically engineered human cells comprising: (a) genomic disruption in at least one human leukocyte antigen (HLA) gene or at least one transcription regulator of an HLA gene; (b) a nucleic acid encoding an exogenous cell surface protein that binds to a protein expressed on the surface of phagocytic or cytolytic immune cells, or a functional fragment or functional variant of the exogenous cell surface protein, wherein the binding results in activation of the phagocytic or cytolytic activity of the immune cells; and (c) a nucleic acid encoding an exogenous antigen protein or an antigenic fragment thereof.

[0032] In some embodiments, the exogenous antigen protein or its antigenic fragment is a microbial protein or its antigenic fragment. In some embodiments, the exogenous antigen protein comprises a nucleocapsidrin protein having at least about 85% sequence identity with respect to SEQ ID NO: 54.

[0033] In some embodiments, the microbial protein is secreted by the genetically modified human cell, expressed on the surface of the genetically modified human cell, or expressed in the cytoplasm of the genetically modified human cell.

[0034] In some embodiments, the microbial protein is a virus, bacterium, parasite, or protist protein. In some embodiments, the microbial protein is a viral protein. In some embodiments, the viral protein is from a virus of the order Nidovirales. In some embodiments, the viral protein is from a virus of the family Coronaviridae. In some embodiments, the viral protein is from the subfamily Orthocoronamiae. In some embodiments, the viral protein is from viruses of the genera Alphacoronavirus, Betacoronavirus, Gammacoronavirus, and Deltacoronavirus. In some embodiments, the viral protein is from a virus of the genus Betacoronavirus. In some embodiments, the viral protein is from a virus of the subgenus Salvecovirus. In some embodiments, the viral protein is from the virus of two Severe Acute Respiratory Syndrome-associated Coronaviruses. In some embodiments, the viral protein is from the virus of two strains of Severe Acute Respiratory Syndrome Coronavirus 2. In some embodiments, the viral protein is the spike protein of Severe Acute Respiratory Syndrome Coronavirus 2. In some embodiments, the viral protein is the spike protein of SEQ ID NO: 1. In some embodiments, the viral protein is the spike protein encoded by SEQ ID NO: 53.

[0035] In some embodiments, the viral protein is derived from a virus selected from the group including influenza, Epstein-Barr virus (EBV), megavirus, Norwalk virus, coxsackievirus, Middle East Respiratory Syndrome-related coronavirus, Severe Acute Respiratory Syndrome-related coronavirus, SARS-CoV-2 virus, hepatitis B, varicella-zoster virus, parvovirus, adenovirus, Marburg virus, Ebola virus, rabies, smallpox, HIV, hantavirus, dengue fever, rotavirus, MERS-CoV, mumps virus, cytomegalovirus (CMV), herpesvirus, papillomavirus, chikungunya virus, or any combination thereof.

[0036] In some embodiments, the genetically modified human cells are differentiated from stem cells. In some embodiments, the stem cells are induced pluripotent stem cells (iPSCs), embryonic stem cells (ESCs), adult stem cells (ASCs), pluripotent stem cells (PSCs), or hematopoietic stem and progenitor cells (HSPCs). In some embodiments, the stem cells are induced pluripotent stem cells (iPSCs).

[0037] In some embodiments, the genetically modified human cells are epithelial cells or endothelial cells. In some embodiments, the genetically modified human cells are not cancer cells. In some embodiments, the genetically modified human cells are irradiated.

[0038] In some embodiments, the immune cells are innate immune cells. In some embodiments, the innate immune cells are NK cells, macrophages, dendritic cells, neutrophils, or eosinophils. In some embodiments, the innate immune cells are NK cells.

[0039] Some embodiments provide populations of genetically modified human cells as disclosed herein.

[0040] Some embodiments provide pharmaceutical compositions comprising the genetically modified human cells and excipients disclosed herein. Some embodiments provide unit dosage forms comprising the compositions or genetically modified human cells disclosed herein.

[0041] Some embodiments provide a method for producing a population of genetically modified human stem cells, the method comprising obtaining a population of human stem cells, inducing genomic disruption in at least one HLA gene or at least one transcription factor of the HLA gene, and introducing a nucleic acid encoding an exogenous cell surface protein that binds to a protein expressed on the surface of phagocytic or cytolytic immune cells, or a functional fragment or functional variant of the exogenous cell surface protein, wherein the binding results in activation of the phagocytic or cytolytic activity of the immune cells, thereby producing a population of genetically modified stem cells.

[0042] In some embodiments, the genome disruption inhibits the expression of the HLA protein encoded by the at least one HLA gene on the surface of the cell. In some embodiments, the genome disruption inhibits the expression of the HLA protein encoded by the at least one HLA gene on the surface of the cell for a period of time sufficient to interact with proteins expressed on the surface of immune cells.

[0043] In some embodiments, the disruption of at least one genome is mediated by an endonuclease. In some embodiments, the endonuclease is a CRISPR endonuclease, a zinc finger nuclease (ZFN), or a transcription activator-like effector nuclease (TALEN). In some embodiments, the disruption of at least one genome is mediated by a CRISPR system comprising an endonuclease and a guide RNA (gRNA), wherein the gRNA comprises an RNA sequence complementary to the DNA sequence of the at least one HLA gene or at least one transcription regulator of the HLA gene.

[0044] In some embodiments, the genome disruption is a single-strand DNA break or a double-strand DNA break.

[0045] In some embodiments, the method further includes introducing nucleic acids encoding a microbial protein or an antigenic fragment thereof.

[0046] In some embodiments, the microbial protein comprises a nucleocapsidrin protein having at least about 85% sequence identity with respect to SEQ ID NO: 54. In some embodiments, the microbial protein is secreted by the genetically engineered human cell, expressed on the surface of the genetically engineered human cell, or expressed in the cytoplasm of the genetically engineered human cell.

[0047] In some embodiments, the microbial protein is a virus, bacterium, or parasite protein. In some embodiments, the microbial protein is a viral protein.

[0048] In some embodiments, the stem cells are induced pluripotent stem cells (iPSCs), embryonic stem cells (ESCs), adult stem cells (ASCs), pluripotent stem cells (PSCs), or hematopoietic stem and progenitor cells (HSPCs). In some embodiments, the stem cells are induced pluripotent stem cells (iPSCs).

[0049] In some embodiments, the method includes differentiating the population of genetically modified human stem cells. In some embodiments, the cells differentiate into epithelial cells or endothelial cells.

[0050] In some embodiments, the immune cells are innate immune cells. In some embodiments, the innate immune cells are NK cells, macrophages, dendritic cells, neutrophils, or eosinophils. In some embodiments, the innate immune cells are NK cells.

[0051] Some embodiments provide a method for producing a population of terminally differentiated genetically engineered human cells, the method comprising: obtaining a population of human stem cells; inducing genomic disruption in at least one HLA gene or at least one transcription factor of the HLA gene; introducing an exogenous cell surface protein that binds to a protein expressed on the surface of phagocytic or cytolytic immune cells, or a nucleic acid encoding a functional fragment or functional variant of the exogenous cell surface protein, the binding resulting in activation of the phagocytic or cytolytic activity of the immune cells, thereby producing a population of genetically engineered human stem cells; and differentiating the population of genetically engineered human stem cells into a population of terminally differentiated genetically engineered human cells.

[0052] In some embodiments, the population of genetically modified human stem cells differentiates into epithelial cells or endothelial cells.

[0053] Some embodiments provide methods for immunizing a human subject with microorganisms, the methods comprising administering the genetically engineered human cells disclosed herein, the compositions disclosed herein, or the pharmaceutical compositions disclosed herein to the subject.

[0054] Some embodiments provide a method for immunizing a human subject with a microorganism, the method comprising administering a population of genetically modified human cells to the subject, the population of genetically modified human cells comprising (a) a genomic disruption in at least one HLA gene or at least one transcription regulator of an HLA gene, (b) a nucleic acid encoding an exogenous cell surface protein that binds to a protein expressed on the surface of phagocytic or cytolytic immune cells, or a functional fragment or functional variant of the exogenous cell surface protein, wherein the binding results in activation of the phagocytic or cytolytic activity of the immune cells, and (c) a nucleic acid encoding a microbial protein or an antigenic fragment thereof.

[0055] In some embodiments, the binding results in immune cell-mediated lysis or phagocytosis of at least a portion of the population of genetically modified human cells.

[0056] In some embodiments, the administration results in the subject initiating an adaptive immune response to the microorganism.

[0057] In some embodiments, the administration results in increased activation and / or proliferation of T cells expressing T cell receptors that specifically bind to the microbial protein or its antigenic fragment.

[0058] In some embodiments, the administration results in increased activation and / or proliferation of B cells expressing B cell receptors that specifically bind to the microbial protein or its antigenic fragment.

[0059] In some embodiments, the administration results in an increase in circulating antibodies that specifically bind to the microbial protein or its antigenic fragment.

[0060] In some embodiments, the microbial protein or its antigenic fragment is secreted by the genetically engineered human cell, expressed on the surface of the genetically engineered human cell, or expressed in the cytoplasm of the genetically engineered human cell.

[0061] In some embodiments, the microbial protein is a virus, bacterium, or parasite protein. In some embodiments, the microbial protein is a viral protein. In some embodiments, the viral protein is from a virus of the Coronaviridae family. In some embodiments, the viral protein is from a virus of the alphacoronavirus, betacoronavirus, gammacoronavirus, and deltacoronavirus gene. In some embodiments, the viral protein is from a virus of the betacoronavirus gene. In some embodiments, the viral protein is from two viruses associated with severe acute respiratory syndrome (SPR). In some embodiments, the viral protein is from two strains of SPR.

[0062] In some embodiments, the viral protein is derived from a virus selected from the group including influenza, Epstein-Barr virus (EBV), megavirus, Norwalk virus, coxsackievirus, Middle East Respiratory Syndrome-related coronavirus, Severe Acute Respiratory Syndrome-related coronavirus, SARS-CoV-2 virus, hepatitis B, varicella-zoster virus, parvovirus, adenovirus, Marburg virus, Ebola virus, rabies, smallpox, HIV, hantavirus, dengue fever, rotavirus, MERS-CoV, mumps virus, cytomegalovirus (CMV), herpesvirus, papillomavirus, chikungunya virus, and any combination thereof.

[0063] In some embodiments, the population of genetically modified human cells is administered intramuscularly or subcutaneously.

[0064] In some embodiments, the immune cells are innate immune cells. In some embodiments, the innate immune cells are NK cells, macrophages, dendritic cells, neutrophils, or eosinophils. In some embodiments, the innate immune cells are NK cells.

[0065] In some embodiments, the genetically modified human cells further include a suicide gene.

[0066] In some embodiments, the microbial protein comprises a nucleocapsidrin protein having at least about 85% sequence identity with respect to SEQ ID NO: 54.

[0067] Some embodiments provide a method for immunizing a subject, the method comprising administering a population of genetically modified mammalian cells to the subject, the population of genetically modified mammalian cells comprising: (a) a genomic disruption in at least one MHC gene or at least one transcription regulator of an MHC gene, the disruption resulting in reduced activation of T cell proliferation compared to the genetically modified human cells without the disruption; and (b) a nucleic acid encoding an exogenous cell surface protein that binds to a protein expressed on the surface of phagocytic or cytolytic immune cells, or a functional fragment or functional variant of the exogenous cell surface protein, the binding resulting in activation of the phagocytic or cytolytic activity of the immune cells.

[0068] In some embodiments, the immunization is antigen-specific, and the genetically engineered mammalian cells further comprise nucleic acids encoding the antigen or a fragment thereof.

[0069] In some embodiments, the immunization is antigen-specific, and the genetically engineered mammalian cells further comprise the antigen or a fragment thereof.

[0070] In some embodiments, the activation results in immune cell-mediated lysis or phagocytosis of at least a portion of the population of genetically engineered mammalian cells.

[0071] In some embodiments, the administration results in the subject initiating an adaptive immune response to the antigen. In some embodiments, the administration results in increased activation and / or proliferation of T cells expressing T cell receptors that specifically bind to the peptide of the antigen. In some embodiments, the administration results in increased activation and / or proliferation of B cells expressing B cell receptors that specifically bind to the peptide of the antigen. In some embodiments, the administration results in an increase in circulating antibodies that specifically bind to the antigen.

[0072] In some embodiments, the antigen is secreted by the genetically modified mammalian cell, expressed on the surface of the genetically modified mammalian cell, or expressed in the cytoplasm of the genetically modified mammalian cell.

[0073] In some embodiments, the antigen is a viral, bacterial, fungal, or parasitic protein. In some embodiments, the viral protein is from a virus of the Coronaviridae family. In some embodiments, the viral protein is from a virus of the alphacoronavirus, betacoronavirus, gammacoronavirus, or deltacoronavirus genus. In some embodiments, the viral protein is from a virus of the betacoronavirus genus.

[0074] In some embodiments, the viral protein is from the virus of two severe acute respiratory syndrome-associated coronaviruses. In some embodiments, the viral protein is from the virus of two strains of severe acute respiratory syndrome coronavirus. In some embodiments, the viral protein is the spike protein of severe acute respiratory syndrome coronavirus 2. In some embodiments, the viral protein is the spike protein of SEQ ID NO: 1. In some embodiments, the viral protein is the spike protein encoded by SEQ ID NO: 53.

[0075] In some embodiments, the viral protein is derived from a virus selected from the group including influenza, Epstein-Barr virus (EBV), megavirus, Norwalk virus, coxsackievirus, Middle East Respiratory Syndrome-related coronavirus, Severe Acute Respiratory Syndrome-related coronavirus, SARS-CoV-2 virus, hepatitis B, varicella-zoster virus, parvovirus, adenovirus, Marburg virus, Ebola virus, rabies, smallpox, HIV, hantavirus, dengue fever, rotavirus, MERS-CoV, mumps virus, cytomegalovirus (CMV), herpesvirus, papillomavirus, chikungunya virus, or at least one of any combination thereof.

[0076] In some embodiments, the antigen comprises a protein or peptide associated with cancer or tumor.

[0077] In some embodiments, the antigen includes a neoantigen.

[0078] In some embodiments, the population of genetically modified cells is administered intramuscularly or subcutaneously.

[0079] In some embodiments, the immune cells are innate immune cells. In some embodiments, the innate immune cells are NK cells, macrophages, dendritic cells, neutrophils, or eosinophils. In some embodiments, the innate immune cells are NK cells. In some embodiments, the genetically engineered mammalian cells further include suicide genes.

[0080] In some embodiments, the genetically modified mammalian cells include genetically modified human cells, and the MHC genes include HLA genes. [Brief description of the drawing]

[0081] Novel features of the present invention are described in detail in the appended claims. A further understanding of the features and advantages of the present invention will be obtained by referring to the following detailed description, which describes explanatory embodiments in which the principles of the present invention are utilized, and to the appended drawings. [Brief explanation of the drawing]

[0082] [Figure 1] The amino acid sequence of the SARS-CoV-2 Spike(S) protein and its individual domains are shown (SEQ ID NO: 1).

[0083] [Figure 2] This flowchart shows an exemplary workflow of the cell vaccine platform described herein.

[0084] [Figure 3] This represents the vaccine cells described herein.

[0085] [Figure 4A] This is an illustrative diagram of the immune response to a viral infection. [Figure 4B]This specification describes exemplary responses resulting from vaccination using the compositions provided herein. Universal vaccine cells (UVCs) are antigen-loaded live cells delivered in vivo, genetically engineered to induce a natural, physiological, and potent activation of the immune system for neutralizing antibody production and sustained cellular immunity. UVCs possess self-supporting properties through robust lysis by innate immune cells, activating cellular and antibody immune responses similar to the natural host response to viral infection, thereby replicating natural physiological immunity.

[0086] [Figure 5] Exemplary inhibitory and activating receptors on NK cells, as well as their related ligands on target cells, are shown. Any one of these receptors may be expressed ectopically or endogenously by the vaccine cells described herein.

[0087] [Figure 6] This is an illustrative schematic diagram illustrating how NK cells recognize platform cells, such as those lacking MHC-I components, as either foreign bodies, virally infected, or pathogenic, and target them for cytolysis.

[0088] [Figure 7] This study demonstrates that platform cells, such as CRISPR knockout B2M (a component of the MHC class I complex) iPSC cells, exhibit elimination expression of MHCI even after IFNg stimulation.

[0089] [Figure 8] A demonstrates the efficacy of the cells described herein by showing that the B2M-deficient platform cells described herein are unable to activate the proliferation of MHC mismatch T cells compared to control iPS cells. B shows a flow cytometry plot of platform cells differentiated into CD31+CD144+ endothelial cells at day 7.

[0090] [Figure 9]Table 5 shows flow cytometry plots obtained 48 hours after transfection of endothelial cells derived from the platform cells described herein that overexpress the NK-activating ligands listed in Table 5.

[0091] [Figure 10] This study shows that when endothelial cells expressing variants of the SARS-CoV-2 spike protein (full-length and spike S1 subunit) were lysed, both protein antigen variants were abundantly detected, indicating a dose-dependent increase in the number of vaccinated cells.

[0092] [Figure 11] This is a schematic diagram of the SARS-CoV-2 virus and its spike protein structure.

[0093] [Figure 12] This shows a natural killer (NK) cell killing assay. It shows the percentage of dead target cells, either K562 cells or iPSC-derived endothelial cells (differentiated from platform cells), when the effector-to-target (E:T) ratio is increased.

[0094] [Figure 13] This is a schematic diagram of a universal vaccine cell (UVC). UVCs contain a deletion at the B2M gene locus (KO-B2M) and are MHC-I deficient. UVCs also contain two knock-in (KI) constructs. One KI construct expresses the NK ligand MICA on the cell surface of UVCs, and the other KI construct expresses the SARS-CoV-2 spike protein and nucleocapsidrin protein intracellularly.

[0095] [Figure 14A] The full-length amino acid sequence (SEQ ID NO: 54) of the SARS-CoV-2 nucleocapsidrin protein is shown.

[0096] [Figure 14B]A schematic diagram shows the expression cassette of SARS-CoV-2 spike (SPIKE) protein and nucleocapsidrin protein (N) in UVC, linked by a T2A peptide cleavage sequence. The construct is driven by the EF1a promoter.

[0097] [Figure 14C] This shows that the nucleocapsidrin protein has the highest density of epitopes across the entire SARS-CoV-2 genome. The distribution of functional epitopes across the entire SARS-CoV-2 genome is plotted. Each bar represents one validated epitope. The X-axis indicates the location in the SARS-CoV-2 ORFeome (open readin frame-ome). The fill of a bar indicates its MHC limitation, and the height of the bar indicates the percentage of MHC-matched patients who recognize the epitope. Patients were considered positive for the epitope if the agglutination ability of the epitope in the screen data exceeded the threshold (mean enrichment of all SARS-CoV-2 fragments in healthy controls + 2SD). For clarity, overlapping epitopes are plotted as adjacent bars.

[0098] [Figure 14D] This shows that ORF1ab has the most epitopes of all SARS-CoV-2 ORFs. The number of epitopes for each SARS-CoV-2 ORF is plotted. The stacked bar graph shows the number of immunodominant epitopes per ORF, and the bar fills indicate the MHC limit for each epitope. The signs of the MHC fills are the same as those in Figure 14C.

[0099] [Figure 15]This shows that UVC cells express high levels of the NK ligand MICA but no MHC-I. Panel A shows flow cytometry analysis of the NK ligand MICA in UVC cells and parental induced pluripotent stem cells (iPSCs). The X-axis represents the fluorescence intensity of the MICA protein. The Y-axis represents the number of cells. The filled area indicating cell type is shown to the right of the plot. Panel B shows flow cytometry analysis of MHC-I expression in UVC cells and parental iPSCs. The X-axis represents the fluorescence intensity of MHC-1 (HLA subtype A, B, or C). The Y-axis represents the number of cells.

[0100] [Figure 16] This study demonstrates that MHC-I-deficient UVC cells induce robust cytolysis by monkey NK cells in vitro. The amount of UVC cytotoxicity in the presence of NK cells was measured using a natural killer cytotoxicity assay by flow cytometry, including calcein AM (CAM) staining. The X-axis represents the effector-to-target (E:T) ratio. The Y-axis represents the percentage of cytotoxic activity of NK cells. MHC-I-deficient endothelial cells (KO ECs) showed higher cytotoxicity when mixed with macaque NK cells compared to wild-type ECs (WT ECs). Both KO ECs and WT ECs were differentiated from UVC iPSCs.

[0101] [Figure 17A]Figures 17A and 17B show that additional NK ligands increase the NK cell response to MHC-I deficient UVC. Figure 17A shows that additional NK ligands may increase cytokine expression in NK cells in response to MHC-I deficient UVC. Expression of CD107a, MIP1-β, IFN-γ, or TNF-α in NK cells was measured using intracellular cytokine staining assays. A summary of all responsive NK cells is shown at the far right. The X-axis lists KO-UVC (KO), MICA (KO-MICA), MICB (KO-MICB), or ULBP1 (KO-ULBP1) that do not express the ligand. The Y-axis shows the percentage (%) of NK cells that respond to KO-UVC. Each point represents an individual animal tested. [Figure 17B] Figures 17A and 17B show that additional NK ligands increase the NK cell response to MHC-I deficient UVCs. Figure 17B shows that additional NK ligands can induce the expression of multiple cytokines in NK cells. Multidimensional cytokine responses in NK cells were analyzed using Simplified Presentation of Incredibly Complex Evaluations (SPICE). Legends for arc and pie chart fills are shown below.

[0102] [Figure 18A] Figures 18A and 18B show robust expression of the SARS-CoV-2 spike protein in UVC iPSCs. Figure 18A shows that approximately half of the UVC iPSCs expressed the spike protein. Flow cytometry was used to measure the expression of the SARS-CoV-2 spike protein. The X and Y axes represent the fluorescence staining intensity and forward scatter high FSC-H of the spike protein, respectively. Approximately 48.5% of MHC-I deficient (B2M- / -) IPSCs engineered to express both MICA (MICA+) and SARS-CoV-2 spike protein (spike+) had high levels of spike protein expression compared to only about 0.41% of control IPSCs engineered to express only MICA. [Figure 18B] Figures 18A and 18B show strong expression of the SARS-CoV-2 spike protein in UVC iPSCs. Figure 18B shows that the expression level of the spike protein in UVC iPSCs was equivalent to that in HEK293 cells transiently transfected with a spike protein expression plasmid. SARS-CoV-2 spike protein expression in HEK293 cells and UVC iPSCs was measured using cell surface flow analysis. The X and Y axes represent the fluorescence staining intensity and cell number of the spike protein, respectively.

[0103] [Figure 19A] Figures 19A and 19B show the results of antibody ELISA for six monkeys at weeks 0, 2, 6, and 8 after vaccination with UVC expressing either the receptor-binding domain of the SARS-CoV-2 spike protein (Figure 19A) or the full-length spike protein (Figure 19B), demonstrating the functional testing of UVC in an NHP model. [Figure 19B] Figures 19A and 19B show the results of antibody ELISA at weeks 0, 2, 6, and 8 after vaccination with UVC expressing either the SARS-CoV-2 spike protein (Figure 19A) or the full-length spike protein (Figure 19B) in six monkeys, demonstrating the functional testing of UVC in an NHP model. [Modes for carrying out the invention]

[0104] The following description and examples illustrate embodiments of the present invention in detail. It should be understood that the present invention is not limited to the specific embodiments described herein and is therefore subject to change. Those skilled in the art will understand that there are numerous variations and modifications of the present invention that fall within the scope of the invention. definition

[0105] The terms “about” and their grammatical equivalents as used herein with respect to reference numbers and their grammatical equivalents may include a range of values ​​plus or minus 10% from that value. For example, the quantity “about 10” includes quantities from 9 to 11. The term “about” in relation to reference numbers may also include a range of values ​​plus or minus 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% from that value.

[0106] As used herein, the term "vaccine" and its grammatical equivalent refer to a drug that induces a host immune response to an infectious disease.

[0107] As used herein, the term "cell vaccine" and its grammatical equivalent refer to a vaccine that utilizes cells to expose the host immune system to an antigen.

[0108] As used herein, the terms “target cell” or “target cell line” and their grammatical equivalents refer to selected cell lines described herein as carriers of a particular type of pathogenic antigen.

[0109] As used herein, the terms “activation” or “to activate” and their grammatical equivalents may refer to the process by which a cell transitions from a dormant state to an active state.

[0110] As used herein, the term “antigen” and its grammatical equivalent refer to a molecule comprising one or more epitopes or binding sites that can be bound by one or more receptors or antibodies. For example, when an antigen is presented, it can stimulate the host’s immune system to induce a cellular antigen-specific immune response or a humoral antibody response. Antigens may also have the ability to induce cellular and / or humoral responses, either by themselves or in combination with other molecules.

[0111] As used herein, “manipulated cell” and its grammatical equivalent refer to a cell containing an exogenous nucleic acid or amino acid sequence, or a cell containing a change, addition, or deletion in an endogenous nucleic acid sequence.

[0112] As used herein, the “innate immune system” refers to the first line of defense against non-self pathogens, which is the innate or nonspecific immune response to the target pathogen. The innate immune response consists of physical, chemical, and cellular defenses against the pathogen. As used herein, “innate immune cells” generally refer to phagocytic or cytolytic immune cells involved in the innate immune response. Specifically, these phagocytic or cytolytic immune cells include monocytes (which develop into macrophages), macrophages, neutrophils, eosinophils, basophils, natural killer (NK) cells, and mast cells.

[0113] As used herein, the term “construction” and its grammatical equivalent refer to a macromolecule or molecular complex containing polynucleotides that is delivered to a host cell either in vitro or in vivo.

[0114] As used herein, the term “vector” and its grammatical equivalent refer to any nucleic acid construct that can direct the delivery or transfer of foreign genetic material to a target cell, where it can be replicated and / or expressed. As used herein, the term “vector” includes the construct to be delivered. A vector may be a linear or cyclic molecule. A vector may be either embedded or non-embedded.

[0115] As used herein, the term “integrated” and its grammatical equivalent refer to one or more nucleotides of a construct that is stably inserted into the cellular genome, i.e., nucleotides covalently linked to a nucleic acid sequence in the chromosomal DNA of a cell.

[0116] As used herein, the term “transgene” and its grammatical equivalent refer to a gene or genetic material that is introduced into a cell. For example, a transgene may be a stretch or segment of DNA containing a gene that is introduced into a cell. A transgene may retain the ability to produce RNA or polypeptides (e.g., proteins) in the engineered cell. A transgene may consist of different nucleic acids, e.g., RNA or DNA. A transgene may include recombinant arms. A transgene may include engineered sites.

[0117] The terms "CRISPR," "CRISPR system," or "CRISPR nuclease system" and their grammatical equivalents may include non-coding RNA molecules (e.g., guide RNA) that bind to DNA and Cas proteins (e.g., Cas9) possessing nuclease functionality (e.g., two nuclease domains). See, for example, Sander, JD, et al., “CRISPR-Cas systems for editing, regulating and targeting genomes,” Nature Biotechnology, 32:347-355 (2014), and also see, for example, Hsu, PD, et al., “Development and applications of CRISPR-Cas9 for genome engineering,” Cell 157(6):1262-1278 (2014).

[0118] As used herein, the term “sequence” and its grammatical equivalents refer to a nucleotide sequence that may be DNA or RNA, may be linear, circular, or branched, and may be single-stranded or double-stranded. Sequences may mutate. Sequences may be of any length, for example, 2 to 1,000,000 nucleotides or longer (or any integer value between or greater than these), for example, about 100 to 10,000 nucleotides, or about 200 to 500 nucleotides.

[0119] As used herein, the terms “reprogramming,” “dedifferentiation,” “increased differentiation potential,” and “increased developmental potential” refer to methods of increasing a cell’s differentiation potential or dedifferentiating a cell to a less differentiated state. For example, a cell with increased differentiation potential has greater developmental flexibility (i.e., can differentiate into more cell types) compared to the same cell that has not been reprogrammed. In other words, a reprogrammed cell is a cell that is less differentiated than the same cell that has not been reprogrammed.

[0120] As used herein, the term “differentiation” is the process by which unspecialized (”uncommitted”) or poorly specialized cells acquire the characteristics of specialized cells, such as blood cells or muscle cells. Differentiated or induced-differentiation cells are cells that have taken on a more specialized (”committed”) position within a cell lineage. When applied to the process of differentiation, the term “committed” refers to cells that, under normal circumstances, continue to differentiate into a particular cell type or subset of cell types, and have progressed in the differentiation pathway to a point where, under normal circumstances, they cannot differentiate into a different cell type or revert to a less differentiated cell type. As used herein, the term “pluripotency” refers to the ability of a cell to form the body or all lineages of somatic cells (i.e., the embryonic body). For example, embryonic stem cells are a type of pluripotent stem cell that can form cells from each of the three germ layers: the ectoderm, mesoderm, and endoderm. Pluripotency is a continuum of developmental potential, ranging from incomplete or partial pluripotent cells (e.g., epiblast stem cells or EpiSCs) that cannot produce a complete organism to more primitive and pluripotent cells (e.g., embryonic stem cells) that can produce a complete organism.

[0121] As used herein, the term “induced pluripotent stem cell,” or iPSC, means that stem cells are produced from induced or altered differentiated adult, neonatal, or fetal cells, i.e., reprogrammed into cells capable of differentiating into any of the three germ layers—mesoderm, endoderm, and ectoderm—or any tissue in the dermis. Produced iPSCs do not refer to cells that exist in nature.

[0122] As used herein, the terms “universal vaccine cell” and “UVC” refer to the vaccine composition described herein. The vaccine composition may include the cells provided herein.

[0123] As used herein, the term “embryonic stem cell” refers to the naturally occurring pluripotent stem cells in the inner cell mass of a blastocyst. Embryonic stem cells are pluripotent and arise during the development of all three primary germ layers: the ectoderm, endoderm, and mesoderm. They do not contribute to the extraembryonic membrane or placenta; that is, they are not totipotent.

[0124] As used herein, the term “multipotent stem cell” refers to a cell that has the developmental capacity to differentiate into cells of one or more germ layers (ectoderm, mesoderm, and endoderm), but not all three. Therefore, multipotent cells are also called “partially differentiated cells.” Multipotent cells are well known in the art, and examples of multipotent cells include adult stem cells such as hematopoietic stem cells and neural stem cells. “Multipotency” means that a cell can form many types of cells in a particular lineage, but not cells in other lineages. For example, a multipotent hematopoietic cell can form various types of blood cells (red blood cells, white blood cells, platelets, etc.), but cannot form neurons. Therefore, the term “multipotency” refers to a state of a cell that has a lower degree of developmental capacity than totipotency and pluripotency.

[0125] Pluripotency can be determined in part by evaluating the pluripotent properties of cells. Features of pluripotency include, but are not limited to, (i) the morphology of pluripotent stem cells, (ii) the potential for unlimited self-renewal, (iii) the expression of pluripotent stem cell markers including, but not limited to, SSEA1 (mouse only), SSEA3 / 4, SSEA5, TRA1-60 / 81, TRA1-85, TRA2-54, GCTM-2, TG343, TG30, CD9, CD29, CD133 / prominin, CD140a, CD56, CD73, CD90, CD105, OCT4, NANOQ SOX2, CD30 and / or CD50, (iv) the ability to differentiate into all three somatic cell lineages (ectoderm, mesoderm, and endoderm), (v) the formation of teratomas consisting of cells from the three somatic cell lineages, and (vi) the formation of embryoid bodies consisting of cells from the three somatic cell lineages.

[0126] Two types of pluripotency have been described: a "primed" or "metastable" state of pluripotency similar to the epiblast stem cells (EpiSCs) of late blastocysts, and a "naive" or "basal" state of pluripotency similar to the inner cell mass of early / preimplantation blastocysts. While both pluripotent states exhibit the above characteristics, the naive or basal state further exhibits (i) pre-inactivation or reactivation of the X chromosome in female cells, (ii) improved clonality and viability in single-cell culture, (iii) overall reduction in DNA methylation, (iv) reduced deposition of H3K27me3 repressive chromatin marks on developmental regulatory gene promoters, and (v) decreased expression of differentiation markers compared to primed pluripotent cells. Standard cell reprogramming methodologies, which involve introducing exogenous pluripotency genes into somatic cells, expressing them, and then silencing or removing them from the resulting pluripotent cells, generally appear to exhibit the characteristics of a primed pluripotent state. Under standard pluripotent cell culture conditions, such cells remain in a primed state unless exogenous transgene expression is maintained, and thus the characteristics of the basal state are observed.

[0127] "Pluripotency factors" or "reprogramming factors" refer to agents that can increase the developmental potential of cells, either alone or in combination with other agents. Pluripotency factors include, but are not limited to, polynucleotides, polypeptides, and small molecules that can increase the developmental potential of cells. Exemplary pluripotency factors include, for example, transcription factors and small molecule reprogramming agents.

[0128] As used herein, the term “pluripotent stem cell morphology” refers to the classic morphological features of embryonic stem cells. The morphology of normal embryonic stem cells is characterized by their round and small size, high nucleus-to-cytoplasmic ratio, prominent presence of nucleoli, and typical intercellular spacing.

[0129] overview This disclosure provides a novel cell vaccine platform that offers clear advantages over current systems, particularly enabling the development of robust, safe, and highly scalable cell vaccines against any pathogen. Standard vaccines used for immunization against microorganisms utilize viruses, lipid nanoparticles, or nucleic acids. Unlike these standard vaccines, cell vaccines offer a clear advantage in that they deliver immunogenic antigens in a physiologically relevant manner, allowing host immune cells to bind to the antigens as if the target were naturally infected. Furthermore, the cellular components are likely to act as intrinsic adjuvants through the in vivo creation of apoptotic bodies that stimulate, attract, and mobilize cells of the innate immune system to promote the development of a robust immune response and immunological memory. Because cell vaccines actually "mimic" the natural process of immunocytolysis of infected cells, the antigens are delivered to the immune system in exactly the same way as they would be done through naturally acquired immunity against invading pathogens. Thus, some embodiments are autoadjuvant.

[0130] Cancer cell line-based cell vaccines are currently under development. However, unlike these other cell vaccines, this disclosure provides a novel genetically engineered cell vaccine that enables the precise creation of “ideal” target cells designed to be killed, in contrast to the natural chance of cancer cell line biology. The cell surface receptors expressed by the target cells are specifically designed for “targeted” lysis by defined cells of the host innate immune system (i.e., the absence of MHC and acquisition of “missing-self” signals, as well as targeted expression of “kill-me” signals to cytolytic and phagocytic cells).

[0131] Embodiments of this disclosure provide a cell vaccine platform utilizing stem cells (e.g., induced pluripotent stem cells) that can be differentiated in vitro. The use of stem cells (e.g., iPSCs) is beneficial because it does not require the use of any type of transformed cancer cells, while retaining the ability to permanently grow a stock of vaccines engineered for the mass production of stable and consistent cell products. Differentiation into a defined, terminally differentiated, stable cell lineage (such as epithelial cells or cutaneous dendritic "Langerhans" cells) allows the vaccine to further detach from cancer cells and design the same cell type as the cell type from the recipient tissue to which it is delivered.

[0132] Further provided herein are vaccines comprising genetically engineered cells differentiated from stem cells (e.g., iPSCs) into epithelial (dendritic) antigen-presenting cells (APCs) such that “APC mimicry” is present. Some embodiments of the vaccine include MHC nulls and NK / Mo innate immunity + APCs presented to the host patient’s natural MHC-specific APC / innate immune system. Therefore, the vaccine needs to generate a superior and safer immune antigen response and neutralizing antibody production to confer sustained immunity to the frontline sites of APCs in the body after intradermal / SQ injection of the universal vaccine cells (UVCs) of the present invention and after vaccine injection.

[0133] Table 1 provides a further comparison between cell-based vaccines and viral vector-based vaccines, as well as exemplary advantages of cell-based vaccines. [Table 1]

[0134] In some embodiments, this specification provides genetically engineered human cells comprising: (a) genomic disruption in at least one human leukocyte antigen (HLA) gene or at least one transcription regulator of an HLA gene; and (b) an exogenous nucleic acid encoding a cell surface protein that binds to a protein expressed on the surface of phagocytic or cytolytic immune cells, or a functional fragment or functional variant of the cell surface protein, wherein the binding results in activation of the phagocytic or cytolytic activity of the immune cells.

[0135] In some embodiments, the genome disruption inhibits the expression of the HLA protein encoded by the at least one HLA gene on the surface of the cell. In some embodiments, the genome disruption inhibits the expression of the HLA protein encoded by the at least one HLA gene on the surface of the genetically engineered human cell for a period of time sufficient to interact with the protein expressed on the surface of the NK cell.

[0136] In some embodiments, the genome disruption is located in an HLA class I gene. In some embodiments, the HLA class I gene is an HLA-A gene, an HLA-B gene, an HLA-C gene, or a β-microglobulin gene. In some embodiments, the HLA class I gene is a β-microglobulin gene.

[0137] In some embodiments, the genome disruption is located in an HLA class II gene. In some embodiments, the HLA class II gene is the HLA-DP gene, HLA-DM gene, HLA-DOA gene, HLA-DOB gene, HLA-DQ gene, or HLA-DR gene.

[0138] In some embodiments, the at least one transcription factor of the HLA gene is the CIITA gene, the RFX5 gene, the RFXAP gene, or the RFXANK gene. In some embodiments, the HLA gene is the CIITA gene.

[0139] In some embodiments, the genetically engineered human cells include genomic disruption of at least one HLA class I gene or at least one transcription factor of the HLA class I gene, and genomic disruption of at least one HLA class II gene or at least one transcription factor of the HLA class II gene.

[0140] In some embodiments, the genetically engineered human cells include genomic disruption in at least one HLA class I transcription factor gene and genomic disruption in at least one HLA class II transcription factor.

[0141] In some embodiments, the immune cells are innate immune cells. In some embodiments, the innate immune cells are NK cells, macrophages, dendritic cells, neutrophils, or eosinophils. In some embodiments, the innate immune cells are NK cells.

[0142] In some embodiments, the binding results in activation of the cytolytic activity of the NK cells. In some embodiments, the cell surface protein is a ligand that specifically binds to natural killer (NK) cell activation receptors expressed on the surface of NK cells. In some embodiments, the cell surface protein is selected from the group consisting of MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, CD155, CD112 (nectin-2), B7-H6, Necl-2, and immunoglobulin Fc. In some embodiments, the cell surface protein is a natural killer (NK) cell activation ligand. In some embodiments, the natural killer cell activation ligand is selected from the group consisting of MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, CD155, CD112 (nectin-2), B7-H6, and Necl-2.

[0143] In some embodiments, the cells comprise a secretory protein that binds to a receptor expressed on the surface of a phagocytic or cytolytic immune cell, or an exogenous nucleic acid encoding a functional fragment or functional variant of the secretory protein, the protein attracting the immune cell toward the genetically engineered human cell.

[0144] In some embodiments, the genetically engineered human cells further comprise an exogenous protein, an antigenic fragment thereof, or a nucleic acid encoding a suicide gene. In some embodiments, the exogenous protein comprises a nucleocapsidrin protein having at least about 85% sequence identity to SEQ ID NO: 54. In some embodiments, the exogenous protein comprises an exogenous antigen protein.

[0145] In some embodiments, the genetically engineered human cells further comprise nucleic acids encoding microbial proteins or antigenic fragments thereof. In some embodiments, the genetically engineered human cells further comprise cancer or tumor-associated proteins or antigenic fragments thereof. In some embodiments, the cancer or tumor-associated proteins comprise nascent antigens or antigenic fragments thereof.

[0146] In some embodiments, the microbial protein is secreted by the genetically modified human cell, expressed on the surface of the genetically modified human cell, or expressed in the cytoplasm of the genetically modified human cell.

[0147] In some embodiments, the microbial protein is a protein of a virus, bacterium, parasite, or protist. In some embodiments, the microbial protein is a viral protein.

[0148] In some embodiments, the viral protein is from a virus of the order Nidovirales. In some embodiments, the viral protein is from a virus of the family Coronaviridae. In some embodiments, the viral protein is from a virus of the subfamily Orthocoronamiae. In some embodiments, the viral protein is from viruses of the genera Alphacoronavirus, Betacoronavirus, Gammacoronavirus, and Deltacoronavirus. In some embodiments, the viral protein is from a virus of the genus Betacoronavirus. In some embodiments, the viral protein is from a virus of the subgenus Salvecovirus. In some embodiments, the viral protein is from two viruses associated with severe acute respiratory syndrome. In some embodiments, the viral protein is from two strains of severe acute respiratory syndrome coronavirus. In some embodiments, the viral protein is the spike protein of severe acute respiratory syndrome coronavirus 2. In some embodiments, the viral protein is the spike protein of SEQ ID NO: 1. In some embodiments, the viral protein is the spike protein encoded by SEQ ID NO: 53.

[0149] In some embodiments, the viral protein is from a virus selected from the group including influenza, Epstein-Barr virus (EBV), megavirus, Norwalk virus, coxsackievirus, Middle East Respiratory Syndrome-related coronavirus, Severe Acute Respiratory Syndrome-related coronavirus, SARS-CoV-2 virus, hepatitis B, varicella-zoster virus, parvovirus, adenovirus, Marburg virus, Ebola virus, rabies, smallpox, HIV, hantavirus, dengue fever, rotavirus, MERS-CoV, mumps virus, cytomegalovirus (CMV), herpesvirus, papillomavirus, chikungunya virus, or any combination thereof.

[0150] In some embodiments, the genetically modified human cells are differentiated from stem cells. In some embodiments, the stem cells are induced pluripotent stem cells (iPSCs), embryonic stem cells (ESCs), adult stem cells (ASCs), pluripotent stem cells (PSCs), or hematopoietic stem and progenitor cells (HSPCs). In some embodiments, the stem cells are induced pluripotent stem cells (iPSCs).

[0151] In some embodiments, the genetically modified human cells are epithelial cells or endothelial cells. In some embodiments, the genetically modified human cells are not cancer cells. In some embodiments, the genetically modified human cells are irradiated. In some embodiments, the genetically modified cells are stem cells. In some embodiments, the stem cells are induced pluripotent stem cells (iPSCs), embryonic stem cells (ESCs), adult stem cells (ASCs), pluripotent stem cells (PSCs), or hematopoietic stem and progenitor cells (HSPCs). In some embodiments, the stem cells are induced pluripotent stem cells (iPSCs). In some embodiments, the genetically modified human cells cannot grow in vitro, in vivo, or both.

[0152] In some embodiments, the disruption of at least one genome is mediated by an endonuclease. In some embodiments, the endonuclease is a CRISPR endonuclease, a zinc finger nuclease (ZFN), or a transcription activator-like effector nuclease (TALEN). In some embodiments, the disruption of at least one genome is mediated by a CRISPR system comprising an endonuclease and a guide RNA (gRNA), wherein the gRNA comprises an RNA sequence complementary to the DNA sequence of the at least one HLA gene or at least one transcription regulator of the HLA gene.

[0153] In some embodiments, this specification provides genetically engineered human cells comprising: (a) genomic disruption in at least one human leukocyte antigen (HLA) gene or at least one transcription regulator of an HLA gene; (b) a nucleic acid encoding an exogenous cell surface protein that binds to a protein expressed on the surface of phagocytic or cytolytic immune cells, or a functional fragment or functional variant of the exogenous cell surface protein, wherein the binding results in activation of the phagocytic or cytolytic activity of the immune cells; and (c) a nucleic acid encoding an exogenous protein or an antigenic fragment thereof.

[0154] In some embodiments, the exogenous protein or its antigenic fragment is a microbial protein or its antigenic fragment.

[0155] In some embodiments, the microbial protein is secreted by the genetically engineered human cell, expressed on the surface of the genetically engineered human cell, or expressed in the cytoplasm of the genetically engineered human cell. In some embodiments, the microbial protein is inserted into the genetically engineered human cell by microinjection, electroporation, or other means using techniques known in the art.

[0156] In some embodiments, the microbial protein is a protein from a virus, bacterium, parasite, or protist.

[0157] In some embodiments, the microbial protein is a viral protein. In some embodiments, the viral protein is from a virus of the order Nidovirales. In some embodiments, the viral protein is from a virus of the family Coronaviridae. In some embodiments, the viral protein is from the subfamily Orthocoronamiae. In some embodiments, the viral protein is from viruses of the genera Alphacoronavirus, Betacoronavirus, Gammacoronavirus, and Deltacoronavirus. In some embodiments, the viral protein is from a virus of the genus Betacoronavirus. In some embodiments, the viral protein is from a virus of the subgenus Salvecovirus. In some embodiments, the viral protein is from two viruses associated with severe acute respiratory syndrome. In some embodiments, the viral protein is from two strains of severe acute respiratory syndrome coronavirus. In some embodiments, the viral protein is the spike protein of severe acute respiratory syndrome coronavirus 2. In some embodiments, the viral protein is the spike protein of SEQ ID NO: 1. In some embodiments, the viral protein is the spike protein encoded by SEQ ID NO: 53.

[0158] In some embodiments, the viral protein is from a virus selected from the group including influenza, Epstein-Barr virus (EBV), megavirus, Norwalk virus, coxsackievirus, Middle East Respiratory Syndrome-related coronavirus, Severe Acute Respiratory Syndrome-related coronavirus, SARS-CoV-2 virus, hepatitis B, varicella-zoster virus, parvovirus, adenovirus, Marburg virus, Ebola virus, rabies, smallpox, HIV, hantavirus, dengue fever, rotavirus, MERS-CoV, mumps virus, cytomegalovirus (CMV), herpesvirus, papillomavirus, chikungunya virus, or any combination thereof.

[0159] In some embodiments, the genetically modified human cells are differentiated from stem cells. In some embodiments, the stem cells are induced pluripotent stem cells (iPSCs), embryonic stem cells (ESCs), adult stem cells (ASCs), pluripotent stem cells (PSCs), or hematopoietic stem and progenitor cells (HSPCs). In some embodiments, the stem cells are induced pluripotent stem cells (iPSCs). In some embodiments, the genetically modified human cells are epithelial cells or endothelial cells. In some embodiments, the genetically modified human cells are not cancer cells. In some embodiments, the genetically modified human cells are irradiated.

[0160] In some embodiments, the immune cells are innate immune cells. In some embodiments, the innate immune cells are NK cells, macrophages, dendritic cells, neutrophils, or eosinophils. In some embodiments, the innate immune cells are NK cells.

[0161] In some embodiments, compositions comprising a population of genetically modified human cells as described herein are provided herein.

[0162] In some embodiments, pharmaceutical compositions comprising genetically modified human cells as disclosed herein are provided.

[0163] In some embodiments, a unit dosage form is provided comprising a pharmaceutical composition containing genetically modified human cells as disclosed herein.

[0164] In some embodiments, this specification provides a method for producing a population of genetically modified human stem cells, the method comprising obtaining a population of human stem cells, inducing genomic disruption in at least one HLA gene or at least one transcription factor of the HLA gene, and introducing an exogenous cell surface protein that binds to a protein expressed on the surface of phagocytic or cytolytic immune cells, or a nucleic acid encoding a functional fragment or functional variant of the exogenous cell surface protein, wherein the binding results in activation of the phagocytic or cytolytic activity of the immune cells, thereby producing a population of genetically modified stem cells.

[0165] In some embodiments, the genome disruption inhibits the expression of the HLA protein encoded by the at least one HLA gene on the surface of the cell. In some embodiments, the genome disruption inhibits the expression of the HLA protein encoded by the at least one HLA gene on the surface of the cell for a period of time sufficient to interact with proteins expressed on the surface of immune cells.

[0166] In some embodiments, the at least one genome disruption is mediated by an endonuclease. In some embodiments, the endonuclease is a CRISPR endonuclease, a zinc finger nuclease (ZFN), or a transcription activator-like effector nuclease (TALEN). In some embodiments, the at least one genome disruption is mediated by a CRISPR system comprising an endonuclease and a guide RNA (gRNA), wherein the gRNA comprises an RNA sequence complementary to the DNA sequence of the at least one HLA gene or at least one transcription regulator of the HLA gene. In some embodiments, the genome disruption is a single-strand DNA break or a double-strand DNA break.

[0167] In some embodiments, the method further includes introducing a nucleic acid encoding a microbial protein or an antigenic fragment thereof. In some embodiments, the microbial protein comprises a nucleocapsidrin protein having at least about 85% sequence identity to SEQ ID NO: 54. In some embodiments, the microbial protein is secreted by the genetically engineered human cell, expressed on the surface of the genetically engineered human cell, or expressed in the cytoplasm of the genetically engineered human cell. In some embodiments, the microbial protein is a viral, bacterial, or parasitic protein. In some embodiments, the microbial protein is a viral protein.

[0168] In some embodiments, the method further includes introducing nucleic acids encoding cancer or tumor-associated proteins, neoantigens, or antigenic fragments thereof.

[0169] In some embodiments, the stem cells are induced pluripotent stem cells (iPSCs), embryonic stem cells (ESCs), adult stem cells (ASCs), pluripotent stem cells (PSCs), or hematopoietic stem and progenitor cells (HSPCs). In some embodiments, the stem cells are induced pluripotent stem cells (iPSCs). In some embodiments, the method further includes differentiating the population of genetically modified human stem cells. In some embodiments, the cells differentiate into epithelial cells or endothelial cells.

[0170] In some embodiments, the genetically engineered human cell according to any one of claims 79 to 93, wherein the immune cell is an innate immune cell. In some embodiments, the innate immune cell is an NK cell, macrophage, dendritic cell, neutrophil, or eosinophil. In some embodiments, the innate immune cell is an NK cell.

[0171] In some embodiments, this specification provides a method for producing a population of genetically engineered human differentiated cells, the method comprising: obtaining a population of human stem cells; inducing genomic disruption in at least one HLA gene or at least one transcription factor of the HLA gene; introducing an exogenous cell surface protein that binds to a protein expressed on the surface of phagocytic or cytolytic immune cells, or a nucleic acid encoding a functional fragment or functional variant of the exogenous cell surface protein, wherein the binding results in activation of the phagocytic or cytolytic activity of the immune cells; producing a population of genetically engineered human stem cells; and differentiating the population of genetically engineered human stem cells into a population of terminally differentiated genetically engineered human cells.

[0172] In some embodiments, the population of genetically modified human stem cells differentiates into epithelial cells or endothelial cells.

[0173] In some embodiments, this specification provides a method for immunizing a human subject with a microorganism, the method comprising administering to the subject the genetically modified human cells described herein, a composition comprising the genetically modified human cells described herein, or a pharmaceutical composition comprising the genetically modified human cells described herein.

[0174] In some embodiments, this specification provides a method for immunizing a human subject with a microorganism, the method comprising administering a population of genetically modified human cells to the subject, the population of genetically modified human cells comprising (a) a genomic disruption in at least one HLA gene or at least one transcription regulator of an HLA gene, (b) a nucleic acid encoding an exogenous cell surface protein that binds to a protein expressed on the surface of phagocytic or cytolytic immune cells, or a functional fragment or functional variant of the exogenous cell surface protein, wherein the binding results in activation of the phagocytic or cytolytic activity of the immune cells, and (c) a nucleic acid encoding a microbial protein or an antigenic fragment thereof.

[0175] In some embodiments, the binding results in immune cell-mediated lysis or phagocytosis of at least a portion of the population of genetically modified human cells.

[0176] In some embodiments, the administration results in the subject initiating an adaptive immune response to the microorganism. In some embodiments, the administration results in increased activation and / or proliferation of T cells expressing T cell receptors that specifically bind the peptide of the microbial protein. In some embodiments, the administration results in increased activation and / or proliferation of B cells expressing B cell receptors that specifically bind the peptide of the microbial protein. In some embodiments, the administration results in an increase in circulating antibodies that specifically bind the microbial protein. In some embodiments, the microbial protein is secreted by the genetically engineered human cell, expressed on the surface of the genetically engineered human cell, or expressed in the cytoplasm of the genetically engineered human cell. In some embodiments, the microbial protein is a virus, bacterium, or parasite protein.

[0177] In some embodiments, the microbial protein is a viral protein. In some embodiments, the viral protein is from a virus of the Coronaviridae family. In some embodiments, the viral protein is from viruses of the alphacoronavirus, betacoronavirus, gammacoronavirus, and deltacoronavirus genera. In some embodiments, the viral protein is from a virus of the betacoronavirus gene. In some embodiments, the viral protein is from two viruses associated with severe acute respiratory syndrome (SPR). In some embodiments, the viral protein is from two strains of SPR.

[0178] In some embodiments, the viral protein is from a virus selected from the group including influenza, Epstein-Barr virus (EBV), megavirus, Norwalk virus, coxsackievirus, Middle East Respiratory Syndrome-related coronavirus, Severe Acute Respiratory Syndrome-related coronavirus, SARS-CoV-2 virus, hepatitis B, varicella-zoster virus, parvovirus, adenovirus, Marburg virus, Ebola virus, rabies, smallpox, HIV, hantavirus, dengue fever, rotavirus, MERS-CoV, mumps virus, cytomegalovirus (CMV), herpesvirus, papillomavirus, chikungunya virus, or any combination thereof.

[0179] In some embodiments, the population of genetically modified cells is administered intramuscularly or subcutaneously.

[0180] In some embodiments, the immune cells are innate immune cells. In some embodiments, the innate immune cells are NK cells, macrophages, dendritic cells, neutrophils, or eosinophils. In some embodiments, the innate immune cells are NK cells.

[0181] In some embodiments, the human cells further comprise a suicide gene, for example, a cleaved EGFR or HER2 gene that lacks or exhibits low levels of intracellular activity but can be targeted by administering a drug such as an EGFR or HER2-binding antibody. In some embodiments, the microbial protein comprises a nucleocapsidrin protein having at least about 85% sequence identity to SEQ ID NO: 54.

[0182] platform This specification provides, in particular, a vaccine platform including genetically modified platform cells for use in vaccines. Specifically, the platform cells described herein are stem cells, such as embryonic stem cells or pluripotent stem cells, that have been genetically modified by disrupting one or more MHC genes (or, in particular, HLA genes in the case of human cells) to facilitate their use as an allogeneic vaccine platform. The platform cells described herein can be modified to express exogenous proteins or antigenic fragments thereof that are associated with a specific vaccine and are tailored to a specific antigen, such as a viral antigen.

[0183] In some embodiments, the platform cells include genomic disruption in at least one human leukocyte antigen (HLA) gene or at least one transcription regulator of an HLA gene, and express exogenous proteins that bind to phagocytic or cytolytic immune cells, such as innate immune cells, and stimulate the activity of the immune cells (e.g., phagocytosis, cytolytic activity, pro-inflammatory cytokine secretion). In some embodiments, the platform cells express secreted exogenous proteins that attract phagocytic or cytolytic immune cells to the platform cells (or vaccine cells designed from platform cells). In some embodiments, the platform cells express and / or secrete exogenous cell surface proteins on their surface that bind to phagocytic or cytolytic immune cells.

[0184] Platform cells In some embodiments, the platform cells described herein are engineered stem cells. In some embodiments, the stem cells are induced pluripotent stem cells (iPSCs), embryonic stem cells (ESCs), adult stem cells (ASCs), pluripotent stem cells (PSCs), or hematopoietic stem and progenitor cells (HSPCs). In some embodiments, the stem cells are induced pluripotent stem cells (iPSCs). In some embodiments, the cells are mammalian cells. In some embodiments, the cells are human cells. In some embodiments, the cells are mouse or non-human primate cells. In some cases, cells such as iPSCs can differentiate into epithelium (ectoderm derived from iPSCs), APCs (Langerhans, dendritic cells), or a combination thereof.

[0185] HLA modification: In some embodiments, the platform cells described herein include genomic disruption in at least one human leukocyte antigen (HLA) gene or at least one transcription regulator of an HLA gene. In some embodiments, the genomic disruption inhibits the expression of the HLA protein encoded by the at least one HLA gene on the surface of the cell. In some embodiments, the genomic disruption inhibits the expression of the HLA protein encoded by the at least one HLA gene on the surface of the genetically engineered human cell for a period sufficient to interact with the protein expressed on the surface of the innate immune cell.

[0186] In some embodiments, the genome disruption results in reduced HLA or MHC-mediated T cell activation and / or proliferation in subjects administered with the manipulated cells described herein, or in an ex vivo assay, compared to other cells expressing the HLA gene. In some embodiments, the genome disruption results in a less HLA or MHC-mediated T cell response in subjects administered with the manipulated cells described herein, or in an ex vivo assay, compared to equivalent cells lacking the genome disruption.

[0187] In some cases, platform cells may be stem cells engineered to be HLA-deficient. HLA-deficient cells may be HLA class I-deficient, HLA class II-deficient, or both. In certain embodiments, HLA-deficient cells refer to cells that lack, no longer maintain, or have reduced levels of surface expression of the complete MHC complex containing the HLA class I protein heterodimer and / or HLA class II heterodimer, where the reduced or decreased levels are lower than those naturally detectable by other cells or synthetic methods.

[0188] HLA class I deficiency can be achieved by functional deletion or genomic disruption of any region of the HLA class I locus (chromosome 6p21), or by deletion, disruption, or reduced expression levels of HLA class I-related genes, including but not limited to the β-2 microglobulin (B2M) gene, TAP1 gene, TAP2 gene, and tapasin. In some embodiments, the disrupted HLA class I genes are the HLA-A gene, HLA-B gene, and HLA-C gene.

[0189] HLA class II deficiency can be achieved by functional deletion, disruption, or reduction of HLA-II related genes, including but not limited to RFXANK, CIITA, RFX5, and RFXAP. In some embodiments, the disrupted HLA class II genes are HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, and HLA-DR.

[0190] In some embodiments, this specification provides platform cells which are HLA-deficient stem cells such as iPSCs, which are further modified by introducing genes that express proteins (e.g., non-classical HLA class I proteins (e.g., HLA-E and HLA-G), chimeric antigen receptors (CARs), T cell receptors (TCRs), CD16Fc receptors, BCL11b, NOTCH, RUNX1, IL15, 41BB, DAPIO, DAP12, CD24, CD3z, 41BBL, CD47, CD113, and PDL1) associated with improved differentiation potential, antigen targeting, antigen presentation, antibody recognition, persistence, immune evasion, resistance to suppression, proliferation, co-stimulation, cytokine stimulation, cytokine production (autocrine or paracrine), chemotaxis, and cytotoxic activity.

[0191] In some embodiments, the genetically engineered human cells include genomic disruption of at least one HLA class I gene or at least one transcription factor of the HLA class I gene, and genomic disruption of at least one HLA class II gene or at least one transcription factor of the HLA class II gene. In some embodiments, the genetically engineered human cells include genomic disruption of at least one HLA class I transcription factor gene, and genomic disruption of at least one HLA class II transcription factor.

[0192] In some embodiments, the platform cells do not express any HLA I protein on their cell surface. In some embodiments, the cells do not express any HLA II protein on their cell surface. In some embodiments, the cells do not express any HLA I or HLA II protein on their cell surface. In some embodiments, the platform cells described herein do not express enough HLA I protein on their cell surface to initiate an immune response by a subject when administered to an HLA-incompatible subject. In some embodiments, the platform cells do not express enough HLA II protein on their cell surface to initiate an immune response by a subject when administered to an HLA-incompatible subject.

[0193] Stimulation of innate immune cell activity: The platform cells described herein are engineered to express exogenous proteins that bind to innate immune cells such as NK cells, dendritic cells, neutrophils, macrophages, or mast cells, thereby stimulating the activity of innate immune cells (e.g., cytolytic activity, pro-inflammatory cytokine secretion).

[0194] In some embodiments, the platform cells contain nucleic acids encoding exogenous proteins that bind to innate immune cells such as NK cells, dendritic cells, neutrophils, macrophages, or mast cells, and stimulate the activity of these innate immune cells (e.g., trogocytosis).

[0195] Innate immune cell activation can be determined by analyzing the levels of at least one of the following: degranulation / activation markers (CD107a, CD63, CD107b, CD69), granzyme B, IFNg, MIP-1b, perforin, TNFa, or any combination thereof. Activation can also be determined by imaging, flow cytometry, ELISA, quantitative PCR, or any combination thereof.

[0196] NK cells: NK cells express multiple activating and inhibitory receptors that recognize proteins expressed on the surface of other cells. Normal, healthy cells express MHC class I molecules on their surface that act as ligands for inhibitory receptors on NK cells, contributing to NK cell self-resistance. Pathogen-infected cells lose surface MHC class I expression, resulting in reduced inhibitory signaling in NK cells. Cellular stress associated with viral infection, such as DNA damage responses or senescence programs, upregulates ligands for activating receptors on infected cells. As a result, signaling from activating receptors in NK cells shifts the balance toward NK cell activation and target cell elimination, either directly through NK cell-mediated cytotoxicity or indirectly through the secretion of pro-inflammatory cytokines.

[0197] In some embodiments, the platforms and vaccine cells described herein express one or more NK cell activating ligands. In some embodiments, the platforms and vaccine cells described herein are genetically engineered to reduce or eliminate the expression of NK cell inhibitory ligands.

[0198] Complete NK cell activation requires recognition of the NK cell activation receptor by one or more NK cell ligands expressed on the surface of target cells. In some embodiments, the platform cells described herein are engineered to enhance the recognition and lysis of the platform cells by NK cells. In some cases, the platform cells are engineered to express (or overexpress) one or more NK cell activation ligands. For example, in one embodiment, cells can be engineered to express cell MICA / B, Necl-2, or any other ligand listed in Table 2 on their cell surface, or to express one or more of their functional domains sufficient to bind to NK cells. Cells can be genetically engineered to introduce an exogenous gene encoding an NK cell activation ligand or an NK cell binding domain therefrom (e.g., using the method described herein or methods known in the art). In some embodiments, the genetically engineered cells described herein express at least one ligand from Table 2, or a variant thereof, or a domain therefrom. [Table 2]

[0199] Furthermore, NK cells express inhibitory receptors that bind to inhibitory ligands on target cells to inhibit NK cell activation. NK cell inhibitory receptors transmit signals via an immunoreceptor tyrosine inhibitory motif (ITIM) located in their cytoplasmic tail. Upon ligand binding, ITIM undergoes phosphorylation, recruiting phosphatases such as Src homology-containing tyrosine phosphatase 1 (SHP-1), SHP-2, and lipid phosphatase SH2 domain-containing inositol-5-phosphatase (SHIP) to further neutralize the activation signal. During NK cell inhibitory signaling, phosphatases SHP-1 and SHP-2 dephosphorylate the ITAM-carrying Vav-1 molecule, preventing downstream signaling. Table 3 provides an exemplary list of inhibitory receptors and their corresponding ligands expressed on target cells. Such receptors and ligands are well known in the art.

[0200] In some embodiments, the target cells described herein are engineered to enhance their recognition and lysis by NK cells by manipulating the cells to not express (or to reduce the expression of) one or more NK cell inhibitory ligands. For example, in one embodiment, cells may be engineered to induce genomic disruption at one or more HLA class I molecules on the cell surface or any other ligand listed in Table 3. The platforms and vaccine cells described herein may be engineered to knock out any one or any combination of genes encoding NK cell inhibitory ligands, e.g., those listed in Table 3. [Table 3]

[0201] Increase or mimic antibody opsonization to enhance ADCC and / or phagocytosis. In some embodiments, platform cells are opsonized ex vivo to mediate increased phagocytosis and / or ADCC activity in vivo. In some embodiments, cells are engineered to express additional exogenous proteins on their surface. In some embodiments, cells are engineered to express a number of exogenous proteins on their cell surface. In some embodiments, engineered cells are ex vivo contacted with an antibody (e.g., containing an antigen-binding domain and an Fc domain) that binds to the exogenous protein so that the cells are coated with the antibody. Opsonization of cells can mediate increased phagocytosis and / or ADCC by phagocytic cells (e.g., macrophages) and NK cells, respectively. In some embodiments, cells are engineered ex vivo to increase in vivo opsonization. In some embodiments, the cells are engineered to express an Fc domain on their surface such that the CH2 domain is proximal to the cell membrane and the CH3 domain is distal to the cell membrane.

[0202] Stimulation of phagocytosis In some embodiments, the cells described herein express exogenous proteins that bind to phagocytic cells and stimulate the activity of phagocytic cells (e.g., phagocytosis). In some embodiments, the cells contain exogenous nucleic acids encoding proteins that bind to phagocytic cells and stimulate the activity of phagocytic cells (e.g., phagocytosis). In some embodiments, the phagocytic cells are macrophages, dendritic cells, eosinophils, or neutrophils. In some embodiments, the exogenous proteins are selected from the group consisting of phosphatidylserine, calreticulin, and c1q.

[0203] In some cases, apoptotic cells secrete molecules, so-called "find-me" signals (also referred to as "come-to-get-me" signals), to attract phagocytic cells toward them. Any or all of these signals can be incorporated into the cells provided herein. Four representative "find-me" signals released by apoptotic cells have been identified, including S1P (sphingosine-1-phosphate), LPC (lysophosphatidylcholine), nucleotides (ATP or UTP), and CX3CL1 (CX3C motif chemokine ligand 1; fractalkine). They bind to S1PR, G2A, P2Y2, and CX3CR on the surface of phagocytic cells, respectively, and promote phagocytic cell migration to apoptotic cells.

[0204] In some embodiments, the platform cells described herein include genomic disruption in at least one gene that inhibits cellular phagocytosis. In some embodiments, the disruption is in a gene selected from the group consisting of CD47 and CD31.

[0205] Recruitment of immune cells In some embodiments described herein, platform cells are provided that express and secrete exogenous proteins that bind to immune cells and attract the immune cells to platform cells. In some embodiments, the cells include exogenous nucleic acids that bind to immune cells and encode secretions that attract immune cells, such as innate immune cells or adaptive immune cells, to platform cells.

[0206] In some embodiments, the exogenous protein is a cytokine or chemokine. In some embodiments, the protein is selected from the group consisting of S1P (sphingosine-1-phosphate), LPC (lysophosphatidylcholine), nucleotides (ATP or UTP), and CX3CL1 (CX3C motif chemokine ligand 1; fractalkine), CX3CL1, and ICAM3. The IL-8 / CXCL8 chemokine appears to be important for neutrophil migration to CCL2, CXCL9, and CXCL10, which are thought to recruit CTLs and monocytes.

[0207] Methods of genetic modification Genetic modification of platform cells or vaccine cells (e.g., knock-in of an introduced gene or knock-out of an undesirable gene) can be achieved by any known genetic engineering technique, including, but not limited to, endonucleases (zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and RNA-induced CRISPR-Cas9 nucleases (CRISPR / Cas9; clustered regular, spaced short palindromic repeat-related 9)).

[0208] CRISPR system The methods for producing genetically modified cells described herein may utilize a CRISPR system, including but not limited to knockout of NK cell inhibitory ligands and knock-in of NK cell activating ligands.

[0209] There are at least five types of CRISPR systems, all of which incorporate RNA and Cas proteins. Types I, III, and IV assemble multi-Cas protein complexes that can cleave nucleic acids complementary to crRNA. Both types I and III require pre-crRNA treatment before the processed crRNA can be assembled into the multi-Cas protein complex. Types II and V CRISPR systems consist of a single Cas protein that has formed a complex with at least one guide RNA.

[0210] The general mechanisms and recent advances of the CRISPR system are discussed in the following publications: Cong, L. et al., “Multiplex genome engineering using CRISPR systems,” Science, 339(6121):819-823(2013); Fu, Y. et al., “High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells,” Nature Biotechnology, 31,822-826(2013); Chu, VT et al., “Increasing the efficiency of homology-directed repair for CRISPR-Cas9-induced precise gene editing in mammalian cells,”Nature Biotechnology 33,543-548(2015);Shmakov,S.et al,“Discovery and functional characterization of diverse Class2 CRISPR-Cas systems,”Molecular Cell,60,1-13(2015);Makarova,KS et al,“An updated evolutionary classification of CRISPR-Cas systems,”,Nature Reviews This is discussed in Microbiology, 13, 1-15 (2015). Site-specific cleavage of target DNA occurs at a location determined by both 1) base pairing complementarity between guide RNA and target DNA (also called protospacer), and 2) a short motif within the target DNA called a protospacer adjacent motif (PAM). For example, engineered cells can be generated using a CRISPR system, e.g., a type II CRISPR system. The Cas enzyme used in the methods disclosed herein may be Cas9, which catalyzes DNA cleavage. Enzymatic action by Cas9 derived from Streptococcus pyogenes or any closely related Cas9 can generate double-strand breaks at target site sequences that hybridize to 20 nucleotides of the guide sequence and have a protospacer adjacent motif (PAM) 20 nucleotides after the target sequence.

[0211] The CRISPR system can be introduced into cells or cell populations by any means. In some embodiments, the CRISPR system can be introduced by electroporation or nucleofection. Electroporation can be performed using, for example, the Neon® Transfection System (ThermoFisher Scientific) or the AMAXA® Nucleofector (AMAXA® Biosystems). The parameters of electroporation can be adjusted to optimize transfection efficiency and / or cell viability. Electroporation devices can have multiple electrical waveform pulse settings, such as exponential decay, time constant, and square wave. Every cell type has its own optimal electric field strength (E) that depends on the applied pulse parameters (e.g., voltage, capacitance, and resistance). The application of the optimal electric field strength triggers an electrical permeation reaction via the induction of a transmembrane voltage, allowing nucleic acids to pass through the cell membrane. In some embodiments, the electroporation pulse voltage, electroporation pulse width, number of pulses, cell density, and chip type can be adjusted to optimize transfection efficiency and / or cell viability.

[0212] Cas protein The vector can be operably ligated to an enzyme-coding sequence encoding a CRISPR enzyme, such as a Cas protein (CRISPR-related protein). In some embodiments, the nuclease or nuclease-encoding polypeptide is derived from the CRISPR system (e.g., a CRISPR enzyme). In some embodiments, the CRISPR enzyme directs single-strand or double-strand cleavage in the target sequence. In some embodiments, the CRISPR enzyme mediates double-strand cleavage in the target DNA sequence (e.g., creating a double-strand break in the target DNA sequence).

[0213] Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 or Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Examples include Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx1S, Csf1, Csf2, CsO, Csf4, Cpf1, c2c1, c2c3, Cas9HiFi, their homologs, or modified versions thereof. In some embodiments, catalytically dead Cas proteins can be used (e.g., catalytically dead Cas9 (dCas9)). Unmodified CRISPR enzymes, such as Cas9, may have DNA cleavage activity. In some embodiments, the nuclease is Cas9. In some embodiments, the polypeptide encodes Cas9. In some embodiments, the nuclease or the polypeptide encoding the nuclease is catalytically dead. In some embodiments, the nuclease is catalytically dead Cas9 (dCas9). In some embodiments, the polypeptide encodes catalytically dead Cas9 (dCas9). The Cas protein may be a high-fidelity Cas protein such as Cas9HiFi.

[0214] S. pyogenes Cas9 (SpCas9) is commonly used as a CRISPR endonuclease for genome engineering, but it may not be the best endonuclease for all target excision sites. For example, while the PAM sequence (5'NGG3') of SpCas9 is abundant throughout the human genome, the NGG sequence may not be correctly positioned to target the desired gene for modification. In some embodiments, different endonucleases may be used to target specific genomic targets. In some embodiments, synthetic SpCas9-derived mutants containing non-NGG PAM sequences may be used. Furthermore, other Cas9 orthologues from various species have been identified, and these “non-SpCas9” bind to a variety of PAM sequences that may also be useful in this disclosure. For example, the relatively large size of SpCas9 (approximately 4kb of coding sequence) is a factor in SpCas9 This means that plasmids containing cDNA may not be efficiently expressed in cells. Conversely, the coding sequence of Staphylococcus aureus Cas9 (SaCas9) is approximately 1 kilobase shorter than that of SpCas9 and may be efficiently expressed in cells. Similar to SpCas9, the SaCas9 endonuclease can modify target genes in vivo in mammalian cells and in vivo in mice.

[0215] Alternatives to S. pyogenes Cas9 may include RNA-guided endonucleases of the Cpf1 family that exhibit cleavage activity in mammalian cells. Unlike Cas9 nucleases, Cpf1-mediated DNA cleavage yields double-strand breaks with short 3' overhangs. The alternating cleavage pattern of Cpf1 may open up the possibility of targeted gene transfer, similar to conventional restriction enzyme cloning, which could increase the efficiency of gene editing. Similar to the Cas9 variants and orthologues described above, Cpf1 may also expand the number of sites that can be targeted by CRISPR to AT-rich regions or AT-rich genomes that lack the NGG PAM sites preferred by SpCas9.

[0216] A vector encoding a CRISPR enzyme containing one or more nuclear localization sequences (NLSs) (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 NLSs) can be used. For example, a CRISPR enzyme may contain at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 NLSs at or near the amino terminus, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 NLSs at or near the carboxyl terminus, or any combination thereof (e.g., one or more NLSs at the amino terminus and one or more NLSs at the carboxyl terminus). If more than one NLS is present, each may be selected independently of the others so that a single NLS may exist in more than one copy and / or in combination with one or more other NLSs present in one or more copies. NLSs may be located anywhere in the polypeptide chain, e.g., near the N-terminus or C-terminus. For example, NLS may be located within 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, or 50 amino acids from the N-terminus or C-terminus along the polypeptide chain, or within approximately that distance. In some cases, NLS may be located within 50 amino acids or more from the N-terminus or C-terminus, for example, within 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 amino acids, or within approximately that distance.

[0217] Any functional concentration of Cas protein can be introduced into cells. For example, 15 micrograms of Cas mRNA can be introduced into cells. In other cases, Cas mRNA may be introduced in amounts ranging from 0.5 micrograms to 100 micrograms. Cas mRNA may be introduced in amounts of 0.5, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 micrograms.

[0218] In some embodiments, the double nickase technique can be used to introduce double-strand breaks or genomic breaks. The Cas protein can be mutated at a known amino acid within either nuclease domain, thereby deleting the activity of one nuclease domain and creating a nickase Cas protein capable of generating single-strand breaks. Using nickase with two specific guide RNAs targeting opposing strands can generate double-strand breaks (DSBs) within the target site (often referred to as the "double nick" or "double nickase" CRISPR system). Because it is unlikely that two off-target nicks will be generated close enough to cause a DSB, this technique can increase target specificity.

[0219] Guiding polynucleic acid (gRNA or gDNA) The guiding polynucleic acid (or guide polynucleic acid) may be DNA (gDNA) or RNA (gRNA). The guiding polynucleic acid may be single-stranded or double-stranded. In some embodiments, the guiding polynucleic acid may include single-stranded and double-stranded regions. The guiding polynucleic acid may also form a secondary structure.

[0220] In some embodiments, the guide nucleic acid is a gRNA. In some embodiments, the gRNA includes a guide sequence that identifies a target site and guides the RNA / Cas complex to the identified target DNA for cleavage. Site-specific cleavage of the target DNA occurs at a location determined by both 1) base pairing complementarity between the gRNA and the target DNA (also called protospacer), and 2) a short motif within the target DNA called a protospacer-adjacent motif (PAM). Similarly, the gRNA is specific to the target DNA and can form a complex with a nuclease to direct its nucleic acid cleavage activity.

[0221] In some embodiments, the gRNA comprises two RNAs, for example, CRISPR RNA (crRNA) and transactivating crRNA (tracrRNA). In some embodiments, the gRNA comprises a single guide RNA (sgRNA) formed by the fusion of a portion (e.g., a functional portion) of the crRNA and the tracrRNA. In some embodiments, the gRNA comprises a biRNA containing both crRNA and tracrRNA. In some embodiments, the gRNA contains crRNA but lacks tracrRNA. In some embodiments, the crRNA hybridizes with target DNA or a protospacer sequence.

[0222] In some embodiments, the gRNA targets a nucleic acid sequence of 20 nucleotides or approximately 20 nucleotides. In some embodiments, the gRNA targets a nucleic acid sequence of 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30 or more nucleotides, or approximately 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30 or more nucleotides. In some embodiments, the gRNA ligates to a genomic region located approximately 1 to 20 base pairs away from the PAM. In some embodiments, the gRNA ligates to a genomic region located approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or up to approximately 20 base pairs away from the PAM. In some embodiments, the gRNA ligates to a genomic region located approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 base pairs away from the PAM.

[0223] The guide RNA may also contain a dsRNA double-stranded region that forms a secondary structure. For example, the secondary structure formed by the guide RNA may include a stem (or hairpin) and a loop. The lengths of the loop and stem can vary. For example, the loop may be in the range of about 3 to about 10 nucleotides in length, and the stem may be in the range of about 6 to about 20 base pairs in length. The stem may contain one or more bulges of 1 to about 10 nucleotides. The total length of the second region may be in the range of about 16 to about 60 nucleotides in length. For example, the loop may be 4 nucleotides or about that length, and the stem may be 12 base pairs or about that length. The dsRNA double-stranded region may contain a protein-binding segment that can form a complex with RNA-bound proteins such as RNA-induced endonucleases, such as Cas proteins.

[0224] In some embodiments, a Cas protein, such as the Cas9 protein, or any derivative thereof, is pre-complexed with gRNA to form a ribonucleoprotein (RNP) complex. In some embodiments, the RNP complex is introduced into cells to mediate editing.

[0225] In some embodiments, the gRNA is modified. Modifications may include chemical changes, synthetic modifications, nucleotide addition, and / or nucleotide removal. Modifications may also enhance CRISPR genomic manipulation. Modifications can alter the chirality of the gRNA. In some embodiments, the chirality may be uniform or sterically pure after modification. In some embodiments, modifications increase the stability of the gRNA.

[0226] In some embodiments, the modification is a chemical modification. The modifications include 5'-adenylic acid, 5'-guanosine triphosphate cap, 5'N7-methylguanosine triphosphate cap, 5'-triphosphate cap, 3'-phosphate, 3'-thiophosphate, 5'-phosphate, 5'-thiophosphate, Cis-Syn thymidine dimer, trimer, C12 spacer, C3 spacer, C6 spacer, d spacer, PC spacer, r spacer, spacer 18, spacer 9, 3'-3' modification, 5'-5' modification, debase, acridine, azobenzene, biotin, biotin BB, biotin TEG, cholesteryl TEG, desthiobiotin TEG, DNP TEG, DNP-X, DOTA, dT-biotin, bibiotin, PC biotin, psoralen C2, psoralen C6, TINA, 3'DABCYL, black hole quencher 1, black hole quencher 2, DABCYL SE, dT-DABCYL, IRDye QC-1, QSY-21, QSY-35, QSY-7, QSY-9, carboxyl linker, thiol linker, 2'-deoxyribonucleoside analog purine, 2'-deoxyribonucleoside analog pyrimidine, ribonucleoside analog, 2'-O-methylribonucleoside analog, sugar-modified analog, fluctuation / universal base, fluorescent dye labeling, 2'-fluoroRNA, 2'O-methylRNA, methylphosphonate, phosphodiester DNA, phosphodiester RNA, phosphothioate DNA, phosphorothioate RNA, UNA, pseudouridine-5'-triphosphate, and 5-methylcytidine-5'-triphosphate, as well as any combination thereof, can be selected.

[0227] In some embodiments, the modification includes phosphorothioate nucleotide linkages. In some embodiments, the gRNA contains 1-10, 1-5, or 1-3 phosphorothioates. In some embodiments, the gRNA contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 phosphorothioate linkages. In some embodiments, the gRNA contains phosphorothioate nucleotide linkages at the N-terminus, C-terminus, or both. For example, in some embodiments, the gRNA contains phosphorothioate nucleotide linkages between 3-5 nucleotides at the N-terminus, 3-5 nucleotides at the C-terminus, or both.

[0228] In some embodiments, the modification is the addition of 2'-O-methylphosphorothioate. In some embodiments, the gRNA contains 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, or 1-2 2'-O-methylphosphorothioate molecules. In some embodiments, the gRNA contains 1-10, 1-5, or 1-3 2'-O-methylphosphorothioate molecules. In some embodiments, the gRNA contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 2'-O-methylphosphorothioate molecules. In some embodiments, the gRNA contains a 2'-O-methylphosphorothioate nucleotide linkage at the N-terminus, C-terminus, or both the N-terminus and C-terminus. For example, in some embodiments, the gRNA includes a 2'-O-methylphosphorothioate internucleotide bond between the N-terminal 3-5 nucleotides, the C-terminal 3-5 nucleotides, or both.

[0229] The gRNA can be introduced at any functional concentration. In some embodiments, 0.5 to 100 micrograms of the gRNA are introduced into the cells. In some embodiments, 0.5, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 micrograms of the gRNA are introduced into the cells.

[0230] Other Endonucleases The manipulated cells described herein can be produced using other endonuclease-based gene editing systems known in the art, such as zinc finger nuclease systems and TALEN systems.

[0231] ZFNs are targeted nucleases containing a nuclease fused to a zinc finger DNA-binding domain. A "zinc finger DNA-binding domain" or "ZFBD" is a polypeptide domain that binds sequence-specifically to DNA via one or more zinc fingers. A zinc finger is a domain consisting of approximately 30 amino acids within a zinc finger-binding domain, and its structure is stabilized by the coordination of a zinc ion. Examples of zinc fingers include, but are not limited to, C2H2, C3H, and C4 zinc fingers. A "designed" zinc finger domain is a non-naturally occurring domain whose design / construction is primarily derived from rational criteria (e.g., substitution rules, as well as the application of computer algorithms to process information in databases storing information on existing ZFN designs and binding data). A "selected" zinc finger domain is a non-naturally occurring domain whose production is primarily derived from empirical processes such as phage display, interaction trapping, or hybrid selection. The best-known example of a ZFN in this field is a fusion of the FokI nuclease and a zinc finger DNA-binding domain.

[0232] TALENs are targeted nucleases containing nucleases fused to the TAL effector DNA-binding domain. The "transcription activator-like effector DNA-binding domain," "TAL effector DNA-binding domain," or "TALE DNA-binding domain" is the polypeptide domain of the TAL effector protein responsible for binding the TAL effector protein to DNA. TAL effector proteins are secreted by plant pathogens of the genus Xanthomonas during infection. These proteins enter the nucleus of plant cells and bind to effector-specific DNA sequences via their DNA-binding domains, activating gene transcription at these sequences via their transactivation domains. The specificity of the TAL effector DNA-binding domain depends on the effector variable number of incomplete 34-amino acid repeats, which include polymorphisms at selected repeat sites called repeat variable diresidues (RVDs). The best-known example of a TALEN in the art is the fusion of the FokI nuclease to the TAL effector DNA-binding domain.

[0233] Another example of a targeted nuclease used in the method described herein is a targeted spoilylnuclease, which is a polypeptide comprising a spoilyl polypeptide having nuclease activity fused to a DNA-binding domain (e.g., zinc finger DNA-binding domain, TAL effector DNA-binding domain) that is specific to the DNA sequence of interest. Further examples of targeted nucleases suitable for the present invention include, but are not limited to, Bxb1, phiC31, R4, PhiBTi, and WO / SPBc / TP9O1-1.

[0234] Any of the aforementioned methods, including genome editing via the use of endonucleases, can result in genomic disruption. Genomic disruption may be sufficient to result in a decrease or elimination of the expression of the protein encoded by the gene. In some cases, genomic disruption can also refer to the incorporation of an exogenous transgene into the cellular genome. In such cases, the exogenous transgene can also be detected. Genomic disruption can be detected in at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the cells tested. Detection can be performed by evaluating disruption at the genomic, mRNA, or protein levels by sequencing. Appropriate methods include PCR, qPCR, flow cytometry, imaging, ELISA, NGS, and any combination thereof. In some cases, protein expression can be reduced by approximately 1x, 2x, 3x, 5x, 10x, 20x, 30x, 50x, 70x, 100x, 125x, 150x, 200x, 250x, 300x, 350x, 500x, or even up to 1000x compared to equivalent methods that do not use gene editing such as CRISPR.

[0235] Transgene The transgene polynucleic acid encoding an exogenous protein or polypeptide to be knocked into the platform or vaccine cells described herein may be single-stranded or double-stranded DNA or RNA and may be introduced into the cell in a linear or circular form. The transgene sequence(s) may be contained within a DNA minicircle that may be introduced into the cell in a circular or linear form. When introduced in a linear form, the ends of the transgene sequence may be protected by any means (e.g., from degradation by exonucleases). For example, one or more dideoxynucleotide residues may be added to the 3' end of the linear molecule, and / or self-complementary oligonucleotides may be ligated to one or both ends. Further methods for protecting the exogenous polynucleotide from degradation include, but are not limited to, the addition of terminal amino groups(s) and the use of modified nucleotide bonds, e.g., phosphorothioates, phosphoramidates, and O-methylribose or deoxyribose residues.

[0236] A transgene can be adjacent to a recombination arm. In some examples, the recombination arm may include a complementary region that targets the transgene to a desired integration site. A transgene can also be integrated into a genomic region such that the insertion disrupts an endogenous gene. A transgene can be integrated by any method, e.g., by non-recombination end joining and / or recombination-directed repair. A transgene can also be integrated during a recombination event in which a double-strand break is repaired. A transgene can also be integrated using a homologous recombination enhancer. For example, an enhancer can block non-homologous end joining so that homology-directed repair occurs to repair a double-strand break.

[0237] A transgene can be adjacent to a recombinant arm if the degree of homology between the arm and its complementary sequence is sufficient to enable homologous recombination between the two. For example, the degree of homology between the arm and its complementary sequence may be 50% or greater. The two homologous-non-identical sequences may be of any length, and their degree of non-homonymy may be as small as a single nucleotide (e.g., for correcting genomic point mutations by targeted homologous recombination) or as large as 10 kilobases or more (e.g., for inserting a gene at a predetermined ectopic site on a chromosome). The two polynucleotides containing the homologous-non-identical sequences do not need to be of the same length.

[0238] Polynucleotides can be introduced into cells as part of a vector molecule having further sequences, such as genes encoding origins of replication, promoters, and antibiotic resistance. Furthermore, the transgene polynucleotide can be introduced as a naked nucleic acid, or as a nucleic acid complexed with a drug such as a liposome or poloxamer, or delivered by a virus (e.g., adenovirus, AAV, herpesvirus, retrovirus, lentivirus, and integrase-deficient lentivirus (IDLV)). AAV viruses are one example of viruses capable of delivering transgenes.

[0239] Transgenes can generally be inserted such that their expression is driven at the integration site by an endogenous promoter, i.e., by a promoter that drives the expression of the endogenous gene into which the transgene is inserted. Transgenes may include promoters and / or enhancers, such as constitutive promoters or inductive or tissue / cell-specific promoters. Minicircle vectors can encode transgenes.

[0240] A transgene can be inserted into an endogenous gene in such a way that all or part of the endogenous gene is expressed, or is not expressed at all. For example, a transgene as described herein can be inserted into an endogenous locus, for example, as a fusion with the transgene, in such a way that part of the endogenous sequence (the N-terminal and / or C-terminal side of the transgene) or neither is expressed. In other cases, a transgene (with or without additional coding sequences, e.g., for an endogenous gene) is incorporated into any endogenous locus, e.g., a safe harbor locus.

[0241] When an endogenous sequence (either endogenous or part of a transgene) is expressed with a transgene, the endogenous sequence can be a full-length sequence (wild-type or mutant) or a partial sequence. Endogenous sequences are functional. Non-limiting examples of the functions of these full-length or partial sequences include extending the serum half-life of a polypeptide expressed by a transgene (e.g., a therapeutic gene) and / or acting as a carrier.

[0242] Furthermore, although not required for expression, the exogenous sequence may include transcriptional or translational regulatory sequences, such as promoters, enhancers, insulators, internal ribosome entry sites, 2A peptides, and / or sequences encoding polyadenylation signals.

[0243] Cell composition: The platform cells described herein can be stored for extended periods for use in vaccine cells as needed. Specifically, these cells can be incorporated into suitable compositions that are stable when frozen or cryopreserved. In some cases, compositions are provided for maintaining the pluripotency of engineered induced pluripotent stem cells (iPSCs) used as platform cells. In some cases, the composition comprises (i) engineered iPSC platform cells, and (ii) a pluripotency maintenance composition, e.g., a small molecule composition comprising a MEK inhibitor, a GSK3 inhibitor, and a ROCK inhibitor. In some embodiments, the platform cells are obtained from reprogrammed non-pluripotent cells, and the resulting iPSCs contain the same target incorporation and / or in / del at selected sites within the engineered non-pluripotent cells. In some embodiments, the engineered iPSCs are obtained by manipulating clonal iPSCs or a pool of iPSCs by introducing one or more target incorporations and / or in / dels at one or more selected sites. In some other embodiments, the genomically engineered iPSCs are obtained by genomically engineering by introducing one or more target inclusions and / or in / dels at one or more selected sites into a pool of reprogramming non-pluripotent cells that are in contact with one or more reprogramming factors and a small molecule composition comprising a TGFP receptor / ALK inhibitor, a MEK inhibitor, a GSK3 inhibitor and / or a ROCK inhibitor.

[0244] The manipulated platform cells of the composition may contain one or more exogenous polynucleotides encoding safety switch proteins, target modalities, receptors, signaling molecules, transcription factors, pharmaceutically active proteins and peptides, drug target candidates, or proteins that promote the engraftment, trafficking, homing, viability, self-renewal, persistence, and / or survival of reprogrammed iPSCs or their derivative cells, and / or in one or more endogenous genes related to target modalities, receptors, signaling molecules, transcription factors, drug target candidates, modulation and regulation of immune responses, or proteins that suppress the engraftment, trafficking, homing, viability, self-renewal, persistence, and / or survival of reprogrammed iPSCs or their derivative cells.

[0245] In some embodiments, one or more exogenous polypeptides or exogenous polynucleotides encoding proteins are operably ligated to (1) one or more exogenous promoters comprising CMV, EF1a, PGK, CAQUBC, or other constitutive, inducible, time-specific, tissue-specific, or cell-type-specific promoters, or (2) one or more endogenous promoters contained in a selected site within a platform cell comprising AAVS1, CCR5, ROSA26, collagen, HTRP, HII, beta-2 microglobulin, GAPDH, TCR, or RUNX1. In some embodiments, the composition further comprises one or more endonucleases capable of selective site recognition for introducing double-strand breaks at the selected site.

[0246] vaccine cells The vaccine cells described herein are prepared by further manipulating the cells to contain nucleic acids encoding an exogenous microbial protein or its antigenic fragment, or to express the exogenous microbial protein or its antigenic fragment. The vaccine cells, upon administration to a target, induce a specific immune response to the exogenous microbial protein.

[0247] Accordingly, in one embodiment, the Specified provides genetically engineered cells comprising: i) genomic disruption in at least one human leukocyte antigen (HLA) gene or at least one transcription regulator of an HLA gene; ii) expression of an exogenous protein that binds to phagocytic or cytolytic innate immune cells and stimulates the activity of the innate immune cells (e.g., phagocytosis, cytolytic activity, pro-inflammatory cytokine secretion); and iii) expression of an exogenous microbial protein or its antigenic fragment. In some embodiments, vaccine cells include the antigen expression constructs described herein.

[0248] The vaccine cells described herein may be any cells suitable for administration to a target and delivery of microbial proteins. In some embodiments, the cells are differentiated from the platform cells. In some embodiments, the cells are differentiated from the platform cells, which are stem cells (e.g., iPSCs). In some embodiments, the cells are epithelial cells. In some embodiments, the cells are endothelial cells.

[0249] Differentiation methods In some embodiments, vaccine cells are differentiated from platform cells, which are stem cells. In some embodiments, the stem cells are induced pluripotent stem cells. In some embodiments, the iPSCs differentiate into epithelial or endothelial cells. In certain embodiments, the iPSCs differentiate into cell types that are inherently less immunogenic to recipient T cells, allowing the differentiated cells to become a concentrated target for NK cell-mediated vaccination. In certain further embodiments, the iPSCs are engineered to present kill tag or suicide switch genes that can be activated to target cells by administration of antibodies or small molecules.

[0250] Differentiation of pluripotent stem cells requires changes in the culture system, such as stimulants in the culture medium or changes in the physical state of the cells. The most traditional strategy utilizes the formation of embryoid bodies (EBs) as a common and important intermediate to initiate lineage-specific differentiation. An "embryoid body" is a three-dimensional cluster that has been shown to mimic embryonic development by giving rise to numerous lineages within its three-dimensional region. Through a differentiation process that typically lasts from a few hours to several days, simple EBs (e.g., aggregated pluripotent stem cells induced to differentiate) continue to mature and develop into cystic EBs, at which point they are further processed, typically for several days to several weeks, to continue differentiation. EB formation is initiated by bringing pluripotent stem cells into close proximity to each other in a three-dimensional multilayer cluster of cells, which is typically achieved by one of several methods, including settling the pluripotent cells in droplets, settling the cells in a "U" bottom well plate, or mechanically agitating them. To promote EB development, pluripotent stem cell aggregates require further differentiation cues, as aggregates maintained in pluripotent culture maintenance medium do not form suitable EBs. Therefore, pluripotent stem cell aggregates need to be transferred to a differentiation medium that provides cues to induce differentiation toward a selected lineage. Culture of EB lineage pluripotent stem cells typically results in the generation of differentiated cell populations (ectoderm, mesoderm, and endoderm) with moderate proliferation within EB cell clusters. However, while EB has been shown to promote cell differentiation, it results in heterogeneous cells with different differentiation states because the three-dimensional structure of the cells is not consistently exposed to differentiation cues from the environment. Furthermore, EB is labor-intensive to create and maintain. Also, EB-mediated cell differentiation involves slow cell proliferation, which also contributes to reduced differentiation efficiency.

[0251] The manipulated iPSCs described herein can be differentiated using biomaterial scaffolds. Depending on the inherent properties of the material, as well as the incorporation of specific chemical and physical cues into the material, biomaterial scaffolds promote the viability and differentiation of stem cells seeded within them. Both natural and synthetic biomaterials can serve as starting points for generating bioactive scaffolds to control stem cell differentiation into desired tissue types. These scaffolds can take several different forms, each with its own unique characteristics. These scaffolds can also be combined to obtain novel hybrid materials that enable better cell viability for specific formulations. Suitable biomaterial scaffolds include, but are not limited to, hydrogels, electrospinning scaffolds, nano and microparticles using protein-based biomaterials (e.g., collagen, fibrin, silk, laminin, fibronectin, and vitronectin), polysaccharide-based biomaterials (e.g., agarose, alginate, hyaluronic acid, chitosan, cellulose and its derivatives, and decellularized extracellular matrix), synthetic biomaterials (e.g., lactate-glycolic acid copolymer (PLGA), poly(ethylene glycol) (PEG), polycaprolactone (PCL), polypyrrole (Ppy), and polydimethylsiloxane (PDMS)), and ceramic-based biomaterials.

[0252] In some cases, the methods provided herein can reduce toxicity compared to equivalent methods. In some cases, toxicity can be reduced by approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 15, 20, 25, 50, 100, 300, 500, 800, or 1000 times.

[0253] cell type In some cases, the method may involve differentiating iPS cells. In some embodiments, stem cells (e.g., iPSCs) may be differentiated into epithelial cells. In some cases, iPS cells can be differentiated into skin epithelial cells, lung epithelial cells, gastrointestinal epithelial cells, alveolar epithelial cells, oral epithelial cells, vaginal epithelial cells, renal epithelial cells, renal ductal epithelial cells, airway epithelial cells, bladder epithelial cells, urothelial cells, vascular epithelial cells, brain epithelial cells, cardiac epithelial cells, ear epithelial cells, tongue epithelial cells, cervical epithelial cells, prostate epithelial cells, mammary epithelial cells, uterine epithelial cells, tracheal epithelial cells, colon epithelial cells, small intestinal epithelial cells, colon epithelial cells, or hepatic epithelial cells.

[0254] APC mimicry In some cases, iPS cells can be differentiated into epithelial (dendritic) antigen-presenting cells (APCs). By differentiating iPS cells into dendritic cells, APC mimicry can be achieved, thereby allowing the vaccine to present MHC null and / or NK / Mo innate immunity + APC to the target's native MHC-specific APC / innate immune system. This results in a superior and / or safer immune antigen response and / or neutralizing antibody production, thereby conferring sustained immunity after administration. In other cases, iPS cells can be differentiated into skin, lung, or GI / intestinal epithelial cells, thus enabling the natural physiological presentation of immunogens to the host immune system. This can facilitate and / or enhance vaccine application for pulmonary application and / or oral administration routes (po), in addition to standard subcutaneous, intradermal, and other skin and skin-system vaccine delivery methods.

[0255] Antigen expression construct Provided herein are antigen expression constructs and engineered cells containing such constructs. In some embodiments, the antigen expression construct comprises a nucleic acid encoding an exogenous protein or an antigenic fragment thereof. In some embodiments, the exogenous protein comprises an exogenous antigen protein. In some embodiments, the construct comprises two or more exogenous proteins or antigenic fragments thereof. In some embodiments, the nucleic acid is DNA or RNA. In some embodiments, the nucleic acid is cDNA. In some embodiments, the nucleic acid is mRNA. In some embodiments, the exogenous protein is a microbial protein. In some embodiments, the microbial protein is a virus, bacterium, parasite, or protozoan protein.

[0256] COVID-19 In some embodiments, the microbial protein is a viral protein. In some embodiments, the viral protein is from a virus of the order Nidovirales. In some embodiments, the viral protein is from a virus of the family Coronaviridae. In some embodiments, the viral protein is from the genera Alphacoronavirus, Betacoronavirus, Gammacoronavirus, and Deltacoronavirus. In some embodiments, the viral protein is from a virus of the genus Betacoronavirus. In some embodiments, the viral protein is from a virus of the subgenus Salvecovirus. In some embodiments, the viral protein is from the virus of two Severe Acute Respiratory Syndrome-associated Coronaviruses. In some embodiments, the viral protein is from the virus of two strains of Severe Acute Respiratory Syndrome-associated Coronavirus. In some embodiments, the viral protein is the spike protein of Severe Acute Respiratory Syndrome Coronavirus 2.

[0257] In some embodiments, the cell vaccines described herein are used to treat coronavirus disease 2019 (COVID-19). As a severe respiratory illness first reported in Wuhan, Hubei Province, China, COVID-19 is also known as COVID-2019, 2019 novel coronavirus, or 2019-nCoV.

[0258] COVID-19 is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The SARS-CoV-2 genome has been sequenced, and the gene order (5' to 3') is replicase ORF1ab, spike (S), envelope (E), membrane (M), and nucleocapsid (N). Wu, F., Zhao, S., Yu, B., Chen, Y., Wang, W., Song, Z., Hu, Y., Tao, Z., Tian, ​​J., Pei, Y., Yuan, M., Zhang, Y., Dai, F., Liu, Y., Wang, Q., Zheng, J., Xu, L., Holmes, E., & Zhang, Y., A new coronavirus associated with human respiratory disease in China. Nature 579, 265-269 (2020) (Its entire contents are incorporated herein by reference for all purposes).

[0259] SARS-CoV-2 invades host cells using a highly glycosylated spike protein (S protein). The coronavirus spike protein is a trimer class I fusion protein that exists in a metastable pre-fusion conformation that undergoes substantial structural rearrangement to fuse the viral membrane with the host cell membrane. This process is triggered when the S1 subunit binds to the host cell receptor. Receptor binding destabilizes the pre-fusion trimer, resulting in the detachment of the S1 subunit and the transition to a stable post-fusion conformation of the S2 subunit. To bind to the host cell receptor, the receptor-binding domain (RBD) of S1 undergoes a hinged conformational change that temporarily hides or exposes the determinants of receptor binding. These two states are called "down" conformation and "up" conformation, with down corresponding to a receptor-inaccessible state and up corresponding to a receptor-accessible state, both considered less stable. Due to the essential function of the S protein, it represents a target for antibody-mediated neutralization, and characterizing the pre-fusion S structure would provide atomic-level information to guide vaccine design and development. Wrapp, D., Wang, N., Corbett, K., Goldsmith, J., Hsieh, C., Abiona, O., Graham, B. & McLellan, J., Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation, SCIENCE, 13 MAR 2020:1260-1263 (its entire contents are incorporated herein by reference for all purposes).

[0260] Amino acid sequence of the SARS-CoV-2 S protein (obtained from NCBI): >YP_009724390.1 Surface glycoprotein [Severe acute respiratory syndrome coronavirus 2]

[0261] The spike protein of SARS-CoV-2 consists of approximately 1,273 amino acids and contains several domains. Wu et al., Wrapp et al., and Xia, S., Zhu, Y., Liu, M., Lan, Q., Xu, W., Wu, Y., Ying, T., Liu, S., Shi, Z., Jiang, S. & Lu, L., Fusion mechanism of 2019-nCoV and fusion inhibitors targeting HR1 domain in spike protein. Cell Mol Immunol (2020). https: / / doi.org / 10.1038 / s41423-020-0374-2. The spike protein contains the following: signal sequence (SS); N-terminal domain (NTD, 14-305aa); receptor-binding domain (RBD, 319-541aa); S1 / S2 protease cleavage site (S1 / S2, R685 / S686); fusion peptide (FP, 788-806aa from Zhu et al., 816-833aa from Wrapp et al.); heptad repeat 1 (HR1, 912-984aa); central helix (CH, 986-1035aa from Wrapp et al.); connector domain (CD, 1076-1141aa from Wrapp et al.); heptad repeat 2 (HR2, 1163-1213aa); transmembrane domain (TM, 1214-1237aa); and cytoplasmic tail (CT, 1238-1273aa).

[0262] NTD sequence: QCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKS (SEQ ID NO: 2).

[0263] RBD sequence: RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF (SEQ ID NO: 3).

[0264] The FB sequence: IYKTPPIKDFGGFNFSQIL (SEQ ID NO: 4) (Zhu et al.) or SFIEDLLFNKVTLADAGF (SEQ ID NO: 5) (Wrapp et al.).

[0265] HR1 sequence: TQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRL (SEQ ID NO: 6).

[0266] CH sequence: KVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLG (SEQ ID NO: 7).

[0267] CD arrangement: TTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPL(Sequence ID 8).

[0268] HR2 sequence: DVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWP (Sequence ID 9).

[0269] TM sequence: WYIWLGFIAGLIAIVMVTIMLCCM (Sequence ID 10).

[0270] CT array: TSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT (Sequence ID 11).

[0271] In some embodiments, the antigen expression construct includes a nucleic acid sequence encoding a protein having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11. In some embodiments, the antigen expression construct includes a protein having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11, or a nucleic acid sequence encoding an antigen fragment of at least 10, 15, 20, 25, 30, 40, 50, or 100 amino acids in length.

[0272] Phylogenetic and genetic comparative analyses of the S gene and its region showed slight variability among strains. The RBD sequence of WHCV (WH-Human 1 coronavirus: a SARS-CoV-2 strain identified by Wu et al.) was more closely related to that of SARS-CoV (73.8-74.9% amino acid identity) and SARS-like CoVs, including strains Rs4874, Rs7327, and Rs4231 (75.9-76.9% amino acid identity), which can use the human ACE2 receptor for cell entry. Furthermore, the RBD of the spike protein derived from WHCV was one amino acid longer than the RBD of the spike protein derived from SARS-CoV. The previously determined crystal structure of the RBD of the SARS-CoV spike protein complexed with human ACE2 (Protein Data Bank (PDB) 2AJF) revealed that regions 433-437 and 460-472 may directly interact with human ACE2 and therefore be important in determining species specificity. Thus, the S protein is a primary target for the development of an effective vaccine against SARS-CoV-2. In some embodiments, the inventors of this application develop a cell vaccine against SARS-CoV-2 using living cells transfected with constructs containing the SARS-CoV-2 S protein.

[0273] Nucleic acid sequence encoding the SARS-CoV-2 S protein: >NC_045512.2:21563-25384 Severe Acute Respiratory Syndrome Coronavirus 2 Isolation Wuhan-Hu-1, Whole Genome (Genetic ID: 43740568)

[0274] In some cases, the nucleic acid sequences incorporated into the constructs provided herein can be modified. Modifications may include sequence truncation. For example, modifications may include deletion of the cytoplasmic tail, deletion of the transmembrane domain, deletion of a furin cleavage site, and any combination thereof. Modifications may also include sequence additions. Additions may include trimerization tags, transgene sequences, and both. In some cases, modifications may also include mutations, such as proline mutations. Any number of modifications can be introduced, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to about 10 mutations.

[0275] Other pathogens In some embodiments, the antigen expression construct comprises nucleic acids encoding microbial proteins (or antigenic fragments thereof) of rabies virus, Ebola virus, HIV, influenza virus, avian influenza virus, SARS coronavirus, herpesvirus, calicivirus, hepatitis virus, Zika virus, West Nile virus, Lacrosse encephalitis, California encephalitis, Venezuelan horse encephalitis, Eastern equine encephalitis, Western equine encephalitis, Japanese encephalitis virus, St. Louis encephalitis virus, yellow fever virus, chikungunya virus, or norovirus.

[0276] In some cases, influenza antigen peptides may be used. The influenza virus antigen may be human or non-human. In some cases, the influenza virus antigen used is derived from types A, B, C, and / or D. In some cases, the influenza virus antigen is A(H1N1), A(H3N2), B(Victoria), or B(Yamagata). In some cases, the influenza virus antigen may be derived from non-human species such as pigs, birds, bats, cattle, dogs, horses, poultry, and cats.

[0277] In some embodiments, the antigen expression construct comprises nucleic acids encoding a microbial protein (or its antigenic fragment) derived from a virus from any of the following viridae: Arenaviridae, Arteriviridae, Astroviridae, Baculoviridae, Badnavirus, Varnaviridae, Birnaviridae, Bromoviridae, Bunyaviridae, Caliciviridae, Capylovirus, Carlavirus, Kaurimovirus, Circoviridae, Crosterovirus, Comoviridae, Coronavirusidae (e.g., coronaviruses such as Severe Acute Respiratory Syndrome (SARS) virus), Corticoviridae, Cystoviridae, Deltavirus, Diantvirus, Enamovirus, Filoviridae (e.g., Marburg virus and Ebola virus (EBOV) (e.g., Zaire, Reston, Côte d'Ivoire, or Sudan strains)), Flaviviridae (e.g., Hepatitis C virus, Dengue virus 1, Dengue virus 2, Dengue virus 3, and Dengue virus 4), Hepadnaviridae, He Rupesviridae (e.g., human herpesviruses 1, 3, 4, 5, and 6, and cytomegalovirus (CMV)), chikungunya virus, hantavirus, hypoviridae, iridoviridae, leviviridae, lipotrixviridae, microviridae, orthomyxoviridae (e.g., influenza virus A (e.g., H1N1), B, and C), papovaviridae, paramyxoviridae (e.g., measles, mumps, and human respiratory syncytial virus), parvoviridae, py The following are examples of viral antigens: Cornaviridae (e.g., poliovirus, rhinovirus, hepatovirus, and aftovirus), Poxviridae (e.g., vaccinia and smallpox viruses), Reoviridae (e.g., rotavirus), Retroviridae (e.g., lentiviruses such as human immunodeficiency virus (HIV) 1 and HIV 2), Rhabdoviridae (e.g., rabies virus, measles virus, respiratory syncytial virus, etc.), Togaviridae (e.g., rubella virus, dengue virus, etc.), and Totiviridae. Suitable viral antigens also include all or part of dengue protein M, dengue protein E, dengue D1NS1, dengue D1NS2, and dengue D1NS3.Viral antigens may originate from specific strains of papillomavirus, herpesviruses, i.e., herpes simplex virus types 1 and 2; hepatitis viruses, such as hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus (HDV), hepatitis E virus (HEV), and hepatitis G virus (HGV); tick-borne encephalitis viruses; parainfluenza, varicella-zoster virus, JC virus, West Nile virus, cytomegalovirus, Epstein-Barr virus, rotavirus, rhinovirus, adenovirus, coxsackievirus, equine encephalitis, BK virus, malaria, MuLV, VSV, HTLV, Japanese encephalitis, yellow fever, Rift Valley fever, and lymphocytic choroidal meningitis.

[0278] In some cases, the antigen is derived from a coronavirus, specifically SARS-CoV-2, SARS-CoV, and / or MERS-CoV. In some cases, the viral peptide may be derived from a variant of SARS-CoV-2. In some cases, the SARS-CoV-2 variants may include B.1.1.7 (or UK variant), B.1.1.207, Cluster5, B.1.351 (or RSA variant), P.1 (or Brazil variant), B.1.617 (or India variant), B.1525, NS3, WIV04 / 2019, or CAL.20C. In some embodiments, the B.1.617 variant includes a mutation in the spike protein containing at least one of E154K, E484Q, L452R, P681R, Q1071H, or any combination thereof. In some embodiments, the variants of SARS-CoV-2 include the lineages A.1, A.2, A.3, A.4, A.5, A.6, B.1, B.2, B.3, B.4, B.5, B.6, B.7, B.8, B.9, B.10, B.11, B.12, B.13, B.14, B.15, or B.16.

[0279] In some embodiments, the antigen expression construct comprises a nucleic acid encoding a microbial protein (or an antigenic fragment thereof) derived from one or more of influenza virus A, influenza virus B, influenza virus C, isavirus, togovirus, and quarangavirus. Exemplary influenza A virus subtypes include H1N1, H1N2, H3N2, H3N1, H5N1, H2N2, and H7N7. Exemplary influenza virus antigens include one or more proteins or glycoproteins such as hemagglutinin (such as HA1 and HA2 subunits), neuraminidase, viral RNA polymerase (such as one or more of PB1, PB2, PA, and PB1-F2), reverse transcriptase, capsid protein, nonstructural proteins (such as NS1 and NEP), nucleoprotein, matrix proteins (such as M1 and M2), and pore protein. In some embodiments, the influenza A virus antigen comprises hemagglutinin (HA) or neuraminidase (NA) glycoprotein, or one or more fragments of HA or NA (including the antigenic site of hemagglutinin HAl glycoprotein). In an exemplary embodiment, MDNP comprises RNA encoding influenza A / WSN / 33 HA protein.

[0280] In some embodiments, the antigen expression construct comprises a nucleic acid encoding a microbial protein (or an antigenic fragment thereof) derived from one or more Ebola viruses, such as Zaire Ebola virus (EBOV), Sudan Ebola virus (SUDV), Taï Forest Ebola virus (TAFV), Reston Ebola virus (RESTV), and Bundibugyo Ebola virus (BDBV). In an exemplary embodiment, MDNP comprises RNA, such as repRNA, encoding Zaire Ebola virus glycoprotein (GP), or one or more fragments of Zaire Ebola virus glycoprotein (GP).

[0281] In some embodiments, the antigen expression construct comprises nucleic acids encoding microbial proteins (or antigenic fragments thereof) derived from one or more viruses of the genus Flavivirus, such as Zika virus (ZIKV).

[0282] In some cases, multiple nucleic acids are expressed in cell vaccines. For example, a cell vaccine may contain at least two, at least three, or at least four nucleic acids. In some cases, at least two are expressed by the cell vaccine, and these are derived from SARS-CoV-2 and influenza (H1N1).

[0283] In some embodiments, the antigen expression constructs include Bacillus anthracis, Clostridium botulinum, Yersinia pestis, Variola major, Francisella tularensis, Poxviridae, Burkholderia pseudomallei, and Coxiella. Contains nucleic acids encoding microbial proteins (or antigenic fragments thereof) of burnetiid, Brucella species, Burkholderia mallei, Chlamydia psittaci, Staphylococcus enterotoxin B, diarrheagenic E. coli, pathogenic Vibrio, Sigella species, Salmonella, Listeria monocytogenes, Campylobacter jejuni, Yersinia enterocolitica, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacteriaceae, Enterococcus faecium, Staphylococcus aureus, Helicobacter pylori, Campylobacter species, Salmonella, Neisseria gonorrhoeae, Streptococcus pneumoniae, Haemophilus influenzae, or Sigella species.

[0284] In some embodiments, the antigen expression construct comprises nucleic acids encoding microbial proteins (or antigenic fragments thereof) of Cryptosporidium parvum, Cyclospora cayatanensis, Giardia lamblia, Entamoeba histolytica, Toxoplasma gondii, Naegleria fowleri, or Balamuthia mandrillaris.

[0285] In some embodiments, peptides or fragments derived from another pathogen used in the vaccine may have approximately 50%, 60%, 70%, 75%, 80%, 85%, 88%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, or 100% identity with any sequence from Table 4. [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4] [Table 4-5] [Table 4-6] [Table 4-7] [Table 4-8]

[0286] Cancer peptides In some embodiments, the antigen expression construct comprises a nucleic acid encoding a peptide (or fragment thereof) associated with cancer or tumor. In some embodiments, the nucleic acid encodes a full-length protein or a fragment or derivative thereof. Exemplary peptides may be nascent antigens or oncoproteins. In some embodiments, the peptide or fragment thereof may be 707-AP, biotinylated molecule, α-actinin-4, abl-bcr alb-b3(b2a2), abl-bcr alb-b4(b3a2), adipophilin, AFP, AIM-2, annexin II, ART-4, BAGE, β-catenin, bcr-abl, bcr-abl p190(e1a2), bcr-abl p210(b2a2), bcr-abl p210(b3a2), BING-4, CAG-3, CAIX, CAMEL, CISH, caspase-8, CD171, CD19, CD20, CD22, CD23, CD24, CD30, CD33, C D38, CD44v7 / 8, CDC27, CDK-4, CEA, CLCA2, Cyp-B, DAM-10, DAM-6, DEK-CAN, EGFRvIII, EGP-2, EGP-40, ELF2, Ep -CAM, EphA2, EphA3, erb-B2, erb-B3, erb-B4, ES-ESO-1a, ETV6 / AML, FBP, Fetal Acetylcholine Receptor, FGF-5, FN, G250, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7B, GAGE-8, GD2, GD3, GnT-V, Gp100, gp75, Her-2, HLA-A *0201-R170I, HMW-MAA, HSP70-2M, HST-2(FGF6), HST-2 / neu, hTERT, iCE, IL-11Rα, IL-13Rα2, KDR , KIAA0205, K-RAS, L1 cell adhesion molecule, LAGE-1, LDLR / FUT, Lewis Y, MAGE-1, MAGE-10, MAGE-12, MAGE-2, MAGE- 3, MAGE-4, MAGE-6, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A6, MAGE-B1, MAGE-B2, Malic acid enzyme, Mammaglobin-A, MART-1 / Melan-A, MART-2, MC1R, M-CSF, Mesoserine, MUC1, MUC16, MUC2, MUM-1, MUM-2, MUM-3, Myosin, NA8 The compound comprises at least one of the following: 8-A, Neo-PAP, NKG2D, NPM / ALK, N-RAS, NY-ESO-1, OA1, OGT, carcinoembryonic antigen (h5T4), OS-9, P polypeptide, P15, P53, PRAME, PSA, PSCA, PSMA, PTPRK, RAGE, ROR1, RU1, RU2, SART-1, SART-2, SART-3, SOX10, SSX-2, Survivin, Survivin-2B, SYT / SSX, TAG-72, TEL / AML1, TGFaRII, TGFbRII, TP1, TRAG-3, TRG, TRP-1, TRP-2, TRP-2 / INT2, TRP-2-6b, tyrosinase, VEGF-R2, WT1, α-folate receptor, κ-light chain, or any combination thereof.

[0287] In some embodiments, the peptide includes a neoantigen peptide. For example, the neoantigen may be a peptide resulting from a polypeptide generated from a genomic sequence containing the E805G mutation in ERBB2IP. Neoantigens and neoepitopes can be identified by whole exome sequencing. In some cases, genes that may contain mutations that give rise to neoantigens or neoepitope peptides include ABL1 and ACO1. 1997, ACVR2A, AFP, AKT1, ALK, ALPPL2, ANAPC1, APC, ARID1A, AR, AR-v7, ASCL2, β2Μ, BRAF, BTK, C15ORF40, CDH1, CLDN6, CNOT1, CT45A5, CTAG1B, DCT, DKK4, EEF1B2, EEF 1DP3, EGFR, EIF2B3, env, EPHB2, ERBB3, ESR1, ESRP1, FAM11IB, FGFR3, FRG1B, GAGE1, GAGE10, GATA3, GBP3, HER2, IDH1, JAK1, KIT, KRAS, LMAN1, MABEB16, MAGEA1, MAGE A10, MAGEA4, MAGEA8, MAGEB17, MAGEB4, MAGEC1, MEK, MLANA, MLL2, MMP13, MSH3, MSH6, MYC, NDUFC2, NRAS, NY-ESO, PAGE2, PAGE5, PDGFRa, PIK3CA, PMEL, pol protein, POL E, PTEN, RAC1, RBM27, RNF43, RPL22, RUNX1, SEC31A, SEC63, SF3B1, SLC35F5, SLC45A2, SMAP1, SMAP1, SPOP, TFAM, TGFBR2, THAP5, TP53, TTK, TYR, UBR5, VHL, XPOT.

[0288] In some embodiments, peptides(s) or fragments(s) thereof may be polypeptides, polypeptides produced from nucleic acid sequences, or A1CF, ABI1, ABL1, ABL2, ACKR3, ACSL3, ACSL6, ACVR1, ACVR1B, ACVR2A, AFDN, AFF1, AFF3, AFF4, AKAP9, AKT1, AKT2, AKT3, ALDH2, ALK, AMER1, ANK1, APC, APOBEC3B, AR, ARAF, ARHGAP26, ARHGAP5, ARHGEF10, ARHGEF10L, ARHGEF12, A RID1A, ARID1B, ARID2, ARNT, ASPSCR1, ASXL1, ASXL2, ATF1, ATIC, ATM, ATP1A1, ATP2B3, ATR, ATRX, AXIN1, AXIN2, B2M, BAP1, BARD1, BAX, BAZ1A, BCL10, B CL11A, BCL11B, BCL2, BCL2L12, BCL3, BCL6, BCL7A, BCL9, BCL9L, BCLAF1, BCOR, BCORL1, BCR, BIRC3, BIRC6, BLM, BMP5, BMPR1A, BRAF, BRCA1, BRCA2, BRD3 , BRD4, BRIP1, BTG1, BTK, BUB1B, C15orf65, CACNA1D, CALR, CAMTA1, CANT1, CARD11, CARS, CASP3, CASP8, CASP9, CBFA2T3, CBFB, CBL, CBLB, CBLC, CCDC6, CCNB1IP1, CCNC, CCND1, CCND2, CCND3, CCNE1, CCR4, CCR7, CD209, CD274, CD28, CD74, CD79A, CD79B, CDC73, CDH1, CDH10, CDH11, CDH17, CDK12, CDK4, CDK 6, CDKN1A, CDKN1B, CDKN2A, CDKN2C, CDX2, CEBPA, CEP89, HCHD7, CHD2, CHD4, CHEK2, CHIC2, CHST11, CIC, CIITA, CLIP1, CLP1, CLTC, CLTCL1, CNBD1, CNBP , CNOT3, CNTNAP2, CNTRL, COL1A1, COL2A1, COL3A1, COX6C, CPEB3, CREB1, CREB3L1, CREB3L2, CREBBP, CRLF2, CRNKL1, CRTC1, CRTC3, CSF1R, CSF3R, CSMD3,CTCF, CTNNA2, CTNNB1, CTNND1, CTNND2, CUL3, CUX1, CXCR4, CYLD, CYP2C8,C YSLTR2, DAXX, DCAF12L2, DCC, DCTN1, DDB2, DDIT3, DDR2, DDX10, DDX3X, DDX 5, DDX6, DEK, DGCR8, DICER1, DNAJB1, DNM2, DNMT1, DNMT3A, DROSHA, EBF1, E CT2L, EED, EGFR, EIF1AX, EIF3E, EIF4A2, ELF3, ELF4, ELK4, ELL, ELN, EML4, E P300, EPAS1, EPHA3, EPHA7, EPS15, ERBB2, ERBB3, ERBB4, ERC1, ERCC2, ERCC 3. ERCC4, ERG, ESR1, ETNK1, ETV1, ETV4, ETV5, ETV6, EWSR1, EXT1, EXT2, EZH2 EZR, FAM131B, FAM135B, FAM46C, FAM47C, FANCA, FANCC, FANCD2, FANCE, FA NCF, FANCG, FAS, FAT1, FAT3, FAT4, FBLN2, FBXO11, FBXW7, FCGR2B, FCRL4, FE N1, FES, FEV, FGFR1, FGFR1OP, FGFR2, FGFR3, FGFR4, FH, FHIT, FIP1L1, FKBP 9. FLCN, FLI1, FLNA, FLT3, FLT4, FNBP1, FOXA1, FOXL2, FOXO1, FOXO3, FOXO4 FOXP1、FOXR1、FSTL3、FUBP1、FUS、GAS7、GATA1、GATA2、GATA3、GLI1、GMPS、G NA11, GNAQ, GNAS, GOLGA5, GOPC, GPC3, GPC5, GPHN, GRIN2A, GRM3, H3F3A, H3 F3B, HERPUD1, HEY1, HIF1A, HIP1, HIST1H3B, HIST1H4I, HLA-A, HLF, HMGA1 HMGA2、HNF1A、HNRNPA2B1、HOOK3、HOXA11、HOXA13、HOXA9、HOXC11、HOXC13、H OXD11, HOXD13, HRAS, HSP90AA1, HSP90AB1, ID3, IDH1, IDH2, IGF2BP2, IKBK B, IKZF1, IL2, IL21R, IL6ST, IL7R, IRF4, IRS4, ISX, ITGAV, ITK, JAK1, JAK2JAK3, JAZF1, JUN, KAT6A, KAT6B, KAT7, KCNJ5, KDM5A, KDM5C, KDM6A, KDR, KD SR, KEAP1, KIAA1549, KIF5B, KIT, KLF4, KLF6, KLK2, KMT2A, KMT2C, KMT2D, K NL1, KNSTRN, KRAS, KTN1, LARP4B, LASP1, LCK, LCP1, LEF1, LEPROTL1, LHFPL 6. LIFR, LMNA, LMO1, LMO2, LPP, LRIG3, LRP1B, LSM14A, LYL1, LZTR1, MAF, MAF B、MALT1、MAML2、MAP2K1、MAP2K2、MAP2K4、MAP3K1、MAP3K13、MAPK1、MAX、MB 21D2, MDM2, MDM4, MDS2, MECOM, MED12, MEN1, MET, MGMT, MITF, MKL1, MLF1 LH1, MLLT1, MLLT10, MLLT11, MLLT3, MLLT6, MN1, MNX1, MPL, MSH2, MSH6, MSI 2. MSN, MTCP1, MTOR, MUC1, MUC16, MUC4, MUTYH, MYB, MYC, MYCL, MYCN, MYD88. MYH11, MYH9, MYO5A, MYOD1, N4BP2, NAB2, NACA, NBEA, NBN, NCKIPSD, NCOA1 NCOA2, NCOA4, NCOR1, NCOR2, NDRG1, NF1, NF2, NFATC2, NFE2L2, NFIB, NFKB2 NFKBIE, NIN, NKX2-1, NONO, NOTCH1, NOTCH2, NPM1, NR4A3, NRAS, NRG1, NSD 1, NSD2, NSD3, NT5C2, NTHL1, NTRK1, NTRK3, NUMA1, NUP214, NUP98, NUTM1, NU TM2A, NUTM2B, OLIG2, OMD, P2RY8, PABPC1, PAFAH1B2, PALB2, PATZ1, PAX3, P AX5, PAX7, PAX8, PBRM1, PBX1, PCBP1, PCM1, PD-1, PDCD1LG2, PDGFB, PDGFRA PDGFRB, PDL1, PER1, PHF6, PHOX2B, PICALM, PIK3CA, PIK3CB, PIK3R1, PIM1 PLAG1, PLCG1, PML, PMS1, PMS2, POLD1, POLE, POLG, POT1, POU2AF1, POU5F1PPARG、PPFIBP1、PPM1D、PPP2R1A、PPP6C、PRCC、PRDM1、PRDM16、PRDM2、PREX2、PRF1、PRKACA、PRKAR1A、PRKCB、PRPF40 B、PRRX1、PSIP1、PTCH1、PTEN、PTK6、PTPN11、PTPN13、PTPN6、PTPRB、PTPRC、PTPRD、PTPRK、PTPRT、PWWP2A、QKI、RABEP1 、RAC1、RAD17、RAD21、RAD51B、RAF1、RALGDS、RANBP2、RAP1GDS1、RARA、RB1、RBM10、RBM15、RECQL4、REL、RET、RFWD3、R GPD3、RGS7、RHOA、RHOH、RMI2、RNF213、RNF43、ROBO2、ROS1、RPL10、RPL22、RPL5、RPN1、RSPO2、RSPO3、RUNX1、RUNX1T1、 S100A7, SALL4, SBDS, SDC4, SDHA, SDHAF2, SDHB, SDHC, SDHD, SEPT5, SEPT6, SEPT9, SET, SETBP1, SETD1B, SETD2, SF3B1, SFPQ, SFRP4, SGK1, SH2B3, SH3GL1, SHTN1, SIRPA, SIX1, SIX2, SKI, SLC34A2, SLC45A3, SMAD2, SMAD3, SMAD4, SMARCA 4、SMARCB1、SMARCD1、SMARCE1、SMC1A、SMO、SND1、SNX29、SOCS1、SOX2、SOX21、SOX9、SPECC1、SPEN、SPOP、SRC、SRGAP3 、SRSF2、SRSF3、SS18、SS18L1、SSX1、SSX2、SSX4、STAG1、STAG2、STAT3、STAT5B、STAT6、STIL、STK11、STRN、SUFU、SUZ12 SYK、TAF15、TAL1、TAL2、TBL1XR1、TBX3、TCEA1、TCF12、TCF3、TCF7L2 、TCL1A、TEC、TERT、TET1、TET2、TFE3、TFEB、TFG、TFPT、TFRC、TGFBR2 、THRAP3、TLX1、TLX3、TMEM127、TMPRSS2、TNC、TNFAIP3、TNFRSF14、T NFRSF17、TOP1、TP53、TP63、TPM3、TPM4、TPR、TRAF7、TRIM24、TRIM27、Derived from neoantibodies derived from at least one of the following: TRIM33, TRIP11, TRRAP, TSC1, TSC2, TSHR, U2AF1, UBR5, USP44, USP6, USP8, VAV1, VHL, VTI1A, WAS, WDCP, WIF1, WNK2, WRN, WT1, WWTR1, XPA, XPC, XPO1, YWHAE, ZBTB16, ZCCHC8, ZEB1, ZFHX3, ZMYM2, ZMYM3, ZNF331, ZNF384, ZNF429, ZNF479, ZNF521, ZNRF3, ZRSR2, or any combination thereof.

[0289] Delivery method The antigen expression constructs described herein can be delivered to target cells by any suitable means, such as potentially stable integration into the cell genome at a site designated via genomic engineering, including safe harbor sites, for constitutive and predictable expression. The antigen expression constructs can be targeted to preferred genomic locations. In some cases, the antigen expression constructs can be stably integrated into the cell genome. In some cases, the antigen expression constructs are integrated into safe harbor sites, MHC loci, TCR loci, HLA loci, inhibitory receptor loci, and any combination thereof. Non-limiting examples of safe harbors include HPRT, AAVS SITE (e.g., AAVS1, AAVS2, ETC.), CCR5, or Rosa26. In some cases, the antigen expression constructs are expressed transiently. Nucleic acids can be introduced into cells using conventional viral and nonviral gene transfer methods. Nonviral methods of nucleic acid delivery include electroporation, lipofection, nucleofection, gold nanoparticle delivery, microinjection, bioristic methods, virosomes, liposomes, immunoliposomes, polycations or lipid-nucleic acid conjugates, naked DNA, mRNA, artificial virions, and drug-enhanced DNA uptake. For example, sonoporation using the Sonitron2000 system (Rich-Mar) can also be used for nucleic acid delivery.

[0290] Nonviral vector delivery systems may include the delivery of DNA plasmids, naked nucleic acids, nucleic acids complexed with delivery vehicles such as liposomes or poloxamers, and mRNA.

[0291] In one embodiment, the antigen expression construct is electroporated into the cell. In some embodiments, the antigen expression construct comprises mRNA, which is electroporated into the cell. Further exemplary nucleic acid delivery systems include those offered by AMAXA Biosystems (Cologne, Germany), Life Technologies (Frederick, Md.), MAXCYTE, Inc. (Rockville, Md.), BTX Molecular Delivery Systems (Holliston, Mass.), and Copernicus Therapeutics Inc. (see, for example, U.S. Patent No. 6,008,336). Lipofection reagents are commercially available (e.g., TRANSFECTAM® and LIPOFECTIN®). Delivery may be to cells (ex vivo administration) or target tissue (in vivo administration).

[0292] Polynucleotides encoding pathogen proteins as described herein and compositions containing such polynucleotides can be delivered using a vector containing a sequence encoding one or more proteins. Any vector system can be used, including but not limited to plasmid vectors, retroviral vectors, lentiviral vectors, adenovirus vectors, poxvirus vectors, herpesvirus vectors, and adeno-associated virus vectors. The viral vector delivery system may contain DNA and RNA viruses having either an episomal genome or an embedded genome after delivery to cells.

[0293] Suicide gene In some cases, the cells provided herein may include the genomic integration of a “kill switch” suicide gene. The suicide gene can be removed by treating the cells containing it with a drug that selectively kills them. Including a suicide gene in the cells can also enhance the safety of using the cells provided herein for therapeutic purposes.

[0294] In some cases, suicide genes can be incorporated into cell products. Suicide genes allow for the elimination of genetically modified cells in cases of adverse events, autoreactivity of injected cells, or eradication of infection. In some embodiments, suicide genes are introduced at random genomic locations or at target loci (e.g., metabolic loci, DNA / RNA replication loci, safe harbors, MHC loci, HLA loci, TCR loci, depletion loci, inhibitory receptor loci (PD-1, CTLA-4, Tim3, CISH, etc.)). Non-limiting examples of safe harbors include HPRT, AAVS SITE (e.g., AAVS1, AAVS2, ETC.), CCR5, or Rosa26. In some cases, suicide genes may be driven by exogenous promoters or utilize endogenous promoters at the integration loci.

[0295] Various suicide genes are known in the art and can be used in the cell compositions provided herein. Exemplary suicide genes may include thymidine kinase / ganciclovir, cytosine deaminase / 5-fluorocytosine, nitroreductase / CB1954, carboxypeptidase G2 / nitrogen mustard, cytochrome P450 / oxazaphosphorine, purine nucleoside phosphorylase / 6-methylpurine deoxyriboside (PNP / MEP), (HRP / IAA), and combinations thereof. In certain embodiments, the suicide gene is the inducible caspase-9 gene (see U.S. pre-grant publication number US2013 / 0071414, which is incorporated herein by reference). Other suicide genes include genes encoding one or more of the following: a structurally intact conjugating epitope for a pharmaceutical-grade anti-EGFR monoclonal antibody, cetuximab (Erbitux), as described herein; EGFRt, a caspase polypeptide (e.g., iCasp9; Straathof et al., Blood 105:4247-4254, 2005; Di Stasi et al.) al., N.Engl.. / .Med.365:1673-1683,2011; Zhou and Brenner, Exp.Hematol.pii:S0301-472X(16)30513-6.doi:10.1016 / j.exphem.2016.07.011), RQR8 (Philip et al., Blood 124:1277-1287,2014), a 10-amino acid tag of the human c-myc protein (Myc) (Kieback et al., Proc.Natl.Acad.Sci.USA 105:623-628,2008), and marker / safety switch polypeptides such as RQR (CD20+CD34; Philip et al.,2014). In some embodiments, the suicide gene is sr39TK, enabling the elimination of cells by introduction of ganciclovir. This gene can also be used to image genetically modified cells using positron emission tomography (POST) on recipient / host-localized cells. The suicide gene can also be a chemically induced caspase, or dimerization induced by a small molecule / chemically induced dimerizer (CID). The suicide gene may also be a selectable surface marker (such as CD19 or CD20 or CD34 or EGFR or LNGFR), which allows for the elimination of cells by introduction of antibodies, such as antibody-dependent cell-mediated cytotoxicity or complement cascades.

[0296] In some cases, the suicide gene can be included within a vector containing the viral antigen peptide provided herein. In other cases, the suicide gene is introduced separately into cells using, for example, a CRISPR system, a viral system, electroporation, transfection, transduction, and any combination thereof. In some cases, the suicide gene is knocked into a target locus.

[0297] Vaccination methods for the target group In one embodiment, the Specified herein provides a method for immunizing a subject to a pathogen by administering a population of vaccine cells described herein that have been modified to induce an adaptive immune response to the pathogen in the subject (for example, the vaccine cells contain a protein or antigen fragment of the pathogen).

[0298] In some embodiments, the pathogen is a virus, a bacterium, or a parasite.

[0299] In some embodiments, the pathogen is a virus. In some embodiments, the virus is rabies virus, Ebola virus, HIV, influenza virus, avian influenza virus, SARS coronavirus, herpesvirus, calicivirus, hepatitis virus, Zika virus, West Nile virus, Lacrosse encephalitis, California encephalitis, Venezuelan horse encephalitis, Eastern horse encephalitis, Western horse encephalitis, Japanese encephalitis virus, St. Louis encephalitis virus, yellow fever virus, chikungunya virus, or norovirus.

[0300] In some embodiments, the virus belongs to the order Nidovirales. In some embodiments, the virus belongs to the family Coronaviridae. In some embodiments, the virus belongs to the genera Alphacoronavirus, Betacoronavirus, Gammacoronavirus, and Deltacoronavirus. In some embodiments, the virus belongs to the genus Betacoronavirus. In some embodiments, the virus belongs to the subgenus Salvecovirus. In some embodiments, the virus is one of two Severe Acute Respiratory Syndrome-associated Coronaviruses. In some embodiments, the virus is one of two Severe Acute Respiratory Syndrome Coronavirus strains. In some embodiments, the virus is Severe Acute Respiratory Syndrome Coronavirus 2.

[0301] In some embodiments, the pathogen is a bacterium. In some embodiments, the bacteria include Bacillus anthracis, Clostridium botulinum, Yersinia pestis, Variola major, Francisella tularensis, Poxviridae, Burkholderia pseudomallei, Coxiella burnetiid, Brucella species, Burkholderia mallei, Chlamydia psittaci, Staphylococcus enterotoxin B, diarrheagenic E. coli, pathogenic Vibrio, Sigella species, Salmonella, Listeria monocytogenes, Campylobacter jejuni, Yersinia enterocolitica, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacteriaceae, Enterococcus faecium, Staphylococcus aureus, Helicobacter pylori, Campylobacter species, Salmonella, Neisseria gonorrhoeae, and Streptococcus. These are pneumoniae, Haemophilus influenzae, or bacteria of the genus Shigera.

[0302] In some embodiments, the pathogen is a parasite. In some embodiments, the parasite is Cryptosporidium parvum, Cyclospora cayatanensis, Giardia lamblia, Entamoeba histolytica, Toxoplasma gondii, Naegleria fowleri, or Balamuthia mandrillaris.

[0303] The cell vaccines described herein may be administered by any suitable delivery route known in the art, but are not limited to intramuscular, intradermal, intravenous, or subcutaneous injection. In some embodiments, the vaccine is administered topically. In some embodiments, the vaccine is administered systemically. In some embodiments, the vaccine may be administered using a pen-type injector device, such as those used for home delivery of epinephrine, to enable self-administration of the vaccine. In some cases, the vaccine is administered by intradermal / SQ injection.

[0304] In some embodiments, the vaccine is administered topically. In some embodiments, the vaccine is administered subcutaneously. In some embodiments, the vaccine is self-administered by the patient.

[0305] In some cases, the vaccine is administered via the pulmonary system. In some cases, the vaccine is inhaled. In some cases, the vaccine is administered by inhalation. In some cases, the vaccine can be inhaled and reach the lungs. In some cases, the vaccine can be inhaled and reach the airways. In some cases, the vaccine is administered orally. In some cases, the vaccine can be administered orally and reach the gastrointestinal tract. In some cases, the vaccine can be taken orally via the gastrointestinal system. In some cases, the vaccine is applied to the skin. In some cases, the vaccine is administered through the skin. In some embodiments, the vaccine is administered by subcutaneous injection. In some embodiments, the vaccine is administered by cutaneous injection. In some embodiments, the vaccine is administered by intradermal injection. The use of such delivery devices may be particularly suitable for large-scale immunization campaigns, such as those required during pandemics.

[0306] kit Any of the compositions described herein may be included in the kit. In non-limiting examples, a vaccine may be included in the kit, any type of cells may be included in the kit, and / or reagents for manipulating the vaccine and / or cells may be provided in the kit. The components are provided in appropriate container means.

[0307] The kit may contain appropriately divided compositions. The components of the kit may be packaged in either an aqueous medium or a lyophilized form. The kit's container means generally include at least one vial, test tube, flask, bottle, syringe or other container means in which the components can be placed, preferably appropriately divided. If the kit has multiple components, the kit generally also includes a second, third, or other additional container in which additional components can be placed separately. However, various combinations of components may be contained in the vial. The kit will also typically include means for tightly sealing the components for commercial sale. Such containers may include injection-molded or blow-molded plastic containers in which the desired vials are held.

[0308] However, the components of the kit may be provided as a dry powder(s). If the reagents and / or components are provided as a dry powder, the powder can be reconstituted by the addition of a suitable solvent. The solvent may also be provided in a separate container.

[0309] Preferred embodiments of the Disclosure are shown and described herein, but it will be apparent to those skilled in the art that such embodiments are provided only as examples. Furthermore, those skilled in the art will be able to conceive of numerous variations, modifications, and substitutions without departing from the Disclosure. When implementing the Disclosure, it should be understood that various substitutes for the embodiments of the Disclosure described herein may be used. The following claims define the scope of the Disclosure, and the methods and structures of these claims, as well as their equivalents, are intended to be encompassed thereby. [Examples]

[0310] Example 1. CRISPR gene manipulation of iPSCs to knock out both MHC class I and II genes. Parental iPS cell lines were transfected using the Lonza nucleofection system with Cas9 protein pre-conjugated with gRNAs targeting genes essential for the expression of MHC class I and II (such as B2M and CIITA). After harvesting the cells from transfection and growing them in complete growth medium for 72 hours, target gene loci were analyzed by PCR and sequencing across the entire modified region. Loss of MHC I and MHC II was confirmed by surface expression using flow cytometry in IFNg-stimulated iPSC cells (and differentiated cells generated from these iPS cells). Figure 7.

[0311] Further experiments involved culturing control cells or B2M knockout platform cells from two different donors with T cells. MHCI-deficient iPSC cells were unable to activate MHC mismatch T cell proliferation compared to control iPS cells (Figure 8A). Further experiments measured NK cell death in modified cells to demonstrate increased cell lysis in the absence of MHCI. Example 2. CRISPR gene manipulation of MHC null iPSCs from Example 1, incorporating important NK cell activation ligand genes or lysis-related genes into the genome.

[0312] Activation ligand gene MHC null iPS cells are transfected with a Cas9 and gRNA complex targeting a genomic region to target and incorporate either a plasmid-based DNA donor of an rAAV template carrying an transgene for stimulating, activating, or recruiting innate immune cells. The target site contains either a genome-safe harbor region or a gene that suppresses or inhibits the stimulation, activation, or recruitment of innate immune cells, which is inactivated via genomic cleavage and insertion of the donor template. The donor template is designed to express the cDNA of a ligand having a constitutive promoter and terminator sequence.

[0313] Lysolytic genes MHC-null iPS platform cells are transfected with Cas9 and gRNA complexes targeting genomic regions to target and incorporate one of the plasmid-based DNA donors of the rAAV template, which carries an transgene for a lysis signal recognized by innate immune cells (exemplary signals are in Table 5). In addition to the absence of MHC-I, innate immune cells such as NK cells can be effectively activated by secondary activation signals (such as those in Table 5) in the form of cell surface ligands that interact with the NKG2D receptor on the surface of NK cells, thereby killing the target iPS cells. Target sites include genome-safe harbor sites or genes that suppress or inhibit the stimulation, activation, or recruitment of innate immune cells, which are inactivated via genomic cleavage and insertion of the donor template. The donor templates are designed to express the cDNA of ligands having constitutive promoter and terminator sequences.

[0314] Generation of endothelial cells from platform cells To induce differentiation towards the endothelial lineage, iPS cells were grown on a vitronectin-coated plate and supplied with RPMI base medium containing B27(-insulin), glutamax, and penecillin / streptomycin. On days 0-2, 6 μM CHIR99021, 10 ng / ml BMP4, and 100 μg / ml AA2P (stage 1) were supplied, followed by 50 ng / mL VEGF165 + 20 ng / mL FGF + 10 μM SB431542 (stage 2) on days 2-7. The flow cytometry results for CD31+CD144+ endothelial cells on day 7 are shown in Figure 8B.

[0315] Flow cytometry 48 hours after transfection was performed on iPSC-derived endothelial cells overexpressing NK-activating ligands, and the data are shown in Figure 9. [Table 5] Example 3. Transfection of engineered cell vaccine cells, such as platform cells, with SARS-CoV-2 spike protein or an S1 subunit expression construct having a desired modification.

[0316] Cell-vaccinated cells (iPSCs or differentiated cells) were transfected with DNA donors (plasmids, linear DNA, or rAAV donors) expressing the spike protein or S1 subunit cDNA using Lonza nucleofector, Thermo Neon, or other lipid-based transfection methods. Endothelial cells expressing SARS-CoV-2 spike protein variants were lysed, and the lysates were analyzed for spike protein antigens by ELISA. Both protein antigen variants were abundantly detectable and showed a dose-dependent increase with the number of vaccinated cells (Figure 10).

[0317] In the exemplary strategy, a DNA sequence encoding a spike antigen variant expression construct is inserted into the AAVS1 safe harbor site using CRISPR genetic engineering. Example 4. Co-culture of transfected cells and donor-derived NK cells from Example 3, and analysis of NK-mediated cytolysis of antigen-loaded vaccine cells by a standard ex vivo NK killing assay and / or analysis of killing by confocal imaging.

[0318] Cell vaccine cells are co-cultured with PBMC-derived NK cells for several days at various effector:target (E:T) ratios. NK-mediated cell lysis is measured using the CyQUANT LDH Cytotoxicity Assay, and live and dead cells are identified using a plate reader. NK cell degranulation is also measured by flow cytometry analysis of CD107a expression in NK cells.

[0319] In another assay, iPS-derived endothelial cells (differentiated from platform cells) were harvested using TRYPLE 24 hours before performing the NK cell killing assay and seeded into wells of a geltrex-coated 96-well plate (2x10).4 The cells were incubated overnight in Stage 2 endothelial differentiation medium (2x10 / well). On the day of the assay, K562 cells were seeded in 96-well plates (2x10 / well). 4 Both endothelial cells and K562 cells were stained with Cell Tracker Blue dye (in a well). Primary NK cells were added to wells containing RPMI in 10% FCS with 200 IU / ml IL2 and 10 ng / ml IL15 at 0, 0.25:1, 1.25:1, 2.5:1, or 5:1 ratios and incubated for 4 hours. The samples were then flow-cytometer-treated using 7AAD staining to identify dead cells within the Cell Tracker Blue target population (Figure 12).

[0320] This data demonstrates effective NK cell killing in the absence of MHC-I. An NK cell assay measuring cytolytic killing of MHC-I-deficient, iPSC-derived endothelial cells (differentiated from platform cells) shows robust, dose-dependent lysis comparable to the gold standard K562 cell line for NK killing. Platform cells or cells differentiated or induced therefrom can be engineered to express NK-activating ligands to further enhance this targeted cell lysis and ensure robust and rapid cell lysis when administered in vivo. Example 5. Comparison of manipulated vaccine cells and unmanipulated cells

[0321] Perform NK-mediated killing and degranulation assays as described above, and compare the cell-vaccinated cells (iPSCs and differentiated cells) with the same cell types without manipulation of ligands that activate either the innate immune system or MHC I & II knockout. Example 6. Detection of SARS-CoV-2 spike protein in culture medium during NK cell-mediated lysis.

[0322] The release of the SARS-CoV-2 spike protein expressed by cell-vaccinated cells (exemplary schematic diagram in Figure 11) is detected in the supernatant using a spike protein-specific ELISA kit (such as the one provided at sinobiological.com / elisa-kits / cov-spike-kit40591).

[0323] Non-human primate research Ten adult rhesus monkeys (6-12 years old) received a total of 1.1 × 10 6 PFU (Group 1; N=3), 1.1 × 10 5 PFU (Group 2; N=3), or 1.1 × 10 4 SARS-CoV-2 (PFU, Group 3; N=3) was inoculated at a dose of 1 ml via the intranasal (IN) route and 1 ml via the intratracheal (IT) route. Ten equivalent rhesus monkeys received control inoculation. Following viral challenge, viral RNA levels were evaluated by RT-PCR in multiple anatomical compartments, such as bronchoalveolar lavage fluid and nasal swabs.

[0324] SARS-CoV-2-specific humoral and cellular immune responses are detected in animals by evaluating the binding antibody response to the SARS-CoV-2 spike (S) protein using ELISA, and the neutralizing antibody (NAb) response using both pseudoviral neutralization assays and live virus neutralization assays. Antibody responses are evaluated against the receptor-binding domain (RBD), the pre-fusion S-external domain (S), and the nucleocapsid (N). Furthermore, the presence of various immune responses such as antibody-dependent complement deposition (ADCD), antibody-dependent cell phagocytosis (ADCP), antibody-dependent neutrophil phagocytosis (ADNP), antibody-dependent NK cell degranulation (NK CD107a), and cytokine secretion (NK MIP1β, NK IFNγ) is assessed. Example 7. Universal vaccine cells (UVC) against SAR-CoV-2

[0325] As shown in Figure 13, UVC is MHC-I deficient (B2M KO) and does not express MHC-II. The lack of MHC-I expression enhances UVC lysis by NK cells. Expression of the NK ligand MICA also further enhances NK cell binding and UVC cytolysis. UVC expresses high levels of intracellular SARS-CoV-2 spike protein and nucleocapsid protein. These proteins are released into the immune microenvironment during NK cell lysis.

[0326] UVC does not express MHC-II, preventing the presentation of arbitrary peptides (e.g., SARS-CoV-2 spike protein peptide) to arbitrary recipient immune cells, and thus preventing stimulation by IFNγ.

[0327] At the vaccination site, UVC activates innate immune cells (e.g., NK cells) to induce its own lysis. The spike and nucleocapsid proteins are released following UVC apoptosis. The phagocytic and phagocytic activity of the apoptotic UVC allows APC to present the spike protein and nucleocapsid peptide to the adaptive immune system via MHC presentation. UVC expresses both the full-length SARS-CoV-2 spike protein and the full-length nucleoplasmid protein. To ensure a robust response by the adaptive immune system, UVC is engineered to express the full-length SARS-CoV-2 spike protein with a disrupted furin cleavage site and two proline residue substitutions. The sequence encoding this spike protein is shown in SEQ ID NO: 53.

[0328] cDNA of RSA spike protein without a furin cleavage site - SEQ ID NO: 53

[0329] These modifications allow the spike protein to maintain an intact and natural cell surface active structure because its multiple subunits dissociate within the host. The amino acid sequence of the nucleocapsid protein is shown in Figure 14A or SEQ ID NO: 54.

[0330] GenBank:QHD43423.2 Sequence ID:54 MSDNGPQNQRNAPRITFGGPSDSTGSNQNGERSGARSKQRRPQGLPNNTASWFTALTQHGKEDLKFPRGQGVPINTNSSPDDQIGYYRRATRRIRGGDGKMKDLSPRWYFYYLGTGPEAGLPYGANKDGIIWVATEGALNTPKDHIGTRNPANNAAIVLQLPQGTTLPKGFYAEGSRGGSQASSRSSSRSRNSSRNSTPGSSRGTSPAR MAGNGGDAALALLLLDRLNQLESKMSGKGQQQQGQTVTKKSAAEASKKPRQKRTATKAYNVTQAFGRRGPEQTQGNFGDQELIRQGTDYKHWPQIAQFAPSASAF FGMSRIGMEVTPSGTWLTYTGAIKLDDKDPNFKDQVILLNKHIDAYKTFPPTEPKKDKKKKADETQALPQRQKKQQTVTLLPAADLDDFSKQLQQSMSSADSTQA

[0331] As shown in Figure 14B, the EF1a promoter is used to drive and ensure the maximum expression of the SARS-CoV-2 spike protein and nucleocapsid protein. To maintain a 1:1 expression ratio, the two proteins are expressed from the same transcript using a T2A peptide cleavage sequence to link them. Example 8. Design of a multivalent SARS-CoV-2 UVC system.

[0332] A whole-genome screening technique called T-Scan, described in Kula et al., “T-Scan: A Genome-wide Method for the Systematic Discovery of T Cell Epitopes.” 2019, Cell, 178:1016-1028.e3 (the entire technique is incorporated herein by reference for all purposes), was used to determine the overall landscape of SARS-CoV-2 CD8+ T cell recognition in an unbiased manner. CD8+ T cells were co-cultured with a whole-genome library of target cells (modified HEK293 cells) engineered to express a single HLA allele. Each target cell in the library also expressed a unique coronavirus-derived 61-amino acid (aa) protein fragment. These fragments were spontaneously processed by target cells to display appropriate peptide epitopes on major histocompatibility complex (MHC) class I molecules on the cell surface. Upon encountering the target in co-culture, CD8+ T cells secreted cytotoxic granules into the target cells, inducing apoptosis of the target. Subsequently, early apoptotic cells were isolated from the co-culture, expression cassettes were sequenced, and the identity of the protein fragments was determined. To optimize the sorting and isolation of rarely recognized target cells, the target cells were engineered to express the granzyme B (GzB) activated fluorescent reporter, as described above, as well as a GzB-activated version of the scramblase enzyme XKR8, which drives the rapid and efficient transport of phosphatidylserine to the outer membrane of early apoptotic cells. The early apoptotic cells were then enriched by magnetically activated cell sorting with annexin V, followed by fluorescence-activated sorting with the fluorescent reporter.

[0333] A library of 61aa protein fragments tiled across all 11 open reading frames (ORFs) of SARS-CoV-2 in 20aa steps. To capture the known genetic diversity of SARS-CoV-2, it included 104 isolates reported as of March 15, 2020, and a complete set of SARS-CoV ORFs (ORFeomes), as well as all protein-coding variants from four endemic coronaviruses that cause the common cold (beta-coronaviruses HKU1 and OC43 and alpha-coronaviruses NL63 and 229E). Known immunodominant antigens from CMV, EBV, and influenza viruses were included as positive controls. Each protein fragment with a unique nucleotide barcode to provide internal replication in our screening was represented 10 times for the final library size of 43,420 clones.

[0334] As shown in Figure 14C, broad reactivity of CD8+ T cells to many SARS-CoV-2 proteins, including ORF1ab, S, N, M, and ORF3a, was observed. As shown in Figure 14D, three of the 29 epitopes were located on the spike protein. Most epitopes (15 out of 29) were located on ORF1ab, and the highest density of epitopes was located on the N protein. Shared epitopes were observed in the S protein for HLA-A*02:01, HLA-A*03:01, and HLA-A*24:02, but not for HLA-A*01:01, HLA-A*11:01, or HLA-B*07:02. The only recurrence response of the S protein in RBD was KCY on HLA-A*03:01. Example 9: Gene expression in CRISPR-operated UVC

[0335] We investigated the expression levels of various proteins under UVC.

[0336] Using CRISPR, the NK ligand MICA was knocked out in the UVC genome, and the B2M locus was knocked out to eliminate MHC-I expression. Flow cytometry was used to examine the expression levels of MICA and MHC-I in UVC and parental iPSCs. As shown in Figure 15, most cells in the UVC population showed high levels of MICA expression and minimal MHC-I expression compared to control parental iPSCs. Example 10: Effective lysis of UVC by NK cells

[0337] UVC can induce effective lysis by NK cells.

[0338] To measure UVC-induced cell lysis, a flow cytometry-based NK cell toxicity assay using calcein AM (CAM) staining of NK cells was used, as described in Jang et al., “An Improved Flow Cytometry-Based Natural Killer Cytotoxicity Assay Involving Calcein AM Staining of Effector Cells.” 2012, Ann. Clin. Lab. Sci. Winter; 42(1):42-9, which is incorporated herein by reference in its entirety for all purposes. Macaque NK cells (effectors) were stained with CAM and seeded with a certain number of MHC-I-deficient (B2M KO) endothelial cells (ECs) as target cells derived from UVC IPSc. Cells were mixed in an E:T ratio of 1:1 or 5:1. Wild-type ECs were used as a control. NK cells and EC cells were distinguished using forward scattering profiles obtained by CAM staining. The amount of dead EC cells was detected using propidium iodide. The rate of cytotoxicity was scored as the percentage of dead cells in the total number of EC cells. As shown in Figure 16, NK cells induced increased lysis in B2M KO-ECs compared to WT-ECs at all E:T ratios.

[0339] Therefore, UVC can induce effective lysis in vitro by monkey NK cells. Example 11: Further responses from NK cells to NK ligands

[0340] NK ligands can induce additional responses from NK cells.

[0341] To demonstrate that NK ligands can increase NK cells, intracellular cytokine staining (ICS) was used to determine the expression of CD107a, MIP1-β, IFN-γ, or TNF-α in MHC-I deficient UVC (KO), UVC transfected with a MICA expression construct (KO-MICA), UVC transfected with a MICB expression construct (KO-MICB), or UVC transfected with a ULBP1 expression construct (KO-ULBP1). Nucleofection was used to transfect UVC. Transfection efficiencies were approximately 40–70% for the MICA and MICB constructs. The expression constructs promoted high levels of expression of their respective NK ligands in transfected UVC. As shown in Figure 17A, KO-MICA increased the total number of NK cells expressing CD107a or MIP1-β, while KO-MICA increased the total number of NK cells expressing CD107a compared to KO. As illustrated in Figure 17B, SPICE analysis shows that when responding to UVC, MICA or MICB also increase the amount of NK cells expressing multiple cytokines. Addition of NK ligands increases the NK cell response to MHC-I deficient UVC. Example 12: Cell surface expression of SARS-CoV-2 spike antigen on UVC cells

[0342] UVC has robust expression of the SARS-CoV-2 spike protein.

[0343] The SARS-CoV-2 spike protein knock-in construct and the constructed MICA knock-in were integrated into the genomes of B2M knockout (B2m- / -) UVC iPSCs. As shown in Figure 18A, nearly half of the engineered UVC iPSC population expressed large amounts of spike protein. As shown in Figure 18B, the level of spike protein expression in UVC iPSCs was similar to that of HEK293T cells with transient transfection of the spike protein expression construct.

[0344] Multivalent antigens (e.g., other SARS-CoV-2 variant spike proteins such as the RSA variant listed in SEQ ID NO: 53, or other proteins such as the nucleoplasmid protein listed in SEQ ID NO: 54) can also be manipulated in UVC. Example 13: UVC Non-Human Primate (NHP) Pilot-1 Study

[0345] Six SARS-CoV-2-negative monkeys were administered MICA knock-in UVC expressing B2M knockout, SARS-CoV-2 spike protein, or its variant or domain. Figures 19A and 19B show the results of antibody ELISA performed at 0, 2, 6, and 8 weeks post-vaccination for both the receptor-binding domain (RBD) (Figure 19A) and the full-length SARS-CoV-2 spike protein (Figure 19B).

[0346] RBD-specific and full-length SARS-CoV-2 spike protein-specific binding antibodies were evaluated by ELISA as described in Chandrashekar, A. et al., Science 369, 812-817 (2020) and Yu, J. et al., Science 369, 806-811 (2020). In short, 1 μg ml of ELISA was used to bind 96-well plates in 1× DPBS. -1The plates were coated with SARS-CoV-2 RBD or full-length protein (A. Schmidt, MassCPR) and incubated overnight at 4°C. After incubation, the plates were washed once with washing buffer (0.05% Tween® 20 in 1× DPBS) and blocked with 350 μl of casein block per well for 2-3 hours at room temperature. After incubation, the block solution was discarded and the plates were blot-dried. Serial dilutions of thermo-inactivated serum diluted with casein block were added to the wells, and the plates were incubated at room temperature for 1 hour, followed by three more washes and incubation in the dark at room temperature for 1 hour with a 1:1,000 dilution of anti-macaque IgG HRP (NIH NHP Reagent Program). The plates were then washed three times and 100 μl of SeraCare KPL TMB SureBlue Start solution was added to each well. Plate development was stopped by adding 100 μl of SeraCare KPL TMB stop solution per well. Absorbance at 450 nm was recorded using a VersaMax or Omega microplate reader. In certain embodiments, for example, the following are provided: (Item 1) Genetically modified human cells, a. Genomic disruption in at least one human leukocyte antigen (HLA) gene, or at least one transcription regulator of an HLA gene, b. A cell surface protein that binds to a protein expressed on the surface of a phagocytic or cytolytic immune cell, or an exogenous nucleic acid encoding a functional fragment or functional variant of the cell surface protein, wherein the binding results in activation of the phagocytic or cytolytic activity of the immune cell, The genetically modified human cells, including the aforementioned cells. (Item 2) The genetically engineered human cell according to item 1, wherein the genome disruption inhibits the expression of the HLA protein encoded by the at least one HLA gene on the surface of the genetically engineered human cell. (Item 3) The genetically engineered human cell according to item 2, wherein the genome disruption completely inhibits the expression of the HLA protein encoded by the at least one HLA gene on the surface of the genetically engineered human cell. (Item 4) A genetically modified human cell as described in any one of items 1 to 3, wherein the aforementioned genome disruption is located in an HLA class I gene. (Item 5) The genetically modified human cells described in item 4, wherein the HLA class I gene is the HLA-A gene, HLA-B gene, HLA-C gene, or β-microglobulin gene. (Item 6) The genetically modified human cells described in item 5, wherein the HLA class I gene is the β-microglobulin gene. (Item 7) The aforementioned genome disruption is in an HLA class II gene in a genetically modified human cell as described in any one of items 1 to 3. (Item 8) The genetically modified human cells described in item 7, wherein the aforementioned HLA class II genes are HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, and HLA-DR genes. (Item 9) A genetically modified human cell as described in any of the preceding items, wherein at least one transcription regulator of the HLA gene is the CIITA gene, the RFX5 gene, the RFXAP gene, or the RFXANK gene. (Item 10) A genetically modified human cell as described in item 9, wherein the aforementioned HLA gene is the CIITA gene. (Item 11) The genetically modified human cells described in any of the preceding items, wherein the genetically modified human cells include genomic disruption of at least one HLA class I gene or at least one transcription factor of the HLA class I gene, and genomic disruption of at least one HLA class II gene or at least one transcription factor of the HLA class II gene. (Item 12) The genetically modified human cells described in any of the preceding items, wherein the genetically modified human cells include genomic disruption in at least one HLA class I transcription factor gene and genomic disruption in at least one HLA class II transcription factor. (Item 13) The aforementioned immune cells are innate immune cells, and are genetically modified human cells as described in any of the preceding items. (Item 14) Genetically modified human cells as described in item 13, wherein the aforementioned innate immune cells are NK cells, macrophages, dendritic cells, neutrophils, or eosinophils. (Item 15) Genetically modified human cells as described in item 14, wherein the aforementioned innate immune cells are NK cells. (Item 16) The genetically modified human cells described in item 15, wherein the binding results in the activation of the cytolytic activity of the NK cells. (Item 17) The genetically modified human cells described in item 14 or 15, wherein the cell surface protein is a ligand that specifically binds to natural killer (NK) cell activating receptors expressed on the surface of NK cells. (Item 18) Genetically modified human cells as described in item 14 or 15, wherein the cell surface protein is selected from the group consisting of MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, CD155, CD112 (nectin-2), B7-H6, Necl-2, and immunoglobulin Fc. (Item 19) Genetically modified human cells as described in item 14 or 15, wherein the cell surface protein is a natural killer (NK) cell activation ligand. (Item 20) The genetically modified human cells described in item 14 or 15, wherein the natural killer cell activating ligand is selected from the group consisting of MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, CD155, CD112 (nectin-2), B7-H6, and Necl-2. (Item 21) The cells comprise exogenous nucleic acids encoding a secretory protein that binds to receptors expressed on the surface of phagocytic or cytolytic immune cells, or a functional fragment or functional variant of the secretory protein, wherein the protein attracts the immune cells toward the genetically modified human cells, as described in any of the preceding items. (Item 22) Genetically modified human cells as described in any of the preceding items, further comprising an exogenous protein, an antigenic fragment thereof, or a nucleic acid encoding a suicide gene. (Item 23) Genetically modified human cells as described in item 22, wherein the exogenous protein includes a microbial protein. (Item 24) The genetically modified human cells described in item 23, wherein the microbial protein comprises a nucleocapsidrin protein having at least approximately 85% sequence identity with respect to SEQ ID NO: 54. (Item 25) The genetically modified human cell according to item 23, wherein the microbial protein is secreted by the genetically modified human cell, expressed on the surface of the genetically modified human cell, or expressed in the cytoplasm of the genetically modified human cell. (Item 26) A genetically modified human cell as described in item 23 or 25, wherein the microbial protein is a protein of a virus, bacterium, parasite, or protozoan. (Item 27) A genetically modified human cell as described in item 26, wherein the microbial protein is a viral protein. (Item 28) Genetically modified human cells as described in item 27, wherein the aforementioned viral protein is from a virus of the order Nidovirales. (Item 29) Genetically modified human cells as described in item 27 or 28, wherein the viral protein is from a virus of the Coronaviridae family. (Item 30) A genetically modified human cell as described in any one of items 27-29, wherein the viral protein is from a virus of the subfamily Orthocoronavirus. (Item 31) A genetically modified human cell as described in any one of items 27-30, wherein the viral protein is from a virus of the genus alphacoronavirus, betacoronavirus, gammacoronavirus, or deltacoronavirus. (Item 32) A genetically modified human cell as described in item 31, wherein the viral protein is from a virus of the genus Betacoronavirus. (Item 33) A genetically modified human cell as described in any one of items 27 to 32, wherein the viral protein is from a virus of the subgenus Salvecovirus. (Item 34) A genetically modified human cell as described in any one of items 27-33, wherein the viral protein is from one of two coronaviruses associated with severe acute respiratory syndrome. (Item 35) A genetically modified human cell as described in any one of items 27-34, wherein the viral protein is from two strains of severe acute respiratory syndrome coronavirus. (Item 36) A genetically modified human cell as described in any one of items 27-35, wherein the viral protein is the spike protein of severe acute respiratory syndrome coronavirus 2. (Item 37) A genetically modified human cell as described in any one of items 27 to 36, wherein the viral protein is the spike protein of Sequence ID No. 1. (Item 38) A genetically modified human cell as described in any one of items 27-36, wherein the viral protein is the spike protein encoded by Sequence ID No. 53. (Item 39) Genetically modified human cells as described in item 27, wherein the viral protein is from a virus selected from the group including influenza, Epstein-Barr virus (EBV), megavirus, Norwalk virus, coxsackievirus, Middle East Respiratory Syndrome-related coronavirus, Severe Acute Respiratory Syndrome-related coronavirus, SARS-CoV-2 virus, hepatitis B, varicella-zoster virus, parvovirus, adenovirus, Marburg virus, Ebola virus, rabies, smallpox, HIV, hantavirus, dengue fever, rotavirus, MERS-CoV, mumps virus, cytomegalovirus (CMV), herpesvirus, papillomavirus, chikungunya virus, or any combination thereof. (Item 40) The genetically modified human cells described above are those differentiated from stem cells, as described in any of the preceding items. (Item 41) Genetically modified human cells as described in item 40, wherein the stem cells are induced pluripotent stem cells (iPSCs), embryonic stem cells (ESCs), adult stem cells (ASCs), pluripotent stem cells (PSCs), or hematopoietic stem and progenitor cells (HSPCs). (Item 42) The aforementioned stem cells are induced pluripotent stem cells (iPSCs), which are genetically modified human cells as described in item 40. (Item 43) The genetically modified human cell described in any of the preceding items, wherein the genetically modified human cell is an epithelial cell or an endothelial cell. (Item 44) The genetically modified human cells described above are not cancer cells, and are genetically modified human cells as described in any of the preceding items. (Item 45) The genetically modified human cells described above are irradiated, as described in any of the preceding items. (Item 46) The genetically modified human cells described in any one of items 1 to 44 are stem cells. (Item 47) Genetically modified human cells as described in item 46, wherein the stem cells are induced pluripotent stem cells (iPSCs), embryonic stem cells (ESCs), adult stem cells (ASCs), pluripotent stem cells (PSCs), or hematopoietic stem and progenitor cells (HSPCs). (Item 48) The aforementioned stem cells are induced pluripotent stem cells (iPSCs), which are genetically modified human cells as described in item 47. (Item 49) The genetically modified human cells described in any of the preceding items are unable to proliferate in vitro, in vivo, or both. (Item 50) Genetically modified human cells, as described in any of the preceding items, for use in vaccines. (Item 51) A genetically modified human cell as described in any of the preceding items, wherein at least one of the genome disruptions is mediated by an endonuclease. (Item 52) The genetically modified human cells described in item 51, wherein the endonuclease is a CRISPR endonuclease, a zinc finger nuclease (ZFN), or a transcription activator-like effector nuclease (TALEN). (Item 53) A genetically modified human cell as described in any of the preceding items, wherein the disruption of at least one genome is mediated by a CRISPR system comprising an endonuclease and a guide RNA (gRNA), and the gRNA comprises an RNA sequence complementary to the DNA sequence of the at least one HLA gene or at least one transcription regulator of the HLA gene. (Item 54) Genetically modified human cells, a. Genomic disruption in at least one human leukocyte antigen (HLA) gene, or at least one transcription regulator of an HLA gene, b. An exogenous cell surface protein that binds to a protein expressed on the surface of a phagocytic or cytolytic immune cell, or a nucleic acid encoding a functional fragment or functional variant of the said exogenous cell surface protein, wherein the binding results in activation of the phagocytic or cytolytic activity of the said immune cell, c. Nucleic acids encoding an exogenous antigenic protein or its antigenic fragment, The genetically modified human cells, including the aforementioned cells. (Item 55) The genetically modified human cell described in item 54, wherein the exogenous antigenic protein or its antigenic fragment is a microbial protein or its antigenic fragment. (Item 56) The genetically engineered human cells described in item 55, wherein the exogenous antigenic protein comprises a nucleocapsidrin protein having at least approximately 85% sequence identity with respect to SEQ ID NO: 54. (Item 57) The genetically modified human cell according to item 55, wherein the microbial protein is secreted by the genetically modified human cell, expressed on the surface of the genetically modified human cell, or expressed in the cytoplasm of the genetically modified human cell. (Item 58) A genetically modified human cell as described in item 55 or 57, wherein the microbial protein is a protein of a virus, bacterium, parasite, or protozoan. (Item 59) A genetically modified human cell as described in item 58, wherein the microbial protein is a viral protein. (Item 60) Genetically modified human cells as described in item 59, wherein the aforementioned viral protein is from a virus of the order Nidovirales. (Item 61) Genetically modified human cells as described in item 59 or 60, wherein the viral protein is from a virus of the Coronaviridae family. (Item 62) A genetically modified human cell as described in any one of items 59-61, wherein the viral protein is from a virus of the subfamily Orthocoronavirus. (Item 63) A genetically modified human cell as described in any one of items 59 to 62, wherein the viral protein is from a virus of the genus alphacoronavirus, betacoronavirus, gammacoronavirus, or deltacoronavirus. (Item 64) A genetically modified human cell as described in item 63, wherein the viral protein is from a virus of the genus Betacoronavirus. (Item 65) A genetically modified human cell as described in any one of items 59 to 64, wherein the viral protein is from a virus of the subgenus Salvecovirus. (Item 66) A genetically modified human cell as described in any one of items 59-65, wherein the viral protein is from one of two coronaviruses associated with severe acute respiratory syndrome. (Item 67) A genetically modified human cell as described in any one of items 59 to 66, wherein the viral protein is from two strains of severe acute respiratory syndrome coronavirus. (Item 68) A genetically modified human cell as described in any one of items 59 to 67, wherein the viral protein is the spike protein of severe acute respiratory syndrome coronavirus 2. (Item 69) A genetically modified human cell as described in any one of items 59 to 68, wherein the viral protein is the spike protein of Sequence ID No. 1. (Item 70) A genetically modified human cell as described in any one of items 59-68, wherein the viral protein is the spike protein encoded by Sequence ID No. 53. (Item 71) Genetically modified human cells as described in item 59, wherein the viral proteins are derived from a virus selected from the group including influenza, Epstein-Barr virus (EBV), megavirus, Norwalk virus, coxsackievirus, Middle East Respiratory Syndrome-related coronavirus, Severe Acute Respiratory Syndrome-related coronavirus, SARS-CoV-2 virus, hepatitis B, varicella-zoster virus, parvovirus, adenovirus, Marburg virus, Ebola virus, rabies, smallpox, HIV, hantavirus, dengue fever, rotavirus, MERS-CoV, mumps virus, cytomegalovirus (CMV), herpesvirus, papillomavirus, chikungunya virus, or any combination thereof. (Item 72) The genetically modified human cells described in any one of items 54 to 71 are those differentiated from stem cells. (Item 73) Genetically modified human cells as described in item 72, wherein the stem cells are induced pluripotent stem cells (iPSCs), embryonic stem cells (ESCs), adult stem cells (ASCs), pluripotent stem cells (PSCs), or hematopoietic stem and progenitor cells (HSPCs). (Item 74) The aforementioned stem cells are induced pluripotent stem cells (iPSCs), which are genetically modified human cells as described in item 73. (Item 75) The genetically modified human cells described in any one of items 54 to 74, wherein the genetically modified human cells are epithelial cells or endothelial cells. (Item 76) The genetically modified human cells described in any one of items 54 to 75 are not cancer cells. (Item 77) The genetically modified human cells described in any one of items 54 to 76, wherein the genetically modified human cells are irradiated. (Item 78) Genetically modified human cells, as described in any one of items 54-77, for use in vaccines. (Item 79) Genetically modified human cells as described in any one of items 54-78, wherein the aforementioned immune cells are innate immune cells. (Item 80) Genetically modified human cells as described in item 79, wherein the innate immune cells are NK cells, macrophages, dendritic cells, neutrophils, or eosinophils. (Item 81) Genetically modified human cells as described in item 80, wherein the aforementioned innate immune cells are NK cells. (Item 82) A composition comprising a population of genetically modified human cells as described in any one of items 1 to 81. (Item 83) A pharmaceutical composition comprising the genetically modified human cells described in any one of items 1 to 81, or the composition described in item 82, and an excipient. (Item 84) A unit dosage form comprising the composition described in item 82 or 83. (Item 85) A method for producing a population of genetically modified human stem cells, wherein the method is: Obtaining a population of human stem cells, Inducing genomic disruption in at least one HLA gene or at least one transcription regulator of the HLA gene, Introducing an exogenous cell surface protein that binds to a protein expressed on the surface of phagocytic or cytolytic immune cells, or a nucleic acid encoding a functional fragment or functional variant of the said exogenous cell surface protein, wherein the binding results in activation of the phagocytic or cytolytic activity of the said immune cells, The method comprising, thereby producing a population of genetically modified stem cells. (Item 86) The method according to item 85, wherein the genome disruption inhibits the expression of the HLA protein encoded by the at least one HLA gene on the surface of the cell. (Item 87) The method according to item 85 or 86, wherein the genome disruption inhibits the expression of the HLA protein encoded by the at least one HLA gene on the surface of the immune cell for a period of time sufficient to interact with the protein expressed on the surface of the immune cell. (Item 88) The method according to any one of items 85-87, wherein the aforementioned at least one genome disruption is mediated by an endonuclease. (Item 89) The method according to item 88, wherein the endonuclease is a CRISPR endonuclease, a zinc finger nuclease (ZFN), or a transcription activator-like effector nuclease (TALEN). (Item 90) The method according to any one of items 85 to 89, wherein the disruption of at least one genome is mediated by a CRISPR system comprising an endonuclease and a guide RNA (gRNA), the gRNA comprising an RNA sequence complementary to the DNA sequence of the at least one HLA gene or at least one transcription regulator of the HLA gene. (Item 91) The method according to any one of items 85-90, wherein the genome disruption is a single-strand DNA break or a double-strand DNA break. (Item 92) The method according to any one of items 85 to 91, further comprising introducing nucleic acids encoding a microbial protein or an antigenic fragment thereof. (Item 93) The method according to item 92, wherein the microbial protein comprises a nucleocapsidrin protein having at least about 85% sequence identity with respect to SEQ ID NO: 54. (Item 94) The genetically modified human cell according to item 92, wherein the microbial protein is secreted by the genetically modified human cell, expressed on the surface of the genetically modified human cell, or expressed in the cytoplasm of the genetically modified human cell. (Item 95) The method according to item 92 or 94, wherein the microbial protein is a virus, bacterium, or parasite protein. (Item 96) The method according to item 95, wherein the microbial protein is a viral protein. (Item 97) The method according to any one of items 85 to 96, wherein the stem cells are induced pluripotent stem cells (iPSCs), embryonic stem cells (ESCs), adult stem cells (ASCs), pluripotent stem cells (PSCs), or hematopoietic stem and progenitor cells (HSPCs). (Item 98) The method according to item 97, wherein the stem cells are induced pluripotent stem cells (iPSCs). (Item 99) The method according to any one of items 85 to 98, further comprising differentiating the aforementioned population of genetically modified human stem cells. (Item 100) The method according to item 99, wherein the cells differentiate into epithelial cells or endothelial cells. (Item 101) Genetically modified human cells as described in any one of items 85 to 100, wherein the immune cells are innate immune cells. (Item 102) The method according to item 101, wherein the innate immune cells are NK cells, macrophages, dendritic cells, neutrophils, or eosinophils. (Item 103) The method according to item 102, wherein the innate immune cells are NK cells. (Item 104) A method for producing a population of terminally differentiated, genetically engineered human cells, wherein the method is: Obtaining a population of human stem cells, Inducing genomic disruption in at least one HLA gene or at least one transcription regulator of the HLA gene, The method involves introducing an exogenous cell surface protein that binds to a protein expressed on the surface of phagocytic or cytolytic immune cells, or a nucleic acid encoding a functional fragment or functional variant of the said exogenous cell surface protein, wherein the binding results in activation of the phagocytic or cytolytic activity of the immune cells, thereby producing a population of genetically modified human stem cells. Differentiating the aforementioned population of genetically modified human stem cells into a population of terminally differentiated genetically modified human cells, The method, including the method described above. (Item 105) The method according to item 104, wherein the population of genetically modified human stem cells differentiates into epithelial cells or endothelial cells. (Item 106) A method for immunizing a human subject against microorganisms, the method comprising administering to the subject the genetically modified human cells described in any one of items 1 to 81, the composition described in item 82, or the pharmaceutical composition described in item 83. (Item 107) A method for immunizing a human subject against microorganisms, the method comprising administering a population of genetically modified human cells to the subject, the population of genetically modified human cells is a. Genomic disruption in at least one HLA gene or at least one transcription regulator of an HLA gene, b. An exogenous cell surface protein that binds to a protein expressed on the surface of a phagocytic or cytolytic immune cell, or a nucleic acid encoding a functional fragment or functional variant of the said exogenous cell surface protein, wherein the binding results in activation of the phagocytic or cytolytic activity of the said immune cell, c. Nucleic acids encoding microbial proteins or their antigenic fragments, The method, including the method described above. (Item 108) The method according to item 107, wherein the binding results in immune cell-mediated lysis or phagocytosis of at least a portion of the population of genetically modified human cells. (Item 109) The method according to item 107 or 108, wherein the administration results in the subject initiating an adaptive immune response to the microorganism. (Item 110) The method according to any one of items 107 to 109, wherein the administration results in increased activation and / or proliferation of T cells expressing T cell receptors that specifically bind to the microbial protein or its antigenic fragment. (Item 111) The method according to any one of items 107 to 110, wherein the administration results in increased activation and / or proliferation of B cells expressing B cell receptors that specifically bind to the microbial protein or its antigenic fragment. (Item 112) The method according to any one of items 107 to 111, wherein the administration results in an increase in circulating antibodies that specifically bind to the microbial protein or its antigenic fragment. (Item 113) The method according to any one of items 107 to 112, wherein the microbial protein or its antigenic fragment is secreted by the genetically modified human cell, expressed on the surface of the genetically modified human cell, or expressed in the cytoplasm of the genetically modified human cell. (Item 114) The method according to any one of items 107 to 113, wherein the microbial protein is a virus, bacterium, or parasite protein. (Item 115) The method according to item 114, wherein the microbial protein is a viral protein. (Item 116) The method according to item 115, wherein the viral protein is from a virus of the Coronaviridae family. (Item 117) The method according to any one of items 115 to 116, wherein the viral protein is from a virus of the genus alphacoronavirus, betacoronavirus, gammacoronavirus, and deltacoronavirus. (Item 118) The method according to any one of items 115 to 117, wherein the viral protein is from a virus of the genus Betacoronavirus. (Item 119) The method according to any one of items 115 to 118, wherein the viral protein is from two viruses associated with severe acute respiratory syndrome. (Item 120) The method according to any one of items 115 to 119, wherein the viral protein is from the virus of two strains of severe acute respiratory syndrome coronavirus. (Item 121) The method according to any one of items 115 to 120, wherein the viral protein is the spike protein of severe acute respiratory syndrome coronavirus 2. (Item 122) The method according to any one of items 115 to 121, wherein the viral protein is the spike protein of Sequence ID No. 1. (Item 123) The method according to any one of items 115 to 121, wherein the viral protein is the spike protein encoded by Sequence ID No. 53. (Item 124) The method according to item 115, wherein the viral protein is derived from a virus selected from the group including influenza, Epstein-Barr virus (EBV), megavirus, Norwalk virus, coxsackievirus, Middle East Respiratory Syndrome-related coronavirus, Severe Acute Respiratory Syndrome-related coronavirus, SARS-CoV-2 virus, hepatitis B, varicella-zoster virus, parvovirus, adenovirus, Marburg virus, Ebola virus, rabies, smallpox, HIV, hantavirus, dengue fever, rotavirus, MERS-CoV, mumps virus, cytomegalovirus (CMV), herpesvirus, papillomavirus, chikungunya virus, and any combination thereof. (Item 125) The method according to any one of items 107 to 124, wherein the population of genetically modified human cells is administered intramuscularly or subcutaneously. (Item 126) The aforementioned immune cells are innate immune cells, and are genetically modified human cells as described in any one of items 107 to 125. (Item 127) The method according to item 126, wherein the innate immune cells are NK cells, macrophages, dendritic cells, neutrophils, or eosinophils. (Item 128) The method according to item 127, wherein the innate immune cells are NK cells. (Item 129) The method according to any one of items 107 to 128, wherein the genetically modified human cells further include a suicide gene. (Item 130) The method according to item 107, wherein the microbial protein comprises a nucleocapsidrin protein having at least about 85% sequence identity with respect to SEQ ID NO: 54. (Item 131) A method for immunizing a target, the method comprising administering a population of genetically modified mammalian cells to the target, the population of genetically modified mammalian cells is a. A genome disruption in at least one MHC gene or at least one transcription factor of an MHC gene, which results in a decrease in T cell proliferation activation compared to the genetically modified human cells without the disruption. b. An exogenous cell surface protein that binds to a protein expressed on the surface of a phagocytic or cytolytic immune cell, or a nucleic acid encoding a functional fragment or functional variant of the said exogenous cell surface protein, wherein the binding results in activation of the phagocytic or cytolytic activity of the said immune cell, The method, including the method described above. (Item 132) The method according to item 131, wherein the immunization is specific to the antigen, and the genetically engineered mammalian cells further comprise nucleic acids encoding the antigen or a fragment thereof. (Item 133) The method according to item 131, wherein the immunization is specific to the antigen, and the genetically engineered mammalian cells further comprise the antigen or a fragment thereof. (Item 134) The method according to any one of items 131 to 133, wherein the activation results in immune cell-mediated lysis or phagocytosis of at least a portion of the population of genetically modified mammalian cells. (Item 135) The method according to any one of items 132 to 134, wherein the administration results in the subject initiating an adaptive immune response to the antigen. (Item 136) The method according to any one of items 132 to 135, wherein the administration results in increased activation and / or proliferation of T cells expressing T cell receptors that specifically bind the peptide of the antigen. (Item 137) The method according to any one of items 132 to 134, wherein the administration results in increased activation and / or proliferation of B cells expressing B cell receptors that specifically bind the peptide of the antigen. (Item 138) The method according to any one of items 132 to 137, wherein the administration results in an increase in circulating antibodies that specifically bind to the antigen. (Item 139) The method according to any one of items 132 to 138, wherein the antigen is secreted by the genetically modified mammalian cell, expressed on the surface of the genetically modified mammalian cell, or expressed in the cytoplasm of the genetically modified mammalian cell. (Item 140) The method according to any one of items 132 to 139, wherein the antigen is a virus, bacterium, fungus, or parasitic protein. (Item 141) The method according to item 140, wherein the viral protein is from a virus of the Coronaviridae family. (Item 142) The method according to any one of items 140 to 141, wherein the viral protein is from a virus of the genus alphacoronavirus, betacoronavirus, gammacoronavirus, or deltacoronavirus. (Item 143) The method according to any one of items 140 to 141, wherein the viral protein is from a virus of the genus Betacoronavirus. (Item 144) The method according to any one of items 141 to 143, wherein the viral protein is from two viruses associated with severe acute respiratory syndrome. (Item 145) The method according to any one of items 141 to 144, wherein the viral protein is from the virus of two strains of severe acute respiratory syndrome coronavirus. (Item 146) The method according to any one of items 141 to 145, wherein the viral protein is the spike protein of severe acute respiratory syndrome coronavirus 2. (Item 147) The method according to any one of items 141 to 146, wherein the viral protein is the spike protein of Sequence ID No. 1. (Item 148) The method according to any one of items 141 to 146, wherein the viral protein is the spike protein encoded by Sequence ID No. 53. (Item 149) The method according to item 140, wherein the viral protein is derived from a virus selected from the group including influenza, Epstein-Barr virus (EBV), megavirus, Norwalk virus, coxsackievirus, Middle East respiratory syndrome-associated coronavirus, severe acute respiratory syndrome-associated coronavirus, SARS-CoV-2 virus, hepatitis B, varicella-zoster virus, parvovirus, adenovirus, Marburg virus, Ebola virus, rabies, smallpox, HIV, hantavirus, dengue fever, rotavirus, MERS-CoV, mumps virus, cytomegalovirus (CMV), herpesvirus, papillomavirus, chikungunya virus, or at least one of any combination thereof. (Item 150) The method according to any one of items 132 to 139, wherein the antigen comprises a protein or peptide associated with cancer or tumor. (Item 151) The method according to item 150, wherein the antigen includes a neoantigen. (Item 152) The method according to any one of items 131 to 151, wherein the population of genetically modified cells is administered intramuscularly or subcutaneously. (Item 153) The aforementioned immune cells are genetically modified cells as described in any one of items 131 to 152, wherein the immune cells are innate immune cells. (Item 154) The method according to item 153, wherein the innate immune cells are NK cells, macrophages, dendritic cells, neutrophils, or eosinophils. (Item 155) The method according to item 153, wherein the aforementioned innate immune cells are NK cells. (Item 156) The method according to any one of items 131 to 155, wherein the genetically modified mammalian cells further include a suicide gene. (Item 157) The method according to any one of items 131 to 156, wherein the genetically modified mammalian cells include genetically modified human cells and the MHC genes include HLA genes.

Claims

[Claim 1] The invention described in the specification.

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