Compositions and methods for inhibiting natural killer cells

Abstract

This disclosure relates to novel polynucleotide sequences, constructs, cell hosts for expressing them, and compositions comprising them. This disclosure further relates to methods for using the novel polynucleotide sequences, constructs, cell hosts, and compositions. In some embodiments, the first polynucleotide encodes a polypeptide that may have natural killer (NK) cell inhibitory activity, and the second polynucleotide encodes a polypeptide that may induce an immune response or encode a protein that is naturally endogenously produced.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Application No. 63 / 526,410, filed on 12 July 2023, the entirety of which is incorporated herein by reference.

[0002] Sequence List This application includes a sequence listing in XML format which constitutes part of the present disclosure. The XML file was created on June 24, 2024, is named "103783-0344_SL.xml", and is 17,669 bytes in size.

[0003] Statement on rights to inventions made under federal government-sponsored research and development. This invention was made with government support under 75N91019D00024, awarded by the National Institutes of Health, and under HU00012020067, HU00012120104, and HU00012120094, awarded by the Uniformed Services University of the Health Sciences. The government has certain rights in this invention.

[0004] This specification discloses novel polynucleotides and polynucleotide constructs for inhibiting natural killer cells or preventing other cells from being killed by natural killer cells, as well as cells capable of expressing the novel polynucleotides and polynucleotide constructs described herein. The compositions disclosed herein include one or more additional polynucleotides, e.g., exogenously delivered polynucleotides, and, in specific cases, one or more polynucleotides encoding polypeptides that may have NK cell inhibitory activity, to be used in conjunction with one or more mRNA vaccines. Methods for enhancing the persistence of one or more exogenously delivered polynucleotides, and methods for reducing one or more undesirable side effects associated with the administration of exogenously delivered polynucleotides are further disclosed. [Background technology]

[0005] While early mRNA vaccine formulations have been tested since 1995, the Pfizer-BioNTech COVID-19 vaccine (BNT162b2) was the first mRNA vaccine approved by the U.S. Food and Drug Administration (FDA). Phase III clinical trials demonstrated that BNT162b2 is highly effective and safe in preventing COVID-19 (Chakraborty et al., 2021. From COVID-19 to Cancer mRNA Vaccines: Moving from Bench to Clinic in the Vaccine Landscape. Front Immunol 12:679344, and Polack et al., 2020. Safety and Efficacy of the BNT162b2 mRNA Covid-19 Vaccine. N Engl J Med 383:2603-2615). Subsequent studies have shown that while protection against infection by novel variants may be variable, vaccines still provide significant protection against the development of serious illness (Liu et al., 2021. Effectiveness and safety of SARS-CoV-2 vaccine in real-world studies: a systematic review and meta-analysis. Infectious Diseases of Poverty 10:132). As of February 2023, more than 400 million doses of BNT162b2 vaccine have been administered in the United States (Centers for Disease Control and Prevention. COVID Data Tracker. US Department of Health and Human Services, CDC, Atlanta, GA). While the basic doctrines of how mRNA vaccines work have been characterized, many questions remain regarding the exact factors that cause the immunogenicity and reactiongenicity of mRNA vaccines.

[0006] Reactiveness to BNT162b2 vaccination is quite common, but does not occur in all individuals. According to the Centers for Disease Control and Prevention (CDC), 84.7% of individuals aged 18–55 years reported at least one local injection site reaction, and 77.4% reported at least one systemic reaction within 7 days of vaccination (Centers for Disease Control. 2022. Pfizer-BioNTech COVID-19 Vaccine Reactions & Adverse Events). In a prospective evaluation (PASS) study cohort of SARS-CoV-2 seroconversion, generally healthy adults reported substantial heterogeneity in both the severity and duration of local and systemic symptoms after BNT162b2 vaccination (Coggins et al., 2021. Adverse Effects and Antibody Titers in Response to the BNT162b2 mRNA COVID-19 Vaccine in a Prospective Study of Healthcare Workers. Open Forum Infectious Diseases 9). In addition, most individuals develop detectable IgG antibodies against the SARS-CoV-2 spike protein after BNT162b2 vaccination, but there is a wide range in the level of peak antibody titer (Moncunill et al., 2022. Determinants of early antibody responses to COVID-19 mRNA vaccines in a cohort of exposed and naive healthcare workers. eBioMedicine 75; Laing et al., 2022. Durability of Antibody Response and Frequency of SARS-CoV-2 Infection 6 Months after COVID-19 Vaccination in Healthcare Workers. Emerg Infect Dis 28:828-832).

[0007] One area that has not yet been extensively evaluated is the potential involvement of natural killer (NK) cells in the inflammatory response to mRNA vaccination. NK cells have been found to be activated within a few days of mRNA vaccination in both animal and human studies (Li et al., 2022. Mechanisms of innate and adaptive immunity to the Pfizer-BioNTech BNT162b2 vaccine. Nature Immunology 23:543-555, Kowalczyk et al., 2016). Self-adjuvanted mRNA vaccines induce local innate immune responses that lead to a potent and boostable adaptive immunity. Vaccine 34:3882-3893, Cuapio et al., 2022. NK cell frequencies, function and correlates to vaccine outcome in BNT162b2 mRNA anti-SARS-CoV-2 vaccinated healthy and immunocompromised individuals. Molecular Medicine 28:20, Saresella et al., 2022. Innate immune responses to three doses of the BNT162b2 mRNA SARS-CoV-2 vaccine. Front (Immunol 13:947320). The precise role that NK cells play in contributing to acute inflammatory side effects or in forming mRNA vaccine-induced adaptive immune responses has not yet been characterized.

[0008] NK cells are innate immune cells that make up 5-20% of peripheral blood lymphocytes (Abel et al., 2018. Natural Killer Cells: Development, Maturation, and Clinical Utilization. Frontiers in Immunology 9). Through various activating and inhibitory receptors present on their cell surface, NK cells recognize and target cells that appear to be transformed, infected, or stressed (Abel, 2018). When NK cells are activated, they have two main effector functions: the release of cytotoxic and inflammatory cytokines. Through these functions, NK cells can play a crucial role in inflammatory and regulatory processes. NK cells are well recognized for their ability to both amplify and depress vaccine-induced adaptive immune responses (Cox et al., 2021. Targeting natural killer cells to enhance vaccine responses. Trends in Pharmacological Sciences 42:789-801). After vaccination, NK cells can release cytokines that stimulate antigen-presenting cells, thereby enhancing the adaptive response (Cox, 2021).On the other hand, the cytolytic activity of NK cells may help suppress adaptive immunity by reducing the number of responding T cells, which in turn may reduce the ability of T cells to assist B cells, potentially reducing the quantity and quality of antibodies produced (Cox, 2021; Rydyznski et al., 2015. Generation of cellular immune memory and B-cell immunity is impaired by natural killer cells. Nature Communications 6:6375; and Cook et al., 2015. NK cells inhibit humoral immunity by reducing the abundance of CD4+T follicular helper cells during a chronic virus infection. J Leukoc Biol 98:153-162). Increased activation and release of pro-inflammatory molecules by NK cells is suspected to be responsible for some of the local and systemic symptoms commonly observed after mRNA vaccination.

[0009] In particular, there is substantial individual variability in baseline NK cell frequency, phenotype, and function (Freud et al., 2017. The Broad Spectrum of Human Natural Killer Cell Diversity. Immunity 47:820-833). This disclosure seeks to reduce inflammatory side effects induced by polynucleotide delivery (e.g., BNT162b2 vaccination) and / or increase the magnitude and / or duration of antibody and / or T cell responses by modifying NK cell frequency and / or function.

[0010] Several patent applications and issued patents generally disclose vaccine technologies, and more recently, mRNA vaccines.

[0011] For example, U.S. Patent Application Publication No. 2020 / 0155671 discloses RNA-decorated particles, such as RNA-decorated lipid particles, and methods for producing them.

[0012] U.S. Patent Application Publication No. 2020 / 0197508 discloses an immunostimulatory RNA molecule comprising a sequence derived from an influenza A virus nucleoprotein-coding RNA molecule that acts as an adjuvant and / or immunostimulator for enhancing the host immune response.

[0013] U.S. Patent No. 10,898,574 discloses formulations, compositions, and methods for delivering biological portions, such as modified nucleic acids, to cells to regulate protein expression.

[0014] U.S. Patent No. 10,703,789 discloses compositions, methods, processes, kits, and devices for the design, preparation, manufacture, and / or formulation of polynucleotides, primary constructs, and modified mRNA (mmRNA) molecules.

[0015] Despite the current state of vaccine technology, there is an urgent need to enhance the persistence of existing and future vaccines. Furthermore, there is a need to reduce vaccine-related undesirable side effects after administration to the target population. This disclosure provides one or more nucleic acids encoding proteins having NK cell inhibitory activity, as well as cells and compositions containing them. [Overview of the Initiative]

[0016] According to some aspects, (a) A first polynucleotide encoding a polypeptide that can have natural killer (NK) cell inhibitory activity, (b) an isolated polynucleotide construct comprising a second polynucleotide encoding a polypeptide capable of inducing an immune response or a protein that is naturally endogenously produced, and compositions comprising such constructs are provided.

[0017] According to another aspect, (a) a first polynucleotide encoding a polypeptide capable of having natural killer (NK) cell inhibitory activity, and (b) a second polynucleotide encoding a polypeptide capable of inducing an immune response, or encoding a protein endogenously produced in nature, and a composition comprising the foregoing, as well as such a construct, and (c) a pharmaceutically acceptable carrier, and a composition comprising the foregoing is provided.

[0018] According to any aspect, the polynucleotide construct or the first and second polynucleotides are mRNA molecules.

[0019] In any embodiment, the first polynucleotide may encode a serine protease inhibitor (serpine) polypeptide or fragment thereof that can have natural killer (NK) cell inhibitory activity, for example, SERPINB9 or fragment thereof that can have NK cell inhibitory activity. Additionally or alternatively, the first polynucleotide may encode a major histocompatibility complex (MHC) class polypeptide or fragment thereof that has NK cell inhibitory activity, for example, an MHC class I, MHC class II, or MHC class III polypeptide that has NK cell inhibitory activity, for example, an HLA-E polypeptide, for example, HLA-E*0103 or HLA-E*0101 that can have NK cell inhibitory activity, or fragment thereof. Additionally or alternatively, the first polynucleotide may encode a lectin polypeptide such as CLEC2D or fragment thereof that can have NK cell inhibitory activity. Additionally or alternatively, the first polynucleotide may encode a cadherin polypeptide or fragment thereof that can have NK cell inhibitory activity. Additionally or alternatively, the first polynucleotide may encode a CD155 (poliovirus receptor) polypeptide or a fragment thereof that may have NK cell inhibitory activity. Additionally or alternatively, the first polynucleotide may encode an RTN4 polypeptide or a fragment thereof that may have NK cell inhibitory activity.

[0020] In any embodiment, the second polynucleotide may encode an antigen capable of inducing an immune response, for example, a COVID-19 antigen capable of inducing an immune response to COVID-19. Alternatively, the second polynucleotide may encode a protein normally produced endogenously, for example, factor VIII or a biologically active fragment thereof.

[0021] In other embodiments, a method for enhancing one or both of the magnitude and duration of the immune response to a vaccine, or for reducing one or more undesirable side effects associated with vaccine administration, (a) A step of administering a pharmaceutically effective amount of a first polynucleotide encoding a polypeptide capable of having NK cell inhibitory activity to the target, (b) The step of administering a pharmaceutically effective amount of one or more vaccines to a target, The invention provides a method by which inhibition of NK cell activity enhances the magnitude and / or persistence of the immune response induced by one or more vaccines administered to a subject, and / or reduces one or more undesirable side effects associated with vaccine administration.

[0022] The vaccine may be a polynucleotide vaccine containing a polynucleotide encoding a vaccine antigen, and optionally, the polynucleotide is an mRNA molecule. The first polynucleotide and the polynucleotide encoding the vaccine antigen may be present in a single polynucleotide construct. Alternatively, the first polynucleotide and the polynucleotide encoding the vaccine antigen may be present in separate polynucleotide constructs formulated in a single composition. Alternatively, the first polynucleotide and the polynucleotide encoding the vaccine antigen may be present in separate polynucleotide constructs formulated in different compositions. When used, the single polynucleotide construct or single composition may be any construct or composition described herein.

[0023] In another embodiment, a method for enhancing the persistence of an exogenously administered polynucleotide, or for reducing one or more undesirable side effects associated with the administration of an exogenously administered polynucleotide, (a) A step of administering a pharmaceutically effective amount of a first polynucleotide encoding a polypeptide capable of having NK cell inhibitory activity to the target, (b) a step of administering a second exogenously administered polynucleotide, A method is provided in which inhibition of NK cell activity enhances the persistence of an exogenously administered polynucleotide and / or reduces one or more undesirable side effects associated with the administration of an exogenously administered polynucleotide.

[0024] The second exogenously administered polynucleotide may encode an antigen capable of inducing an immune response, such as a COVID-19 antigen capable of inducing an immune response to COVID-19. The second exogenously administered polynucleotide may encode a protein normally produced endogenously, such as factor VIII or a biologically active fragment thereof.

[0025] According to any such method in which the first polynucleotide and the second exogenously administered polynucleotide are provided separately, step (a) may be carried out before step (b), after step (b), or concurrently with step (b).

[0026] In other embodiments, constructs and compositions described herein are provided for enhancing one or both of the magnitude and duration of the immune response to a vaccine such as a polynucleotide vaccine, or for reducing one or more undesirable side effects associated with the administration of a polynucleotide vaccine, or for enhancing the duration of an exogenously administered polynucleotide, or for reducing one or more undesirable side effects associated with the administration of an exogenously administered polynucleotide.

[0027] In another embodiment, cells expressing the polynucleotide constructs described herein are provided. [Brief explanation of the drawing]

[0028] [Figure 1A] This is a strobe chart. [Figure 1B] This is a sample questionnaire used by participants in a study. [Figure 2] This is a flow cytometry gating strategy for NK cell populations. [Figure 3] Panels a-d are graphs illustrating the frequency and functionality of natural killer (NK) cells in relation to sex. [Figure 4] (Panels a-b) are graphs illustrating the functionality of NK cells in female versus male participants. [Figure 5] Panels a-d are graphs illustrating the frequency and functionality of NK cells in relation to age. [Figure 6] Panels a-e are graphs illustrating the longitudinal frequency of NK cells. [Figure 7A] The breakdown of various local and systemic symptoms observed in the cohort of Example 1 is shown below. [Figure 7B] The breakdown of various local and systemic symptoms observed in the cohort of Example 1 is shown below. [Figure 8] Panels a-f are graphs illustrating post-vaccination symptom scores related to NK cell frequency and functionality. [Figure 9] (Panels a-d) are graphs illustrating the NK cell function in participants with low symptom scores compared to high symptom scores after vaccination 1 and 2. [Figure 10] (Panels a-d) are graphs illustrating IgG levels at 1 month and 6 months after the second vaccination, related to NK cell frequency and functionality. [Figure 11] (Panels a-h) are graphs illustrating IgG levels at 1 month and 6 months after the second vaccination, related to the NK cell toxicity index. [Figure 12] This heatmap illustrates the correlation between NK cell receptors and symptom scores after vaccination, as well as IgG levels at 1 month and 6 months after vaccination. [Figure 13] This demonstrates the protection of 721,211 leukocytes expressing SERPINB or SERPINB and HLA-E from NK cell killing compared to the parental cell line and cells expressing only HLA-E. [Figure 14]This shows the percentage of spike protein-specific CD8+ T cells activated by incubation with spike S1 peptide in splenocytes (Panel A) or lymph node cells (Panel B) of mice vaccinated with different mRNA LNP vaccine formulations. [Modes for carrying out the invention]

[0029] The embodiments disclosed herein include polynucleotide constructs, cells expressing polynucleotide constructs, compositions comprising nucleic acids encoding one or more NK cell inhibitors that can inhibit NK cell activity, and methods for enhancing the persistence of one or more vaccines (e.g., to enhance the magnitude and / or duration of the immune response induced by the vaccine) and for reducing undesirable side effects associated with the vaccine after administration to a subject. In some embodiments, the disclosed constructs, compositions, and methods enhance one or both the magnitude and duration of the antibody response and / or T cell response to a vaccine antigen, such as an exogenously delivered polynucleotide-encoded vaccine antigen, such as an mRNA-encoded antigen in an mRNA vaccine.

[0030] Definition: As used herein, the singular forms "a," "an," and "the" are intended to include the plural form unless otherwise clearly indicated by the context.

[0031] As used herein, the term “about” means, for example, when referring to a measurable value such as quantity or duration of time, to include a variation of plus or minus 10% around the specified value, as such variation is appropriate for carrying out the methods and compositions in which such variation is disclosed.

[0032] As used herein, “administer” and / or “to administer” means giving or providing a composition or compound to a subject (e.g., a patient), including acts of ingestion by the subject or application to the subject, either by the subject itself or by a caregiver such as a medical professional, so that the composition or compound may exert its effects.

[0033] As used herein, the term “antigen” means a substance capable of producing one or more immune responses. Antigens may include, but are not limited to, peptides, proteins, glycoproteins, polysaccharides, and lipids, their portions, and combinations thereof. As used herein, antigens may be natural or synthetic.

[0034] As used herein, the terms “carrier” and / or “pharmaceutically acceptable carrier” mean any and all carriers that are compatible with the other components of the formulation and are bioacceptable for the intended route of administration.

[0035] As used herein, the term "cDNA" refers to DNA that is complementary to and synthesized from an mRNA template using the enzyme reverse transcriptase.

[0036] As used herein, the term “coding sequence” means a polynucleotide that directly specifies the amino acid sequence of a polypeptide. The boundaries of a coding sequence are generally determined by an open reading frame, which typically begins with a start codon and ends with a stop codon.

[0037] As used herein, the term “construction” is used to describe a molecule, such as a polynucleotide (e.g., Serpin B9 polynucleotide), that can optionally be chemically bonded to one or more additional molecular parts (e.g., one or more additional polynucleotides).

[0038] As used herein, the term “control sequence” means a nucleic acid sequence required for the expression of the polynucleotide encoding the polypeptide described herein in the expression system in question (if any). Each control sequence may be native to (i.e., from the same gene) or exogenous to (i.e., from a different gene) the polynucleotide encoding the polypeptide, or may be native to or exogenous to each other. Such control sequences include, but are not limited to, leaders, polyadenylation sequences, propeptide sequences, promoters, signal peptide sequences, and transcriptional terminators. Typically, a control sequence may include at least one promoter, as well as transcription and translation termination signals. Where present, the control sequence may be provided with a linker for the purpose of introducing a specific restriction site to facilitate ligation of the control sequence with the coding region of the polynucleotide encoding the polypeptide.

[0039] As used herein, the terms “effective dose” and / or “pharmaceutical effective dose” refer to the amount administered to a host, or to host cells, problems, or organs, that achieves the intended therapeutic outcome (e.g., inhibition of NK cell activity or induction of an immune response).

[0040] As used herein, the terms “enhance,” “improve,” “increase,” “decrease,” or “reduce,” or their grammatical equivalents, refer to values ​​relative to baseline measurements (e.g., natural killer cell activity, measurements in the same subject before inhibition of natural killer cell activity, and / or measurements in a control individual (or control individuals) in the absence of inhibition of natural killer cell activity).

[0041] As used herein, the term “expression” refers to any step involved in the production of a polypeptide, including but not limited to transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0042] As used herein, the term “exogenous” means that the molecule (e.g., nucleic acid) or activity referred to is introduced or delivered to a living organism or subject (e.g., a patient).

[0043] As used herein, the term “expression vector” refers to any means for cloning a nucleic acid and / or transferring a nucleic acid into a host cell. The term “vector” includes both viral and nonviral means for introducing nucleic acids into cells in vitro, ex vivo, or in vivo. Numerous vectors known in the art may be used to manipulate nucleic acids and incorporate response elements and promoters into genes, etc. Possible vectors include, but are not limited to, plasmids or modified viruses containing bacteriophages.

[0044] As used herein, the term “host cell” means any cell type that is receptive to transformation, transfection, transduction, etc., with nucleic acid constructs or expression vectors for expressing one or more polypeptides. The term “host cell” includes any offspring of a parent cell that are not identical to the parent cell due to mutations that occur during replication.

[0045] As used herein, the term “hybrid polypeptide” means a polypeptide in which a region of one polypeptide is fused directly or through a linker at the N-terminus or C-terminus of a region of another polypeptide. Hybrid polypeptides described herein may have at least 20% of the desired biological activity of a mature polypeptide, or at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% of the desired biological activity of a mature polypeptide, such as NK cell inhibitory activity.

[0046] As used herein, the term “isolated” means a substance in a form or environment not found in nature. Non-limiting examples of isolated substances include: (1) any substance not found in nature; (2) any substance, including but not limited to any nucleic acid, variant, protein, peptide, or cofactor, which has been removed at least partially from one or more of the naturally occurring components to which it relates in nature; (3) any substance modified by human intervention compared to its naturally occurring form; or (4) any substance modified by increasing the amount of the substance compared to other components to which it relates in nature (e.g., multiple copies of the gene encoding the substance, or the use of a promoter stronger than the one associated with the gene encoding the substance in nature).

[0047] As used herein, the term “lipid” refers to a group of organic compounds characterized by being insoluble in water but soluble in certain organic solvents, including, but not limited to, esters of fatty acids. Examples of lipids include “simple lipids” (e.g., fats and oils, and waxes), “complex lipids” (e.g., phospholipids and glycolipids), and “derived lipids” (e.g., steroids).

[0048] The term "lipid particles" includes lipid formulations that can be used to deliver polynucleotides to target sites of interest (e.g., cells, tissues, organs, etc.). Typically, the polynucleotides are completely encapsulated within the lipid particles.

[0049] As used herein, the terms “natural killer cell inhibitory activity” and / or “NK cell inhibitory activity” mean a reduction in the level of natural killer cell activity in the presence of the inhibitors described herein, compared to the level of natural killer cell activity in the absence of the inhibitor. For clarity, any reduction in the level of natural killer cell activity does not necessarily require a reduction in the total number of natural killer cells. For example, a reduction in the level of natural killer cell activity may result not only from a reduction in the total number of natural killer cells, but also from the inhibition of the binding of natural killer cells to specific targets (e.g., cells or cell surface proteins), and / or from protecting and / or isolating cells from natural killer cell activity (e.g., by inhibiting the cytolytic activity of cells by either cell surface molecules or secreted molecules that bind to receptors on the surface of natural killer cells or receptors in internal compartments of natural killer cells, in order to attenuate the killing activity of natural killer cells).

[0050] As used herein, the term “nucleic acid” means polynucleotide and includes single-stranded or double-stranded polymers of deoxyribonucleotide bases (i.e., guanine (G) cytosine I adenine (A) and thymine (T)) or ribonucleotide bases (i.e., guanine (G) cytosine (C) adenine (A) and uracil (U)). Unless otherwise specified, nucleic acid sequences disclosed herein may enumerate “T” in representative DNA sequences, but where the sequence represents RNA (e.g., mRNA), “T” will be substituted with “U”. Nucleic acids may also include fragments and modified nucleotides. For clarity, the terms “polynucleotide,” “polynucleotide sequence,” “nucleic acid sequence,” “nucleotide sequence,” and “nucleic acid fragment” are used interchangeably to refer to single-stranded or double-stranded polymers of RNA and / or DNA that optionally contain synthetic, unnatural, or modified nucleotide bases.

[0051] As used herein, the term “nucleic acid construct” means a single-stranded or double-stranded nucleic acid molecule which is isolated from a naturally occurring gene, modified to contain a nucleic acid segment in a manner otherwise not found in nature, or is a synthetic compound.

[0052] As used herein, the terms “open reading frame” and / or “ORF” refer to a sequence of nucleic acid, either DNA or RNA, that encodes a protein or polypeptide. An ORF may include a translation start signal or start codon, such as ATG or AUG, and a termination codon.

[0053] As used herein, the term “operatably linked” means a configuration in which a control sequence is positioned appropriately relative to a polynucleotide coding sequence so that the control sequence directs the expression of the coding sequence. Nucleic acids are “operatably linked” when they are positioned in a functional relationship with another nucleic acid sequence. The coding sequences do not need to be contiguous with each other, as long as the expressed sequences are ultimately processed to produce the desired protein.

[0054] As used herein, the term “polynucleotide fragment” means a nucleotide sequence having a length reduced relative to that of a reference nucleic acid and having a common portion that is identical to that of the reference nucleic acid. In one embodiment, the fragment is at least 80% of the length of the reference nucleic acid, for example, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.9%, or at least 99.99% of the length of the reference nucleic acid.

[0055] As used herein, the terms “polypeptide,” “protein,” and “peptide” are interchangeable and may refer to polymers of two or more amino acids.

[0056] As used herein, the term “polypeptide fragment” means a length that is relatively reduced with respect to a reference nucleic acid, and an amino acid sequence on the common portion that has the same amino acid sequence as the reference polypeptide. In one embodiment, the fragment is at least 80% of the length of the reference polypeptide, for example, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.9%, or at least 99.99% of the length of the reference polypeptide.

[0057] As used herein, the term “recombinant” means a cell, nucleic acid, polypeptide, expression cassette, or vector that has been modified by the introduction of a new part or by modification of an existing part by a recombinant technique, or a material identical thereto but produced or derived from a synthetic material using a recombinant technique, or a material corresponding to the natural or unmodified form of that material. For example, a recombinant cell expresses a gene (i.e., “exogenous nucleic acid”) not found in the cell’s natural (non-recombinant) form, or a natural gene that is otherwise expressed at different levels, typically underexpressed or not expressed at all.

[0058] As used herein, the term “reference sequence” refers to a defined sequence used as the basis for sequence comparison. A reference sequence may be a subset of a larger sequence, for example, a segment of a full-length gene or polypeptide sequence. Generally, a reference sequence is the length of at least 20 nucleotides or amino acid residues, at least 25 residues, at least 50 residues, or the full length of a nucleic acid or polypeptide. Since each of two polynucleotides or polypeptides may (1) contain sequences that are similar between the two sequences (i.e., parts of the complete sequence) and (2) further contain sequences that are inconsistent between the two sequences, sequence comparison between two (or more) polynucleotides or polypeptides is typically performed by comparing the sequences of the two polynucleotides or polypeptides to identify and compare local regions of sequence similarity.

[0059] As used herein, the term “RNA transcript” refers to the product resulting from the transcription of a DNA sequence catalyzed by RNA polymerase. If an RNA transcript is a complete, complementary copy of a DNA sequence, it is referred to as a primary transcript or premRNA. If an RNA transcript is an RNA sequence derived from a post-transcriptional treatment of a primary transcript or premRNA, it is referred to as mature RNA. “Messenger RNA” or “mRNA” refers to RNA that is free of introns and can be translated into proteins by cells.

[0060] As used herein, the term “undesirable side effect” means one or more effects and / or symptoms associated with the administration of a substance to a subject that are not the desired and / or intended effects and / or are unpleasant to the subject.

[0061] As used herein, the term “vaccine” means an immunogenic composition for administration to a mammal to induce an immune response to a specific antigen, which may be referred to herein as “vaccine antigen.”

[0062] As used herein, the term “variant” means a polypeptide having an amino acid sequence that possesses the desired biological activity of the reference polypeptide (such as NK cell inhibitory activity or the ability to induce an immune response) and that includes one or more modifications (i.e., substitutions, insertions, and / or deletions) relative to the reference polypeptide, i.e., several amino acid positions. Substitution means replacing an amino acid occupying a position with a different amino acid, deletion means removing an amino acid occupying a position, and insertion means adding an amino acid adjacent to and immediately following an amino acid occupying a position.

[0063] As used herein, the term “wild-type” polynucleotide means that the polynucleotide is free from any mutations compared to naturally occurring polynucleotides. “Wild-type” protein means that the protein contains the amino acid sequence of a naturally occurring protein and exhibits the same level of activity as a naturally occurring protein.

[0064] Polynucleotides In some embodiments, this disclosure relates to one or more isolated polynucleotides encoding biologically active peptides. As described herein, it is assumed that one or more polynucleotides can encode any polypeptide of interest. In certain embodiments, one or more polynucleotides described herein are intended to encode a polypeptide having NK cell inhibitory activity. In certain embodiments, one or more polynucleotides described herein are further intended to encode an antigen (e.g., a polynucleotide that can be administered as a vaccine). In certain embodiments, one or more polynucleotides are still intended to encode a protein that is normally endogenously produced (e.g., a polynucleotide that can be administered to subjects having a genetic mutation that results in a deficiency and / or insufficient amount or function of a particular protein that is normally endogenously produced) (see Dolgin, E., The tangled history of mRNA vaccines. Nature, 2021. 597(7876): pp. 318-324).

[0065] The polynucleotides disclosed throughout this specification may be individual polynucleotides or polynucleotide constructs comprising one or more polynucleotides (e.g., one polynucleotide, two polynucleotides, three polynucleotides, four polynucleotides, five polynucleotides, six polynucleotides, seven polynucleotides, eight polynucleotides, nine polynucleotides, ten polynucleotides, etc.). Furthermore, the polynucleotides provided herein may be one or more polynucleotides, one or more polynucleotide fragments, and / or combinations thereof. In certain embodiments, the polynucleotides described herein are wild-type polynucleotides. In other embodiments, the polynucleotides described herein are synthetic, i.e., synthetic polynucleotides described herein are not found in nature, or are made from or contain one or more synthetic, non-natural, and / or modified nucleotide bases. Furthermore, in other embodiments, the polynucleotides described herein are recombinant polynucleotides.

[0066] Furthermore, it should be understood that one or more polynucleotides described herein may or may not contain an ORF. In certain embodiments, one or more polynucleotides described herein contain an ORF. In certain embodiments, one or more polynucleotides provided herein may further contain one or more untranslated regions (UTRs). In more specific embodiments, one or more polynucleotides described contain a 5'UTR. In other specific embodiments, one or more polynucleotides contain a 3'UTR. In yet another specific embodiment, one or more polynucleotides contain a 5'UTR and a 3'UTR. One or more polynucleotides described herein may further contain a poly(A) tail and / or a 5' cap analogue. In certain embodiments, one or more polynucleotides contain a poly(A) tail and / or a 5' cap. In yet another specific embodiment, one or more polynucleotides are RNA transcripts. In yet another specific embodiment, one or more polynucleotides are mature RNA transcripts. In yet another specific embodiment, one or more polynucleotides are RNA molecules. In a more specific embodiment, one or more polynucleotides are mRNA molecules.

[0067] Polynucleotide constructs: In certain embodiments, this disclosure describes polynucleotide constructs. It should be understood that in all embodiments disclosed herein, the polypeptides obtained by encoding a polynucleotide construct may be isolated polypeptides, isolated hybrid polypeptides, isolated synthetic polypeptides, or isolated wild-type polypeptides. It should be further understood that the embodiments disclosed herein intend polypeptides exhibiting biological activity, including NK cell inhibitory activity. It should also be further understood that the embodiments disclosed herein intend polypeptides exhibiting biological activity, including immunogenic activity.

[0068] As described herein, this disclosure describes polynucleotide constructs comprising one or more polynucleotides (e.g., one polynucleotide, two polynucleotides, three polynucleotides, four polynucleotides, five polynucleotides, six polynucleotides, seven polynucleotides, eight polynucleotides, nine polynucleotides, ten polynucleotides, etc.).

[0069] In one aspect, this disclosure is, (a) A first polynucleotide encoding a polypeptide that can have NK cell inhibitory activity, (b) A second polynucleotide encoding a polypeptide that can induce an immune response, or a protein that is normally produced endogenously, Describe a polynucleotide construct containing one or more polynucleotides including [specific polynucleotides].

[0070] In this regard, it should be understood that the first and second polynucleotides can be arranged in any order within the polynucleotide construct. It should also be understood that the first and second polynucleotides can independently encode either a full-length polypeptide having a specified biological activity (e.g., NK cell inhibitory activity or immune response induction or induction of a desired biological activity of a normally endogenously produced protein) or a fragment of a full-length polypeptide.

[0071] Where three or more polynucleotides (e.g., 3 polynucleotides, 4 polynucleotides, 5 polynucleotides, 6 polynucleotides, 7 polynucleotides, 8 polynucleotides, 9 polynucleotides, 10 polynucleotides, etc.) are assumed to carry out the embodiments provided herein, it is intended that these additional polynucleotides may be polynucleotides or polynucleotide fragments.

[0072] In specific embodiments, the first polynucleotide described herein includes an ORF. In certain embodiments, the first polynucleotide may further include one or more untranslated regions (UTRs). In certain embodiments, the first polynucleotide includes a 5'UTR. In other specific embodiments, the first polynucleotide includes a 3'UTR. In other specific embodiments, the first polynucleotide includes a 5'UTR and a 3'UTR. The first polynucleotide may further include a poly(A)tail and / or a 5' cap analogue. In certain embodiments, the first polynucleotide includes a poly(A)tail and / or a 5' cap. In more specific embodiments, the first polynucleotide is an RNA transcript. In even more specific embodiments, the first polynucleotide is a mature RNA transcript. In even more specific embodiments, the first polynucleotide is an RNA molecule. In even more specific embodiments, the first polynucleotide is an mRNA molecule.

[0073] In specific embodiments, the second polynucleotide described herein includes an ORF. In certain embodiments, the second polynucleotide may further include one or more untranslated regions (UTRs). In certain embodiments, the second polynucleotide includes a 5'UTR. In other specific embodiments, the second polynucleotide includes a 3'UTR. In other specific embodiments, the second polynucleotide includes a 5'UTR and a 3'UTR. The second polynucleotide may further include a poly(A) tail and / or a 5' cap analogue. In certain embodiments, the second polynucleotide includes a poly(A) tail and / or a 5' cap. In more specific embodiments, the second polynucleotide is an RNA transcript. In even more specific embodiments, the second polynucleotide is a mature RNA (mRNA) transcript. In even more specific embodiments, the second polynucleotide is an RNA molecule. In even more specific embodiments, the second polynucleotide is an mRNA molecule.

[0074] Where three or more polynucleotides (e.g., 3, 4, 5, 6, 7, 8, 9, 10, etc.) are assumed to perform the embodiments provided herein, the additional polynucleotides are intended to include ORFs. Furthermore, where three or more polynucleotides are assumed to perform the embodiments provided herein, the additional polynucleotides are intended to include, or not include, one or more untranslated regions (UTRs). In certain embodiments, where three or more polynucleotides are assumed to perform the embodiments provided herein, the additional polynucleotides are intended to include a 5'UTR. In other certain embodiments, where three or more polynucleotides are assumed to perform the embodiments provided herein, the additional polynucleotides are intended to include a 3'UTR. In other certain embodiments, where three or more polynucleotides are assumed to perform the embodiments provided herein, the additional polynucleotides are intended to include a 5'UTR and a 3'UTR. Furthermore, in certain other embodiments, if three or more polynucleotides are assumed to perform the embodiments provided herein, those additional polynucleotides are intended to further include poly(A) tails and / or 5' cap analogues, or not. In certain embodiments, if three or more polynucleotides are assumed to perform the embodiments provided herein, those additional polynucleotides are intended to include poly(A) tails and / or 5' caps. In more specific embodiments, if three or more polynucleotides are assumed to perform the embodiments provided herein, those additional polynucleotides are intended to be RNA transcripts or RNA molecules.In more specific embodiments, if three or more polynucleotides are assumed to carry out the embodiments provided herein, those additional polynucleotides are intended to be mature RNA transcripts or mRNA molecules.

[0075] In certain embodiments, the first polynucleotide is a polynucleotide encoding a biologically active serine protease inhibitor (serpine) polypeptide. In other embodiments, the first polynucleotide is a polynucleotide encoding a biologically active major histocompatibility complex (MHC) polypeptide. In yet more specific embodiments, the first polynucleotide is a polynucleotide encoding a biologically active lectin polypeptide. In yet more specific embodiments, the first polynucleotide is a polynucleotide encoding a biologically active cadherin polypeptide. In yet more specific embodiments, the first polynucleotide is a polynucleotide encoding a biologically active CD155 (i.e., poliovirus receptor) polypeptide. In yet another more specific embodiment, the first polynucleotide is a polynucleotide encoding a biologically active RTN4 polypeptide. In one particular embodiment, the first polynucleotide is a polynucleotide encoding a serpine polypeptide having NK cell inhibitory activity. In another embodiment, the first polynucleotide is a polynucleotide encoding an MHC polypeptide having NK cell inhibitory activity. In yet another specific embodiment, the first polynucleotide is a polynucleotide encoding a lectin polypeptide having NK cell inhibitory activity. In a more specific embodiment, the first polynucleotide is a polynucleotide encoding a cadherin polypeptide having NK cell inhibitory activity. In an even more specific embodiment, the first polynucleotide is a polynucleotide encoding a CD155 polypeptide having NK cell inhibitory activity. In yet another more specific embodiment, the first polynucleotide is a polynucleotide encoding an RTN4 polypeptide having NK cell inhibitory activity.

[0076] According to any of these embodiments, the second polynucleotide may encode an antigen. Alternatively, according to any of the embodiments relating to the first polynucleotide, the second polynucleotide may encode a protein that is normally produced endogenously.

[0077] Natural killer cell inhibitors: Natural killer (NK) cells belong to a rapidly expanding family of known innate lymphocytes (ILCs) and are a type of cytotoxic lymphocyte crucial to the innate immune system, representing 5–20% of circulating lymphocytes in humans. The role of NK cells is analogous to that of cytotoxic T cells in the adaptive immune response of vertebrates. NK cells provide a rapid response to virus-infected cells and other intracellular pathogens, acting approximately 1–3 days after infection and responding to tumorigenesis. Unlike other immune cells that detect major histocompatibility complexes (MHC) presented on the surface of infected cells and influence the death of infected cells by lysis or apoptosis, NK cells are unique. NK cells have the ability to recognize and kill stressed cells in the absence of antibodies and MHC. Such a mechanism provides a faster immune response. In addition to natural killer cells being effectors of innate immunity, both activating and inhibitory NK cell receptors play important functional roles (e.g., including self-tolerance and maintenance of NK cell activity). NK cells also play a role in adaptive immune responses (for example, experiments have demonstrated that NK cells can respond to the current environment, form antigen-specific immunological memory, and respond to secondary infections with the same antigen).

[0078] In humans, NK cells are generally classified into two classes: regulatory NK cells (CD56) which have greater cytokine-producing capacity. bright CD16 - NK cells, and CD56, which are antitumor / antiviral NK cells with enhanced cytotoxic function. dim CD16 +They can be classified into NK cells (Poznanski, et al., “What defines NK cell functional fate: phenotype or metabolism.” Frontiers in Immunology, vol.10, 2019, www.frontiersin.org / articles / 10.3389 / fimmu.2019.01414). Furthermore, NK cells express various additional receptors that fine-tune NK cell activity (ibid.). Non-exclusive examples of receptors include, but are not limited to, activated innate cytotoxic receptors (NKp30, NKp44, and NKp46), activating and inhibitory CD94 / NKG2 receptors that recognize non-classical MHC, and inhibitory KIR receptors that recognize classical MHC (ibid.). The expression of one or more of these receptors can be a way to identify specific NK cell developmental stages, effector subsets, and memory populations (ibid.).

[0079] The subject matter provided herein relates to NK cell inhibitors, such as polynucleotides and polynucleotide constructs encoding polypeptides that inhibit NK cells. In certain embodiments disclosed throughout, the NK inhibitor can be any NK inhibitor. In more specific embodiments provided herein, one or more NK inhibitors are selected from the group consisting of serine protease inhibitors (serpines), major histocompatibility complex (MHC) molecules, lectins, cadherins, CD155 (i.e., poliovirus receptor) proteins, RTN4 proteins, and combinations thereof.

[0080] Serpine: Serine protease inhibitors (serpines) are a superfamily of structurally similar proteins whose protease inhibitory activity was first identified, and they are found throughout all life. More than 1000 serpines have been identified in animals, plants, fungi, bacteria, archaea, and viruses, and more than 30 human serpine proteins have been specifically identified (Law et al., 2006. An overview of the serpin superfamily. Genome Biol 7(5):216). Thus, serpines are the largest and most diverse superfamily of protease inhibitors. Serpines are noteworthy for their unusual mechanism of action, in which they inhibit their target proteases by disrupting the active site of the target. Protease inhibition by serpines controls a range of biological processes, including coagulation and inflammation, and therefore these proteins are targets of medical research.

[0081] Most serpines are protease inhibitors that target extracellular chymotrypsin-like serine proteases. These proteases have a nucleophilic serine residue in their catalytic triplicate at their active site. Serpines act as irreversible suicide inhibitors by capturing intermediates in the protease's catalytic mechanism. Some serpines inhibit other protease classes, typically cysteine ​​proteases, and are referred to as “cross-class inhibitors.” These enzymes differ from serine proteases in that they use a nucleophilic cysteine ​​residue, rather than serine, at their active site. Nevertheless, their enzymatic chemistry is similar, and the mechanism of inhibition by serpines is the same for both classes of proteases. While most serpines regulate the proteolytic cascade, some proteins with serpine structures are not enzyme inhibitors but instead perform diverse functions such as storage, transport, and molecular chaperoning. The term serpine is also used to describe these members despite their non-inhibitory functions because they are evolutionarily related. Closely related to this disclosure is the finding that the SERPINB9 protein has been shown to protect cells from lysis by inhibiting granzyme B, a major effector protease used by both natural killer cells and cell-lysing T cells.

[0082] As disclosed herein, in one embodiment, one or more polynucleotides are polynucleotides encoding serpine polypeptides. In another embodiment, one or more polynucleotides encode one or more serpine polypeptides selected from the group consisting of SERPINA1, SERPINA2, SERPINA3, SERPINA4, SERPINA5, SERPINA6, SERPINA7, SERPINA8, SERPINA9, SERPINA10, SERPINA11, SERPINA12, SERPINA13, SERPINB1, SERPINB2, SERPINB3, SERPINB4, SERPINB5, SERPINB6, SERPINB7, SERPINB8, SERPINB9, SERPINB10, SERPINB11, SERPINB12, SERPINB13, SERPINC1, SERPIND1, SERPINE1, SERPINE2, SERPINE3, SERPINF1, SERPINF2, SERPING1, SERPINH1, SERPINI1, SERPINI2, and combinations thereof.

[0083] In another embodiment, one or more polynucleotides encode one or more serpine polypeptide variants selected from the group consisting of SERPINA1, SERPINA2, SERPINA3, SERPINA4, SERPINA5, SERPINA6, SERPINA7, SERPINA8, SERPINA9, SERPINA10, SERPINA11, SERPINA12, SERPINA13, SERPINB1, SERPINB2, SERPINB3, SERPINB4, SERPINB5, SERPINB6, SERPINB7, SERPINB8, SERPINB9, SERPINB10, SERPINB11, SERPINB12, SERPINB13, SERPINC1, SERPIND1, SERPINE1, SERPINE2, SERPINE3, SERPINF1, SERPINF2, SERPING1, SERPINH1, SERPINI1, SERPINI2, and combinations thereof.

[0084] In a more specific embodiment, one or more polynucleotides encode a SERPINB9 polypeptide. In another specific embodiment, one or more polynucleotides encode a SERPINB9 polypeptide variant. In yet another specific embodiment, one or more polynucleotides encode a fragment of a SERPINB9 polypeptide that may have NK cell inhibitory activity.

[0085] In a more specific embodiment, one or more polynucleotides encode a SERPINB9 polypeptide, and one or more polynucleotides have at least 60% sequence identity with respect to the mRNA sequence of SEQ ID NO: 1. In a more specific embodiment, one or more polynucleotides encode a SERPINB9 polypeptide and have at least 65% sequence identity with respect to the sequence of SEQ ID NO: 1. In a more specific embodiment, one or more polynucleotides encode a SERPINB9 polypeptide and have at least 70% sequence identity with respect to the sequence of SEQ ID NO: 1. In a more specific embodiment, one or more polynucleotides encode a SERPINB9 polypeptide and have at least 75% sequence identity with respect to the sequence of SEQ ID NO: 1. In a more specific embodiment, one or more polynucleotides encode a SERPINB9 polypeptide and have at least 80% sequence identity with respect to the sequence of SEQ ID NO: 1. In a more specific embodiment, one or more polynucleotides encode a SERPINB9 polypeptide and have at least 85% sequence identity with respect to the sequence of SEQ ID NO: 1. In a more specific embodiment, one or more polynucleotides encode a SERPINB9 polypeptide and have at least 90% sequence identity with respect to the sequence of SEQ ID NO: 1. In a more specific embodiment, one or more polynucleotides encode a SERPINB9 polypeptide and have at least 91% sequence identity with respect to the sequence of SEQ ID NO: 1. In a more specific embodiment, one or more polynucleotides encode a SERPINB9 polypeptide and have at least 92% sequence identity with respect to the sequence of SEQ ID NO: 1. In a more specific embodiment, one or more polynucleotides encode a SERPINB9 polypeptide and have at least 93% sequence identity with respect to the sequence of SEQ ID NO: 1. In a more specific embodiment, one or more polynucleotides encode a SERPINB9 polypeptide and have at least 94% sequence identity with respect to the sequence of SEQ ID NO: 1. In a more specific embodiment, one or more polynucleotides encode a SERPINB9 polypeptide and have at least 95% sequence identity with respect to the sequence of SEQ ID NO: 1.In a more specific embodiment, one or more polynucleotides encode a SERPINB9 polypeptide and have at least 96% sequence identity to the sequence of SEQ ID NO: 1. In a more specific embodiment, one or more polynucleotides encode a SERPINB9 polypeptide and have at least 97% sequence identity to the sequence of SEQ ID NO: 1. In a more specific embodiment, one or more polynucleotides encode a SERPINB9 polypeptide and have at least 98% sequence identity to the sequence of SEQ ID NO: 1. In a more specific embodiment, one or more polynucleotides encode a SERPINB9 polypeptide and have at least 99% sequence identity to the sequence of SEQ ID NO: 1. In a more specific embodiment, one or more polynucleotides encode a SERPINB9 polypeptide and have at least 99.5% sequence identity to the sequence of SEQ ID NO: 1. In a more specific embodiment, one or more polynucleotides encode a SERPINB9 polypeptide and have at least 99.9% sequence identity to the sequence of SEQ ID NO: 1. In a more specific embodiment, one or more polynucleotides encode a SERPINB9 polypeptide and have at least 99.99% sequence identity to the sequence of SEQ ID NO: 1. In a further specific embodiment, one or more polynucleotides encode a SERPINB9 polypeptide and have 100% sequence identity to the sequence of SEQ ID NO: 1. In a further specific embodiment, one or more polynucleotides encode a SERPINB9 polypeptide, and one or more polynucleotides contain a sequence that has 100% sequence identity to the sequence of SEQ ID NO: 1. In a particular specific embodiment, the SERPINB9 polypeptide has the sequence of SEQ ID NO: 1.

[0086] In a more specific embodiment, one or more polynucleotides encode a SERPINB9 polypeptide, and the SERPINB9 polypeptide has at least 60% sequence identity with respect to the sequence of SEQ ID NO: 5. In a more specific embodiment, one or more polynucleotides encode a SERPINB9 polypeptide, and the SERPINB9 polypeptide has at least 65% sequence identity with respect to the sequence of SEQ ID NO: 5. In a more specific embodiment, one or more polynucleotides encode a SERPINB9 polypeptide, and the SERPINB9 polypeptide has at least 70% sequence identity with respect to the sequence of SEQ ID NO: 5. In a more specific embodiment, one or more polynucleotides encode a SERPINB9 polypeptide, and the SERPINB9 polypeptide has at least 75% sequence identity with respect to the sequence of SEQ ID NO: 5. In a more specific embodiment, one or more polynucleotides encode a SERPINB9 polypeptide, and the SERPINB9 polypeptide has at least 80% sequence identity with respect to the sequence of SEQ ID NO: 5. In a more specific embodiment, one or more polynucleotides encode a SERPINB9 polypeptide, and the SERPINB9 polypeptide has at least 85% sequence identity with respect to the sequence of SEQ ID NO: 5. In a more specific embodiment, one or more polynucleotides encode a SERPINB9 polypeptide, and the SERPINB9 polypeptide has at least 90% sequence identity with respect to the sequence of SEQ ID NO: 5. In a more specific embodiment, one or more polynucleotides encode a SERPINB9 polypeptide, and the SERPINB9 polypeptide has at least 91% sequence identity with respect to the sequence of SEQ ID NO: 5. In a more specific embodiment, one or more polynucleotides encode a SERPINB9 polypeptide, and the SERPINB9 polypeptide has at least 92% sequence identity with respect to the sequence of SEQ ID NO: 5. In a more specific embodiment, one or more polynucleotides encode a SERPINB9 polypeptide, and the SERPINB9 polypeptide has at least 93% sequence identity with respect to the sequence of SEQ ID NO: 5.In a more specific embodiment, one or more polynucleotides encode a SERPINB9 polypeptide, and the SERPINB9 polypeptide has at least 94% sequence identity with respect to the sequence of SEQ ID NO: 5. In a more specific embodiment, one or more polynucleotides encode a SERPINB9 polypeptide, and the SERPINB9 polypeptide has at least 95% sequence identity with respect to the sequence of SEQ ID NO: 5. In a more specific embodiment, one or more polynucleotides encode a SERPINB9 polypeptide, and the SERPINB9 polypeptide has at least 96% sequence identity with respect to the sequence of SEQ ID NO: 5. In a more specific embodiment, one or more polynucleotides encode a SERPINB9 polypeptide, and the SERPINB9 polypeptide has at least 97% sequence identity with respect to the sequence of SEQ ID NO: 5. In a more specific embodiment, one or more polynucleotides encode a SERPINB9 polypeptide, and the SERPINB9 polypeptide has at least 98% sequence identity with respect to the sequence of SEQ ID NO: 5. In a more specific embodiment, one or more polynucleotides encode a SERPINB9 polypeptide, and the SERPINB9 polypeptide has at least 99% sequence identity to the sequence of SEQ ID NO: 5. In a more specific embodiment, one or more polynucleotides encode a SERPINB9 polypeptide, and the SERPINB9 polypeptide has at least 99.5% sequence identity to the sequence of SEQ ID NO: 5. In a more specific embodiment, one or more polynucleotides encode a SERPINB9 polypeptide, and the SERPINB9 polypeptide has at least 99.9% sequence identity to the sequence of SEQ ID NO: 5. In a more specific embodiment, one or more polynucleotides encode a SERPINB9 polypeptide, and the SERPINB9 polypeptide has at least 99.99% sequence identity to the sequence of SEQ ID NO: 5. In a more specific embodiment, one or more polynucleotides encode a SERPINB9 polypeptide, and the SERPINB9 polypeptide has 100% sequence identity to the sequence of SEQ ID NO: 5.In a more specific embodiment, one or more polynucleotides encode a SERPINB9 polypeptide, the SERPINB9 polypeptide containing a sequence having 100% sequence identity with respect to the sequence of SEQ ID NO: 5. In a particular specific embodiment, the encoded SERPINB9 polypeptide has the sequence of SEQ ID NO: 5.

[0087] MHC Protein: The major histocompatibility complex (MHC) is a large locus containing a series of closely linked polymorphic genes that encode cell surface proteins (MHC molecules) essential to the adaptive immune system. The MHC gene family is divided into three subgroups: MHC class I, MHC class II, and MHC class III. Of all the genes present in the MHC, there are two types of genes that encode MHC class I and MHC class II molecules, which are proteins directly involved in antigen presentation.

[0088] MHC class I molecules: MHC class I molecules are a class of major histocompatibility complex (MHC) molecules found on the cell surface of nucleated cells. Their function is to display peptide fragments of intracellular proteins to cytotoxic T cells (CTLs). Because MHC class I molecules present peptides derived from cytosolic proteins, the MHC class I presentation pathway is often called the cytosolic or endogenous pathway. Alternatively, class I MHC itself can serve as inhibitory ligands for natural killer (NK) cells. A decrease in normal levels of surface class I MHC is a mechanism used by some viruses and certain tumors to evade the CTL response and is known to activate NK cell killing. MHC-I molecules include HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, HLA-K, and HLA-L.

[0089] MHC class II molecules: MHC class II molecules are a class of MHC molecules that are typically found only on specialized antigen-presenting cells such as dendritic cells, mononuclear phagocytes, some endothelial cells, thymic epithelial cells, and B cells. These cells are important in initiating the immune response. The antigens presented by class II molecules are derived from extracellular proteins. Class II molecules are primarily found on T helper cells (CD4). + They interact with immune cells such as HLA-DM, HLA-DO, HLA-DP, HLA-DQ, and HLA-DR. The presented peptides (antigens) control how T cells respond to infection. Stable peptide bonds are essential to prevent peptide dissociation and degradation, which can occur without strong attachment to MHC molecules. This would prevent T cell recognition of the antigen, T cell recruitment, and an appropriate immune response. MHC-II molecules include HLA-DM, HLA-DO, HLA-DP, HLA-DQ, and HLA-DR.

[0090] MHC class III molecules: MHC class III is another class of MHC molecules. Unlike other MHC types, MHC class III molecules are not well understood structurally or functionally. MHC class III molecules do not appear to be involved in antigen binding, and only a few seem to be involved in immunology. However, others appear to be signaling molecules involved in cell communication.

[0091] As disclosed herein, in one embodiment, one or more polynucleotides encode one or more MHC molecules (polypeptides) selected from the group consisting of MHC class I, MHC class II, and MHC class III polypeptides, and combinations thereof. In another embodiment, one or more polynucleotides encode one or more MHC class I polypeptides. In yet another embodiment, one or more polynucleotides encode one or more MHC class I polypeptides selected from the group consisting of HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, HLA-K, HLA-L, and combinations thereof. In yet another embodiment, one or more polynucleotides encode one or more MHC class II polypeptides. In yet another embodiment, one or more polynucleotides encode one or more MHC class II polypeptides selected from the group consisting of HLA-DM, HLA-DO, HLA-DP, HLA-DQ, HLA-DR, and combinations thereof. In yet another embodiment, one or more polynucleotides encode one or more MHC class III polypeptides. In yet another embodiment, one or more polynucleotides encode polypeptides of one or more MHC classes selected from the group consisting of HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, HLA-K, HLA-L, HLA-DM, HLA-DO, HLA-DP, HLA-DQ, HLA-DR, and combinations thereof. In yet another embodiment, one or more polynucleotides encode polypeptide variants of one or more MHC classes selected from the group consisting of HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, HLA-K, HLA-L, HLA-DM, HLA-DO, HLA-DP, HLA-DQ, HLA-DR, and combinations thereof.

[0092] In a more specific embodiment, one or more polynucleotides encode an MHC class I polypeptide HLA-E. In yet another specific embodiment, one or more polynucleotides encode an HLA-E polypeptide variant. In yet another specific embodiment, one or more polynucleotides encode a fragment of an HLA-E polypeptide that may have NK cell inhibitory activity.

[0093] In a more specific embodiment, one or more polynucleotides encode the MHC class I polypeptide HLA-E*0103. In another specific embodiment, one or more polynucleotides encode an HLA-E*0103 polypeptide variant. In yet another specific embodiment, one or more polynucleotides encode a fragment of the HLA-E*0103 polypeptide that may have NK cell inhibitory activity.

[0094] In a more specific embodiment, one or more polynucleotides encode an HLA-E*0103 polypeptide, and the one or more polynucleotides have at least 60% sequence identity with respect to the mRNA sequence of SEQ ID NO: 2. In a more specific embodiment, one or more polynucleotides encode an HLA-E*0103 polypeptide, and the one or more polynucleotides have at least 65% sequence identity with respect to the sequence of SEQ ID NO: 2. In a more specific embodiment, one or more polynucleotides encode an HLA-E*0103 polypeptide, and the one or more polynucleotides have at least 70% sequence identity with respect to the sequence of SEQ ID NO: 2. In a more specific embodiment, one or more polynucleotides encode an HLA-E*0103 polypeptide, and the one or more polynucleotides have at least 75% sequence identity with respect to the sequence of SEQ ID NO: 2. In a more specific embodiment, one or more polynucleotides encode an HLA-E*0103 polypeptide, and the one or more polynucleotides have at least 80% sequence identity with respect to the sequence of SEQ ID NO: 2. In a more specific embodiment, one or more polynucleotides encode an HLA-E*0103 polypeptide, and the one or more polynucleotides have at least 85% sequence identity with respect to the sequence of SEQ ID NO: 2. In a more specific embodiment, one or more polynucleotides encode an HLA-E*0103 polypeptide, and the one or more polynucleotides have at least 90% sequence identity with respect to the sequence of SEQ ID NO: 2. In a more specific embodiment, one or more polynucleotides encode an HLA-E*0103 polypeptide, and the one or more polynucleotides have at least 91% sequence identity with respect to the sequence of SEQ ID NO: 2. In a more specific embodiment, one or more polynucleotides encode an HLA-E*0103 polypeptide, and the one or more polynucleotides have at least 92% sequence identity with respect to the sequence of SEQ ID NO: 2.In a more specific embodiment, one or more polynucleotides encode an HLA-E*0103 polypeptide, and the one or more polynucleotides have at least 93% sequence identity with respect to the sequence of SEQ ID NO: 2. In a more specific embodiment, one or more polynucleotides encode an HLA-E*0103 polypeptide, and the one or more polynucleotides have at least 94% sequence identity with respect to the sequence of SEQ ID NO: 2. In a more specific embodiment, one or more polynucleotides encode an HLA-E*0103 polypeptide, and the one or more polynucleotides have at least 95% sequence identity with respect to the sequence of SEQ ID NO: 2. In a more specific embodiment, one or more polynucleotides encode an HLA-E*0103 polypeptide, and the one or more polynucleotides have at least 96% sequence identity with respect to the sequence of SEQ ID NO: 2. In a more specific embodiment, one or more polynucleotides encode an HLA-E*0103 polypeptide, and the one or more polynucleotides have at least 97% sequence identity with respect to the sequence of SEQ ID NO: 2. In a more specific embodiment, one or more polynucleotides encode an HLA-E*0103 polypeptide, and the one or more polynucleotides have at least 98% sequence identity with respect to the sequence of SEQ ID NO: 2. In a more specific embodiment, one or more polynucleotides encode an HLA-E*0103 polypeptide, and the one or more polynucleotides have at least 99% sequence identity with respect to the sequence of SEQ ID NO: 2. In a more specific embodiment, one or more polynucleotides encode an HLA-E*0103 polypeptide, and the one or more polynucleotides have at least 99.5% sequence identity with respect to the sequence of SEQ ID NO: 2. In a more specific embodiment, one or more polynucleotides encode an HLA-E*0103 polypeptide, and the one or more polynucleotides have at least 99.9% sequence identity with respect to the sequence of SEQ ID NO: 2.In a more specific embodiment, one or more polynucleotides encode an HLA-E*0103 polypeptide, and the one or more polynucleotides have at least 99.99% sequence identity with respect to the sequence of SEQ ID NO: 2. In a more specific embodiment, one or more polynucleotides encode an HLA-E*0103 polypeptide, and the one or more polynucleotides have 100% sequence identity with respect to the sequence of SEQ ID NO: 2. In a more specific embodiment, one or more polynucleotides encode an HLA-E*0103 polypeptide, and the one or more polynucleotides contain a sequence that has 100% sequence identity with respect to the sequence of SEQ ID NO: 2. In a particular specific embodiment, one or more polynucleotides encode an HLA-E*0103 polypeptide, and the one or more polynucleotides contain the sequence of SEQ ID NO: 2.

[0095] In a more specific embodiment, one or more polynucleotides encode an HLA-E*0103 polypeptide, and the HLA-E*0103 polypeptide has at least 60% sequence identity with respect to the sequence of SEQ ID NO: 6. In a more specific embodiment, one or more polynucleotides encode an HLA-E*0103 polypeptide, and the HLA-E*0103 polypeptide has at least 65% sequence identity with respect to the sequence of SEQ ID NO: 6. In a more specific embodiment, one or more polynucleotides encode an HLA-E*0103 polypeptide, and the HLA-E*0103 polypeptide has at least 70% sequence identity with respect to the sequence of SEQ ID NO: 6. In a more specific embodiment, one or more polynucleotides encode an HLA-E*0103 polypeptide, and the HLA-E*0103 polypeptide has at least 75% sequence identity with respect to the sequence of SEQ ID NO: 6. In a more specific embodiment, one or more polynucleotides encode an HLA-E*0103 polypeptide, and the HLA-E*0103 polypeptide has at least 80% sequence identity with respect to the sequence of SEQ ID NO: 6. In a more specific embodiment, one or more polynucleotides encode an HLA-E*0103 polypeptide, and the HLA-E*0103 polypeptide has at least 85% sequence identity with respect to the sequence of SEQ ID NO: 6. In a more specific embodiment, one or more polynucleotides encode an HLA-E*0103 polypeptide, and the HLA-E*0103 polypeptide has at least 90% sequence identity with respect to the sequence of SEQ ID NO: 6. In a more specific embodiment, one or more polynucleotides encode an HLA-E*0103 polypeptide, and the HLA-E*0103 polypeptide has at least 91% sequence identity with respect to the sequence of SEQ ID NO: 6. In a more specific embodiment, one or more polynucleotides encode an HLA-E*0103 polypeptide, and the HLA-E*0103 polypeptide has at least 92% sequence identity with respect to the sequence of SEQ ID NO: 6.In a more specific embodiment, one or more polynucleotides encode an HLA-E*0103 polypeptide, and the HLA-E*0103 polypeptide has at least 93% sequence identity with respect to the sequence of SEQ ID NO: 6. In a more specific embodiment, one or more polynucleotides encode an HLA-E*0103 polypeptide, and the HLA-E*0103 polypeptide has at least 94% sequence identity with respect to the sequence of SEQ ID NO: 6. In a more specific embodiment, one or more polynucleotides encode an HLA-E*0103 polypeptide, and the HLA-E*0103 polypeptide has at least 95% sequence identity with respect to the sequence of SEQ ID NO: 6. In a more specific embodiment, one or more polynucleotides encode an HLA-E*0103 polypeptide, and the HLA-E*0103 polypeptide has at least 96% sequence identity with respect to the sequence of SEQ ID NO: 6. In a more specific embodiment, one or more polynucleotides encode an HLA-E*0103 polypeptide, and the HLA-E*0103 polypeptide has at least 97% sequence identity with respect to the sequence of SEQ ID NO: 6. In a more specific embodiment, one or more polynucleotides encode an HLA-E*0103 polypeptide, and the HLA-E*0103 polypeptide has at least 98% sequence identity with respect to the sequence of SEQ ID NO: 6. In a more specific embodiment, one or more polynucleotides encode an HLA-E*0103 polypeptide, and the HLA-E*0103 polypeptide has at least 99% sequence identity with respect to the sequence of SEQ ID NO: 6. In a more specific embodiment, one or more polynucleotides encode an HLA-E*0103 polypeptide, and the HLA-E*0103 polypeptide has at least 99.5% sequence identity with respect to the sequence of SEQ ID NO: 6. In a more specific embodiment, one or more polynucleotides encode an HLA-E*0103 polypeptide, and the HLA-E*0103 polypeptide has at least 99.9% sequence identity with respect to the sequence of SEQ ID NO: 6.In a more specific embodiment, one or more polynucleotides encode an HLA-E*0103 polypeptide, and the HLA-E*0103 polypeptide has at least 99.99% sequence identity with respect to the sequence of SEQ ID NO: 6. In a more specific embodiment, one or more polynucleotides encode an HLA-E*0103 polypeptide, and the HLA-E*0103 polypeptide has 100% sequence identity with respect to the sequence of SEQ ID NO: 6. In a more specific embodiment, one or more polynucleotides encode an HLA-E*0103 polypeptide, and the HLA-E*0103 polypeptide contains a sequence that has 100% sequence identity with respect to the sequence of SEQ ID NO: 6. In a particular specific embodiment, one or more polynucleotides encode an HLA-E*0103 polypeptide, and the encoded HLA-E*0103 polypeptide has the sequence of SEQ ID NO: 6.

[0096] In a more specific embodiment, one or more polynucleotides encode the MHC class I polypeptide HLA-E*0101. In another specific embodiment, one or more polynucleotides encode an HLA-E*0101 polypeptide variant. In yet another specific embodiment, one or more polynucleotides encode a fragment of the HLA-E*0101 polypeptide that may have NK cell inhibitory activity.

[0097] In a more specific embodiment, one or more polynucleotides encode the HLA-E*0101 polypeptide, and the one or more polynucleotides have at least 60% sequence identity with respect to the mRNA sequence of SEQ ID NO: 3. In a more specific embodiment, one or more polynucleotides encode the HLA-E*0101 polypeptide, and the one or more polynucleotides have at least 65% sequence identity with respect to the sequence of SEQ ID NO: 3. In a more specific embodiment, one or more polynucleotides encode the HLA-E*0101 polypeptide, and the one or more polynucleotides have at least 70% sequence identity with respect to the sequence of SEQ ID NO: 3. In a more specific embodiment, one or more polynucleotides encode the HLA-E*0101 polypeptide, and the one or more polynucleotides have at least 75% sequence identity with respect to the sequence of SEQ ID NO: 3. In a more specific embodiment, one or more polynucleotides encode the HLA-E*0101 polypeptide, and the one or more polynucleotides have at least 80% sequence identity with respect to the sequence of SEQ ID NO: 3. In a more specific embodiment, one or more polynucleotides encode the HLA-E*0101 polypeptide, and the one or more polynucleotides have at least 85% sequence identity with respect to the sequence of SEQ ID NO: 3. In a more specific embodiment, one or more polynucleotides encode the HLA-E*0101 polypeptide, and the one or more polynucleotides have at least 90% sequence identity with respect to the sequence of SEQ ID NO: 3. In a more specific embodiment, one or more polynucleotides encode the HLA-E*0101 polypeptide, and the one or more polynucleotides have at least 91% sequence identity with respect to the sequence of SEQ ID NO: 3. In a more specific embodiment, one or more polynucleotides encode the HLA-E*0101 polypeptide, and the one or more polynucleotides have at least 92% sequence identity with respect to the sequence of SEQ ID NO: 3.In a more specific embodiment, one or more polynucleotides encode the HLA-E*0101 polypeptide, and the one or more polynucleotides have at least 93% sequence identity with respect to the sequence of SEQ ID NO: 3. In a more specific embodiment, one or more polynucleotides encode the HLA-E*0101 polypeptide, and the one or more polynucleotides have at least 94% sequence identity with respect to the sequence of SEQ ID NO: 3. In a more specific embodiment, one or more polynucleotides encode the HLA-E*0101 polypeptide, and the one or more polynucleotides have at least 95% sequence identity with respect to the sequence of SEQ ID NO: 3. In a more specific embodiment, one or more polynucleotides encode the HLA-E*0101 polypeptide, and the one or more polynucleotides have at least 96% sequence identity with respect to the sequence of SEQ ID NO: 3. In a more specific embodiment, one or more polynucleotides encode the HLA-E*0101 polypeptide, and the one or more polynucleotides have at least 97% sequence identity with respect to the sequence of SEQ ID NO: 3. In a more specific embodiment, one or more polynucleotides encode the HLA-E*0101 polypeptide, and the one or more polynucleotides have at least 98% sequence identity with respect to the sequence of SEQ ID NO: 3. In a more specific embodiment, one or more polynucleotides encode the HLA-E*0101 polypeptide, and the one or more polynucleotides have at least 99% sequence identity with respect to the sequence of SEQ ID NO: 3. In a more specific embodiment, one or more polynucleotides encode the HLA-E*0101 polypeptide, and the one or more polynucleotides have at least 99.5% sequence identity with respect to the sequence of SEQ ID NO: 3. In a more specific embodiment, one or more polynucleotides encode the HLA-E*0101 polypeptide, and the one or more polynucleotides have at least 99.9% sequence identity with respect to the sequence of SEQ ID NO: 3.In a more specific embodiment, one or more polynucleotides encode the HLA-E*0101 polypeptide, and the one or more polynucleotides have at least 99.99% sequence identity with respect to the sequence of SEQ ID NO: 3. In a more specific embodiment, one or more polynucleotides encode the HLA-E*0101 polypeptide, and the one or more polynucleotides have 100% sequence identity with respect to the sequence of SEQ ID NO: 3. In a more specific embodiment, one or more polynucleotides encode the HLA-E*0101 polypeptide, and the one or more polynucleotides contain a sequence that has 100% sequence identity with respect to the sequence of SEQ ID NO: 3. In a particular specific embodiment, one or more polynucleotides encode the HLA-E*0101 polypeptide, and the one or more polynucleotides contain the sequence of SEQ ID NO: 3.

[0098] In a more specific embodiment, one or more polynucleotides encode an HLA-E*0101 polypeptide, and the HLA-E*0101 polypeptide has at least 60% sequence identity with respect to the sequence of SEQ ID NO: 7. In a more specific embodiment, one or more polynucleotides encode an HLA-E*0101 polypeptide, and the HLA-E*0101 polypeptide has at least 65% sequence identity with respect to the sequence of SEQ ID NO: 7. In a more specific embodiment, one or more polynucleotides encode an HLA-E*0101 polypeptide, and the HLA-E*0101 polypeptide has at least 70% sequence identity with respect to the sequence of SEQ ID NO: 7. In a more specific embodiment, one or more polynucleotides encode an HLA-E*0101 polypeptide, and the HLA-E*0101 polypeptide has at least 75% sequence identity with respect to the sequence of SEQ ID NO: 7. In a more specific embodiment, one or more polynucleotides encode an HLA-E*0101 polypeptide, and the HLA-E*0101 polypeptide has at least 80% sequence identity with respect to the sequence of SEQ ID NO: 7. In a more specific embodiment, one or more polynucleotides encode an HLA-E*0101 polypeptide, and the HLA-E*0101 polypeptide has at least 85% sequence identity with respect to the sequence of SEQ ID NO: 7. In a more specific embodiment, one or more polynucleotides encode an HLA-E*0101 polypeptide, and the HLA-E*0101 polypeptide has at least 90% sequence identity with respect to the sequence of SEQ ID NO: 7. In a more specific embodiment, one or more polynucleotides encode an HLA-E*0101 polypeptide, and the HLA-E*0101 polypeptide has at least 91% sequence identity with respect to the sequence of SEQ ID NO: 7. In a more specific embodiment, one or more polynucleotides encode an HLA-E*0101 polypeptide, and the HLA-E*0101 polypeptide has at least 92% sequence identity with respect to the sequence of SEQ ID NO: 7.In a more specific embodiment, one or more polynucleotides encode an HLA-E*0101 polypeptide, and the HLA-E*0101 polypeptide has at least 93% sequence identity with respect to the sequence of SEQ ID NO: 7. In a more specific embodiment, one or more polynucleotides encode an HLA-E*0101 polypeptide, and the HLA-E*0101 polypeptide has at least 94% sequence identity with respect to the sequence of SEQ ID NO: 7. In a more specific embodiment, one or more polynucleotides encode an HLA-E*0101 polypeptide, and the HLA-E*0101 polypeptide has at least 95% sequence identity with respect to the sequence of SEQ ID NO: 7. In a more specific embodiment, one or more polynucleotides encode an HLA-E*0101 polypeptide, and the HLA-E*0101 polypeptide has at least 96% sequence identity with respect to the sequence of SEQ ID NO: 7. In a more specific embodiment, one or more polynucleotides encode an HLA-E*0101 polypeptide, and the HLA-E*0101 polypeptide has at least 97% sequence identity with respect to the sequence of SEQ ID NO: 7. In a more specific embodiment, one or more polynucleotides encode an HLA-E*0101 polypeptide, and the HLA-E*0101 polypeptide has at least 98% sequence identity with respect to the sequence of SEQ ID NO: 7. In a more specific embodiment, one or more polynucleotides encode an HLA-E*0101 polypeptide, and the HLA-E*0101 polypeptide has at least 99% sequence identity with respect to the sequence of SEQ ID NO: 7. In a more specific embodiment, one or more polynucleotides encode an HLA-E*0101 polypeptide, and the HLA-E*0101 polypeptide has at least 99.5% sequence identity with respect to the sequence of SEQ ID NO: 7. In a more specific embodiment, one or more polynucleotides encode an HLA-E*0101 polypeptide, and the HLA-E*0101 polypeptide has at least 99.9% sequence identity with respect to the sequence of SEQ ID NO: 7.In a more specific embodiment, one or more polynucleotides encode an HLA-E*0101 polypeptide, and the HLA-E*0101 polypeptide has at least 99.99% sequence identity with respect to the sequence of SEQ ID NO: 7. In a more specific embodiment, one or more polynucleotides encode an HLA-E*0101 polypeptide, and the HLA-E*0101 polypeptide has 100% sequence identity with respect to the sequence of SEQ ID NO: 7. In a more specific embodiment, one or more polynucleotides encode an HLA-E*0101 polypeptide, and the HLA-E*0101 polypeptide contains a sequence that has 100% sequence identity with respect to the sequence of SEQ ID NO: 7. In a particular specific embodiment, one or more polynucleotides encode an HLA-E*0101 polypeptide, and the encoded HLA-E*0101 polypeptide has the sequence of SEQ ID NO: 7.

[0099] Lectin polypeptide: Lectins and type C lectins are a heterogeneous group of proteins with various distinct functions. These include two type C lectins found in the natural killer gene complex (NKC): killer cell lectin-like receptor subfamily B, member 1 (also known as KLRB1 or CD161), and type C lectin domain family 2 member D (CLEC2D). LLT1 proteins are particularly closely related to this disclosure in their role in the regulation of NK and B cells (Llibre et al. 2016. Multi-functional lectin-like transcript-1: A new player in human immune regulation. Immunol Lett. 177:62-69). Non-exclusive examples of lectins and type C lectin polypeptides include CD161, Clec2d8 (mouse), Clec2d11 (rat), and CLEC2d (human).

[0100] As disclosed herein, in one embodiment, one or more polynucleotides encode one or more lectin polypeptides. In another embodiment, one or more polynucleotides encode one or more lectin polypeptides selected from the group consisting of Clec2d8 (mouse), Clec2d11 (rat), CLEC2D (human), and combinations thereof.

[0101] In a more specific embodiment, one or more polynucleotides encode one or more lectin polypeptides selected from the group consisting of Clec2d8 (mouse), Clec2d11 (rat), CLEC2D (human), and combinations thereof.

[0102] In a more specific embodiment, one or more polynucleotides encode the lectin polypeptide CLEC2D. In yet another specific embodiment, one or more polynucleotides encode a CLEC2D polypeptide variant. In yet another specific embodiment, one or more polynucleotides encode a fragment of the CLEC2D polypeptide that may have NK cell inhibitory activity.

[0103] In a more specific embodiment, one or more polynucleotides encode a CLEC2D polypeptide, and the one or more polynucleotides have at least 60% sequence identity with respect to the mRNA sequence of SEQ ID NO: 4. In a more specific embodiment, one or more polynucleotides encode a CLEC2D polypeptide, and the one or more polynucleotides have at least 65% sequence identity with respect to the sequence of SEQ ID NO: 4. In a more specific embodiment, one or more polynucleotides encode a CLEC2D polypeptide, and the one or more polynucleotides have at least 70% sequence identity with respect to the sequence of SEQ ID NO: 4. In a more specific embodiment, one or more polynucleotides encode a CLEC2D polypeptide, and the one or more polynucleotides have at least 75% sequence identity with respect to the sequence of SEQ ID NO: 4. In a more specific embodiment, one or more polynucleotides encode a CLEC2D polypeptide, and the one or more polynucleotides have at least 80% sequence identity with respect to the sequence of SEQ ID NO: 4. In a more specific embodiment, one or more polynucleotides encode a CLEC2D polypeptide, and the one or more polynucleotides have at least 85% sequence identity with respect to the sequence of SEQ ID NO: 4. In a more specific embodiment, one or more polynucleotides encode a CLEC2D polypeptide, and the one or more polynucleotides have at least 90% sequence identity with respect to the sequence of SEQ ID NO: 4. In a more specific embodiment, the one or more polynucleotides encode a CLEC2D polypeptide, and the one or more polynucleotides have at least 91% sequence identity with respect to the sequence of SEQ ID NO: 4. In a more specific embodiment, one or more polynucleotides encode a CLEC2D polypeptide, and the one or more polynucleotides have at least 92% sequence identity with respect to the sequence of SEQ ID NO: 4. In a more specific embodiment, one or more polynucleotides encode a CLEC2D polypeptide, and the one or more polynucleotides have at least 93% sequence identity with respect to the sequence of SEQ ID NO: 4.In a more specific embodiment, one or more polynucleotides encode a CLEC2D polypeptide, and the one or more polynucleotides have at least 94% sequence identity with respect to the sequence of SEQ ID NO: 4. In a more specific embodiment, one or more polynucleotides encode a CLEC2D polypeptide, and the one or more polynucleotides have at least 95% sequence identity with respect to the sequence of SEQ ID NO: 4. In a more specific embodiment, one or more polynucleotides encode a CLEC2D polypeptide, and the one or more polynucleotides have at least 96% sequence identity with respect to the sequence of SEQ ID NO: 4. In a more specific embodiment, one or more polynucleotides encode a CLEC2D polypeptide, and the one or more polynucleotides have at least 97% sequence identity with respect to the sequence of SEQ ID NO: 4. In a more specific embodiment, one or more polynucleotides encode a CLEC2D polypeptide, and the one or more polynucleotides have at least 98% sequence identity with respect to the sequence of SEQ ID NO: 4. In a more specific embodiment, one or more polynucleotides encode a CLEC2D polypeptide, and the one or more polynucleotides have at least 99% sequence identity with respect to the sequence of SEQ ID NO: 4. In a more specific embodiment, one or more polynucleotides encode a CLEC2D polypeptide, and the one or more polynucleotides have at least 99.5% sequence identity with respect to the sequence of SEQ ID NO: 4. In a more specific embodiment, one or more polynucleotides encode a CLEC2D polypeptide, and the one or more polynucleotides have at least 99.9% sequence identity with respect to the sequence of SEQ ID NO: 4. In a more specific embodiment, one or more polynucleotides encode a CLEC2D polypeptide, and the one or more polynucleotides have at least 99.99% sequence identity with respect to the sequence of SEQ ID NO: 4. In a more specific embodiment, one or more polynucleotides encode a CLEC2D polypeptide, and the one or more polynucleotides have 100% sequence identity with respect to the sequence of SEQ ID NO: 4.In a more specific embodiment, one or more polynucleotides encode a CLEC2D polypeptide, and the one or more polynucleotides contain a sequence that has 100% sequence identity with respect to the sequence of SEQ ID NO: 4. In a particular specific embodiment, one or more polynucleotides encode a CLEC2D polypeptide, and the one or more polynucleotides contain the sequence of SEQ ID NO: 4.

[0104] In a more specific embodiment, one or more polynucleotides encode a CLEC2D polypeptide, and the CLEC2D polypeptide has at least 60% sequence identity with respect to the sequence of SEQ ID NO: 8. In a more specific embodiment, one or more polynucleotides encode a CLEC2D polypeptide, and the CLEC2D polypeptide has at least 65% sequence identity with respect to the sequence of SEQ ID NO: 8. In a more specific embodiment, one or more polynucleotides encode a CLEC2D polypeptide, and the CLEC2D polypeptide has at least 70% sequence identity with respect to the sequence of SEQ ID NO: 8. In a more specific embodiment, one or more polynucleotides encode a CLEC2D polypeptide, and the CLEC2D polypeptide has at least 75% sequence identity with respect to the sequence of SEQ ID NO: 8. In a more specific embodiment, one or more polynucleotides encode a CLEC2D polypeptide, and the CLEC2D polypeptide has at least 80% sequence identity with respect to the sequence of SEQ ID NO: 8. In a more specific embodiment, one or more polynucleotides encode a CLEC2D polypeptide, and the CLEC2D polypeptide has at least 85% sequence identity with respect to the sequence of SEQ ID NO: 8. In a more specific embodiment, one or more polynucleotides encode a CLEC2D polypeptide, and the CLEC2D polypeptide has at least 90% sequence identity with respect to the sequence of SEQ ID NO: 8. In a more specific embodiment, one or more polynucleotides encode a CLEC2D polypeptide, and the CLEC2D polypeptide has at least 91% sequence identity with respect to the sequence of SEQ ID NO: 8. In a more specific embodiment, one or more polynucleotides encode a CLEC2D polypeptide, and the CLEC2D polypeptide has at least 92% sequence identity with respect to the sequence of SEQ ID NO: 8. In a more specific embodiment, one or more polynucleotides encode a CLEC2D polypeptide, and the CLEC2D polypeptide has at least 93% sequence identity with respect to the sequence of SEQ ID NO: 8.In a more specific embodiment, one or more polynucleotides encode a CLEC2D polypeptide, and the CLEC2D polypeptide has at least 94% sequence identity with respect to the sequence of SEQ ID NO: 8. In a more specific embodiment, one or more polynucleotides encode a CLEC2D polypeptide, and the CLEC2D polypeptide has at least 95% sequence identity with respect to the sequence of SEQ ID NO: 8. In a more specific embodiment, one or more polynucleotides encode a CLEC2D polypeptide, and the CLEC2D polypeptide has at least 96% sequence identity with respect to the sequence of SEQ ID NO: 8. In a more specific embodiment, one or more polynucleotides encode a CLEC2D polypeptide, and the CLEC2D polypeptide has at least 97% sequence identity with respect to the sequence of SEQ ID NO: 8. In a more specific embodiment, one or more polynucleotides encode a CLEC2D polypeptide, and the CLEC2D polypeptide has at least 98% sequence identity with respect to the sequence of SEQ ID NO: 8. In a more specific embodiment, one or more polynucleotides encode a CLEC2D polypeptide, and the CLEC2D polypeptide has at least 99% sequence identity to the sequence of SEQ ID NO: 8. In a more specific embodiment, one or more polynucleotides encode a CLEC2D polypeptide, and the CLEC2D polypeptide has at least 99.5% sequence identity to the sequence of SEQ ID NO: 8. In a more specific embodiment, one or more polynucleotides encode a CLEC2D polypeptide, and the CLEC2D polypeptide has at least 99.9% sequence identity to the sequence of SEQ ID NO: 8. In a more specific embodiment, one or more polynucleotides encode a CLEC2D polypeptide, and the CLEC2D polypeptide has at least 99.99% sequence identity to the sequence of SEQ ID NO: 8. In a more specific embodiment, one or more polynucleotides encode a CLEC2D polypeptide, and the CLEC2D polypeptide has 100% sequence identity to the sequence of SEQ ID NO: 8.In a more specific embodiment, one or more polynucleotides encode a CLEC2D polypeptide, the CLEC2D polypeptide containing a sequence having 100% sequence identity with respect to the sequence of SEQ ID NO: 8. In a particular specific embodiment, one or more polynucleotides encode a CLEC2D polypeptide, the encoded CLEC2D polypeptide having the sequence of SEQ ID NO: 8.

[0105] Cadherin polypeptide: Calcium-dependent adhesion molecules, or cadherins, are important cell adhesion molecules that form adhesion junctions that allow cells to adhere to one another. Cadherins are a class of type I transmembrane proteins that are calcium (Ca 2+ They are ion-dependent. The cadherin superfamily is essential for maintaining cell-cell contact and regulating cytoskeletal complexes, and includes cadherins, protocadherins, and desmosome cadherins.

[0106] Classical cadherins: Generally, classical cadherins play a role in cell lamination and structure formation, while desmosome cadherins focus on resistance to cell damage. Classical cadherins include CDH1 (e-cadherin), CDH2, CDH3, and CDH12.

[0107] Desmosome cadherins: There are two types of desmosome cadherins, desmogleins and desmocolins, both of which play a role in maintaining desmosome function by mitigating mechanical stress on tissues. Desmogleins include DSG1, DSG2, DSG3, and DSG4. Desmocolins include DSC1, DSC2, DSC3, and DSC4.

[0108] Protocadherin: Protocadherins are the largest mammalian subgroup of the cadherin superfamily of alloaffinity cell adhesion proteins, and have been shown to mediate cell-cell adhesion, particularly in nervous system cells. Protocadherins include PCDH1, PCDH7, PCDH8, PCDH9, PCDH10, PCDH11X / 11Y, PCDH12, PCDH15, PCDH17, PCDH18, PCDH19, PCDH20, PCDHA1, PCDHA2, PCDHA3, PCDHA4, PCDHA5, PCDHA6, PCDHA7, PCDHA8, PCDHA9, PCDHA10, PCDHA11, PCDHA12, PCDHA13, PCDHAC1, PCDHAC2, PCDHB1, PCDHB2, PCDHB3, PCDHB4, PCDHB5, PCDHB6, PCDHB7, PCDHB8, PCDHB9, P This includes CDHB10, PCDHB11, PCDHB12, PCDHB13, PCDHB14, PCDHB15, PCDHB16, PCDHB17, PCDHB18, PCDHGA1, PCDHGA2, PCDHGA3, PCDHGA4, PCDHGA5, PCDHGA6, PCDHGA7, PCDHGA8, PCDHGA9, PCDHGA10, PCDHGA11, PCDHGA12, PCDHGB1, PCDHGB2, PCDHGB3, PCDHGB4, PCDHGB5, PCDHGB6, PCDHGB7, PCDHGC3, PCDHGC4, PCDHGC5, FAT, FAT2, and FAT4.

[0109] Ungrouped cadherins: The following are ungrouped cadherins, i.e., cadherins that do not belong to any of the aforementioned cadherin subgroups (i.e., classical cadherins, desmosome cadherins, or protocadherins). Ungrouped cadherins include CDH4, CDH5, CDH6, CDH7, CDH8, CDH9, CDH10, CDH11, CDH13, CDH15, CDH16, CDH17, CDH18, CDH19, CDH20, CDH23, CDH22, CDH24, CDH26, CDH28, CELSR1, CELSR2, CELSR3, CLSTN1, CLSTN2, CLSTN3, DCHS1, DCHS2, LOC389118, PCLKC, RESDA1, and RET.

[0110] As disclosed herein, in one embodiment, one or more polynucleotides encode one or more cadherin polypeptides. In another embodiment, one or more polynucleotides encode one or more cadherin polypeptides selected from the group consisting of classical cadherins, desmosome cadherins, protocadherins, ungrouped cadherins, and combinations thereof.

[0111] In another embodiment, one or more polynucleotides encode one or more classical polypeptides selected from the group consisting of CDH1 (e-cadherin), CDH2, CDH3, CDH12, and combinations thereof.

[0112] In yet another embodiment, one or more polynucleotides encode one or more desmosome cadherin polypeptides selected from the group consisting of desmogleins, desmocholines, and combinations thereof.

[0113] In yet another embodiment, one or more polynucleotides encode one or more desmoglein cadherin polypeptides selected from the group consisting of DSG1, DSG2, DSG3, DSG4, and combinations thereof.

[0114] In yet another embodiment, one or more polynucleotides encode one or more desmocholine cadherin polypeptides selected from the group consisting of DSC1, DSC2, DSC3, DSC4, and combinations thereof.

[0115] In another embodiment, one or more polynucleotides are PCDH1, PCDH7, PCDH8, PCDH9, PCDH10, PCDH11X / 11Y, PCDH12, PCDH15, PCDH17, PCDH18, PCDH19, PCDH20, PCDHA1, PCDHA2, PCDHA3, PCDHA4, PCDHA5, PCDHA6, PCDHA7, PCDHA8, PCDHA9, PCDHA10, PCDHA11, PCDHA12, PCDHA13, PCDHAC1, PCDHAC2, PCDHB1, PCDHB2, PCDHB3, PCDHB4, PCDHB5, PCDHB6, PCDHB7, PCDHB8, PCDHB9, PCDHB10, PCDHB1 Encodes one or more protocadherin polypeptides selected from the group consisting of 1, PCDHB12, PCDHB13, PCDHB14, PCDHB15, PCDHB16, PCDHB17, PCDHB18, PCDHGA1, PCDHGA2, PCDHGA3, PCDHGA4, PCDHGA5, PCDHGA6, PCDHGA7, PCDHGA8, PCDHGA9, PCDHGA10, PCDHGA11, PCDHGA12, PCDHGB1, PCDHGB2, PCDHGB3, PCDHGB4, PCDHGB5, PCDHGB6, PCDHGB7, PCDHGC3, PCDHGC4, PCDHGC5, FAT, FAT2, FAT4, and combinations thereof.

[0116] In another embodiment, one or more polynucleotides encode one or more ungrouped cadherin polypeptides selected from the group consisting of CDH4, CDH5, CDH6, CDH7, CDH8, CDH9, CDH10, CDH11, CDH13, CDH15, CDH16, CDH17, CDH18, CDH19, CDH20, CDH23, CDH22, CDH24, CDH26, CDH28, CELSR1, CELSR2, CELSR3, CLSTN1, CLSTN2, CLSTN3, DCHS1, DCHS2, LOC389118, PCLKC, RESDA1, RET, and combinations thereof.

[0117] In another embodiment, one or more polynucleotides are CDH1 (e-cadherin), CDH2, CDH3, CDH12, DSG1, DSG2, DSG3, DSG4, DSC1, DSC2, DSC3, DSC4, PCDH1, PCDH7, PCDH8, PCDH9, PCDH10, PCDH11X / 11Y, PCDH12, PCDH15, PCDH17, PCDH18, PCDH19, PCDH20, PCDHA1, PCDHA2, PCDHA3, PCDHA4, PCDHA5 , PCDHA6, PCDHA7, PCDHA8, PCDHA9, PCDHA10, PCDHA11, PCDHA12, PCDHA13, PCDHAC1, PCDHAC2, PCDHB1, PCDHB2, PCDHB3, PCDHB4, PC DHB5, PCDHB6, PCDHB7, PCDHB8, PCDHB9, PCDHB10, PCDHB11, PCDHB12, PCDHB13, PCDHB14, PCDHB15, PCDHB16, PCDHB17, PCDHB18, PCD HGA1, PCDHGA2, PCDHGA3, PCDHGA4, PCDHGA5, PCDHGA6, PCDHGA7, PCDHGA8, PCDHGA9, PCDHGA10, PCDHGA11, PCDHGA12, PCDHGB1, PCD HGB2, PCDHGB3, PCDHGB4, PCDHGB5, PCDHGB6, PCDHGB7, PCDHGC3, PCDHGC4, PCDHGC5, FAT, FAT2, FAT4, CDH4, CDH5, CDH6, CDH7, CDH8, It encodes one or more cadherin polypeptides selected from the group consisting of CDH9, CDH10, CDH11, CDH13, CDH15, CDH16, CDH17, CDH18, CDH19, CDH20, CDH23, CDH22, CDH24, CDH26, CDH28, CELSR1, CELSR2, CELSR3, CLSTN1, CLSTN2, CLSTN3, DCHS1, DCHS2, LOC389118, PCLKC, RESDA1, RET, and combinations thereof.

[0118] In another embodiment, one or more polynucleotides are CDH1 (e-cadherin), CDH2, CDH3, CDH12, DSG1, DSG2, DSG3, DSG4, DSC1, DSC2, DSC3, DSC4, PCDH1, PCDH7, PCDH8, PCDH9, PCDH10, PCDH11X / 11Y, PCDH12, PCDH15, PCDH17, PCDH18, PCDH19, PCDH20, PCDHA1, PCDHA2, PCDHA3, PCDHA4, PCDHA5, PCDHA6, PCDHA7, PCDHA8, PCDHA9, PCDHA10, PCDHA11, PCDHA12, PCDHA13, PCDHAC1, PCDHAC2, PCDHB1, PCDHB2, PCDHB3, PCDHB4, PCDH B5, PCDHB6, PCDHB7, PCDHB8, PCDHB9, PCDHB10, PCDHB11, PCDHB12, PCDHB13, PCDHB14, PCDHB15, PCDHB16, PCDHB17, PCDHB18, PCDHGA 1, PCDHGA2, PCDHGA3, PCDHGA4, PCDHGA5, PCDHGA6, PCDHGA7, PCDHGA8, PCDHGA9, PCDHGA10, PCDHGA11, PCDHGA12, PCDHGB1, PCDHGB2 , PCDHGB3, PCDHGB4, PCDHGB5, PCDHGB6, PCDHGB7, PCDHGC3, PCDHGC4, PCDHGC5, FAT, FAT2, FAT4, CDH4, CDH5, CDH6, CDH7, CDH8, CDH9 It encodes one or more cadherin polypeptide variants selected from the group consisting of CDH10, CDH11, CDH13, CDH15, CDH16, CDH17, CDH18, CDH19, CDH20, CDH23, CDH22, CDH24, CDH26, CDH28, CELSR1, CELSR2, CELSR3, CLSTN1, CLSTN2, CLSTN3, DCHS1, DCHS2, LOC389118, PCLKC, RESDA1, RET, and combinations thereof.

[0119] In a more specific embodiment, one or more polynucleotides encode a CDH1(e-cadherin) polypeptide. In another specific embodiment, one or more polynucleotides encode a variant CDH1(e-cadherin) polypeptide.

[0120] CD155 (poliovirus receptor or "PVR"): CD155 (differentiation antigen group 155) is a protein encoded by the CDR gene in humans, and is known as the poliovirus receptor because it is a molecule used by the poliovirus to invade cells. The CD155 gene is specific to primate lineages and encodes a type I transmembrane glycoprotein in the immunoglobulin superfamily. Its normal cellular function is to establish intercellular adhesion junctions between epithelial cells. The role of CD155 in the immune system is unclear, but it may be involved in the enteric humoral immune response. Subsequent data also suggest that CD155 may be used to actively select MHC-independent T cells in the thymus.

[0121] In specific embodiments provided herein, one or more polynucleotides encode a CD155 (poliovirus receptor) polypeptide. In more specific embodiments, one or more polynucleotides encode a variant CD155 (poliovirus receptor) polypeptide. In yet another specific embodiment, one or more polynucleotides encode a fragment of a CD155 (poliovirus receptor) polypeptide that may have NK cell inhibitory activity.

[0122] Nogo receptor 1 (also known as NgR1): The Nogo-66 receptor (NgR) or Nogo receptor 1 (NgR1) is encoded in humans by the RTN4R gene, and NgR1 plays a role in inhibiting axonal growth and, in some cases, regulating axonal regeneration and plasticity in the central nervous system. NgR1 is involved in neural plasticity and regeneration. Closely related to this disclosure is the recent discovery that NgR1 can inhibit natural killer cell activity. Recently, it has been demonstrated that NgR1 plays a role in inhibiting natural killer cell-mediated death by destabilizing immunological synapse formation through binding to its ligand NogoA (RTN4 in humans) on target cells (Oh et al., 2023. NgR1 is an NK cell inhibitory receptor that destabilizes the immunological synapse. Nature Immunology 24:463-473).

[0123] In specific embodiments provided herein, one or more polynucleotides encode an RTN4 polypeptide. In more specific embodiments, one or more polynucleotides encode a variant RTN4 polypeptide. In yet another specific embodiment, one or more polynucleotides encode a fragment of an RTN4 polypeptide that may have NK cell inhibitory activity.

[0124] Antigens and proteins that are normally produced endogenously: As stated above, the second polypeptide (e.g., one or more second polypeptides) encodes a polypeptide that can induce an immune response (e.g., an antigen) or a protein normally produced endogenously (e.g., a polynucleotide that can be administered to a subject with a genetic mutation resulting in a deficiency and / or insufficient amount or function of a particular protein normally produced endogenously) (Dolgin, E., The tangled history of mRNA vaccines. Nature, 2021. 597(7876): p.318-324).

[0125] The polypeptides (such as antigens) that can induce an immune response are not particularly limited. The following examples illustrate embodiments relating to a vaccine against coronavirus-19 (for example, in which the second polynucleotide would encode a COVID-19 antigen), but the disclosure also includes embodiments in which the second polypeptide encodes a polypeptide that can induce an immune response against different pathogens or tumor cells. Non-limiting examples include vaccines against influenza, coronavirus, Clostridium difficile, HIV, malaria, norovirus, etc. In each case, the second polypeptide (e.g., one or more second polypeptides) encodes a polypeptide that can induce an immune response against a target, such as an influenza antigen, coronavirus antigen, Clostridium difficile antigen, HIV antigen, malaria antigen, norovirus antigen, etc.

[0126] Proteins that are normally endogenously produced are not particularly limited. As described above, proteins that are normally endogenously produced may be any proteins that are insufficiently produced in a target subject (patient) due to insufficient amounts of protein or genetic mutations resulting in functionally deficient variants of the protein. A non-limiting example is factor VIII, which can be used to treat hemophilia. Thus, in specific embodiments, the second polynucleotide encodes factor VIII or a biologically active fragment thereof.

[0127] Cell hosts expressing the disclosed polynucleotides and polynucleotide constructs In another embodiment, this disclosure envisions a host cell expressing a polynucleotide and / or polynucleotide construct disclosed herein. In certain embodiments, a cell host would be genetically engineered to express a polynucleotide and / or polynucleotide construct. Using methods well known to those skilled in the art, an expression vector containing the polypeptide of interest or the coding sequence of a polypeptide, as well as any requested and required transcription / translation regulatory sequences and / or signals, can be constructed. These methods include, but are not limited to, in vitro recombinant DNA techniques, synthetic techniques, and in vivo recombinant / genetic engineering. See, for example, the techniques described in Maniatis et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, NY, and Ausubel et al., 1989, Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley Interscience, NY.

[0128] The host cell may be any cell useful for the production of polypeptides and / or polypeptide constructs disclosed herein, such as a prokaryotic cell or a eukaryotic cell. More specifically, the host cell may be, but is not limited to, a microbial cell, a fungal cell, an insect cell, a mammalian cell, and / or a plant cell.

[0129] Microbial expression hosts may be preferred due to their ease of use and the wide range of technological platforms readily available for these organisms. In certain embodiments, the prokaryotic host cell may be any Gram-positive or Gram-negative bacterium. Gram-positive bacteria include, but are not limited to, Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, and Streptomyces. Gram-negative bacteria include, but are not limited to, Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter, Ilyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

[0130] The bacterial host cells may include, but are not limited to, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, and / or Bacillus thuringiensis cells, and may be any Bacillus cells.

[0131] The bacterial host cells may also include, but are not limited to, any Streptococcus cells, including Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, and / or Streptococcus equi subsp. Zooepidemicus cells.

[0132] The bacterial host cells may also include, but are not limited to, Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, and / or Streptomyces lividans cells, and may be any Streptomyces cells.

[0133] In another embodiment, the host cell may also be a eukaryote such as a fungus, insect, plant, and / or mammalian cell.

[0134] In more specific embodiments, the host cell may be a fungal cell. As used herein, “fungus” includes the phyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota, as well as the phylum Oomycota, and all imperfect fungi (mitosporic fungi) (as defined by Hawksworth et al., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CAB International, University Press, Cambridge, UK).

[0135] The fungal host cell may be a yeast cell. As used herein, “yeast” includes ascospore-forming yeasts (endomycetares), basidiospore-forming yeasts, and yeasts belonging to the imperfect fungi (Blastomycetes). Because the classification of yeast may change in the future, for the purposes of this disclosure, yeast shall be defined as described in Biology and Activities of Yeast (Skinner, Passmore, and Davenport, editors, Soc. App. Bacteriol. Symposium Series No. 9, 1980).

[0136] The yeast host cells may be Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia cells, such as Kluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomyces oviformis, and / or Yarrowia lipolytica cells.

[0137] The fungal host cell may be a filamentous fungal cell. "Filamentous fungi" include all filamentous forms of the Eumycota and Oomycota subphyla. Filamentous fungal host cells include, but are not limited to, Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, and / or Trichoderma cells.

[0138] For example, filamentous fungal host cells include Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporium zonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola insolens, Humicoestertiiosa, Mucormiehei, Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum, Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii, Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, and / or Trichoderma viride cells.

[0139] In another embodiment, the host cell may be an insect cell.

[0140] In more specific embodiments, the host cells may include, but are not limited to, cells derived from leipodoptera, hymenoptera, dipterans, and / or beetles.

[0141] Insect host cells include, but are not limited to, cells derived from insects such as Bombyx, Drosophila, Spodoptera, and / or Trichiplusia.

[0142] For example, insect host cells include Bombyx horsfieldi, Bombyx huttoni, Bombyx incomposita, Bombyx lemeepauli, Bombyx mandarina, Bombyx mori, Bombyx rotundapex, Bombyx shini, Drosophila ananassae, Drosophila erecta, Drosophila grimshawi, Drosophila mauritiana, Drosophila melanogaster, Drosophila mojavensis, Drosophila persimilis, Drosophila pseudoobscura, Drosophila sechellia, Drosophila simulans, Drosophila virilis, Drosophila willistoni, Drosophila yakuba, Spodopte estertiinia, Spodopte esterula, Spodoptera androgea, Spodopte estertiata, Spodoptera apertura, Spodoptera cilium, Spodoptera compta, Spodoptera connexa, Spodoptera depravata, Spodoptera dolichos, Spodoptera eridania, Spodoptera evanida, Spodoptera excelsa, Spodoptera exempta, Spodoptera exigua, Spodoptera fasciculata, Spodoptera frugiperda, Spodoptera hipparis, Spodoptera latifascia, Spodoptera littoralis, Spodoptera litura, Spodopte estertiasy, Spodoptera marima, Spodopte estertitia, Spodoptera ochrea, Spodoptera ornithogalli, Spodoptera pecten, Spodoptera pectinicornis, Spodopteraperuviana, Spodoptera picta, Spodoptera praefica, Spodoptera pulchella, Spodoptera roseae, Spodoptera estertiiuna, Spodoptera teferii, Spodoptera triturate, Spodoptera umbraculata, Trichoplusia arachnoides, Trichoplus esternea, Trichoplusia callista, Trichoplusia cinnabarina, Trichoplusia cupreomicans, Trichoplusia elacheia, Trichoplusia epicharis, Trichoplusia glyceia, Trichoplusia gromieri, Trichoplusia lectula, Trichoplusia lampra, Trichoplusia ni, Trichoplusia obtusisigna, Trichoplusia orichalcea, Trichoplusia photeina, Trichoplusia roseoatafasci, Trichoplus estertiitia, Trichoplusia These may be sogai, Trichoplusia telaugea, and / or Trichoplusia tetrastigma cells.

[0143] In specific embodiments, the host cell may be Bombyx mori (i.e., silkworm), Drosophila melanogaster (i.e., fruit fly), Spodoptera frugiperda (i.e., armyworm), Trichoplusia ni (i.e., cabbage looper), and / or Aedes sp (i.e., any species of mosquito, more specifically Aedes aegypti or Aedes aegypti).

[0144] In more specific embodiments, the host cells may be larvae of Bombyx mori (i.e., silkworms), Drosophila melanogaster (i.e., fruit flies), Spodoptera frugiperda (i.e., armyworms), Trichoplusia ni (i.e., cabbage inchworms), and / or Aedes sp. (i.e., any species of mosquito, more specifically Aedes aegypti or Aedes aegypti).

[0145] In more specific embodiments, the host cells are those derived from moths (ATCC CCL 80), armyworms (ATCC CRL 1711), mosquito larvae (ATCC strains CCL 125, CCL 126, CRL 1660, CRL 1591, CRL 6585, CRL 6586), and silkworms (ATCC CRL 8851). In particularly preferred embodiments, the cell line is a Drosophila cell line, e.g., Schneider 2 cell line (see, e.g., Schneider, 1972, J. Embryol. Exp. Morph., Vol. 27, pp. 353-365), a Spodoptera cell line, e.g., Sf9 cells, Sf21 cells, or expressSF + or cell lines derived from Trichoplusia, such as Tn5 cells, H5 cells, and High-Five® (Invitrogen) cells.

[0146] Insect cells expressing the polypeptides disclosed herein can be constructed by methods known in the art (Lindskog, et al., Biopharmaceutical Processing, Elsevier, 2018, Pages 111-130, ISBN 9780081006238, doi:10.1016 / B978-0-08-100623-8.00006-2).

[0147] In specific embodiments, mammalian cells are hosts for the production of polypeptides encoded from the polynucleotide constructs described herein, due to their ability to glycosylate proteins in a form most suitable for human application.

[0148] In specific embodiments, the mammalian host cells are human cells and / or non-human cells.

[0149] In a more specific embodiment, the host cell may be a hamster cell, a mouse cell, or a human cell.

[0150] In more specific embodiments, the host cells are Chinese hamster cells (CHO) cells, baby hamster kidney cells (BHK) cells, and / or mouse myeloma cells (e.g., NS0, SP2 / 0, etc.).

[0151] The host intended herein also includes a plant, e.g., a transgenic plant, plant part, or plant cell, containing the polynucleotides of this disclosure, to express and produce the polypeptides disclosed herein in recoverable amounts. The transgenic plant may be a dicotyledonous or monocotyledonous plant. Examples of monocotyledonous plants include, but are not limited to, grasses, e.g., pasture grass (Poa), fodder grasses, e.g., Festuca, Lolium, cool-season grasses, e.g., Agrostis, and cereals, e.g., wheat, oats, rye, barley, rice, sorghum, and maize (corn).

[0152] Examples of dicotyledonous plants include, but are not limited to, tobacco, legumes such as lupine, potato, sugar beet, pea, bean, and soybean, and cruciferous plants such as cauliflower, rapeseed, and the closely related model organism Arabidopsis thaliana.

[0153] Examples of plant parts include stems, calluses, leaves, roots, fruits, seeds, and tubers, as well as individual tissues containing these parts, such as epidermis, mesophyll, parenchyma, fibrous tissue, and meristem. Certain plant cell compartments, such as chloroplasts, apoplasts, mitochondria, vacuoles, peroxisomes, and cytoplasm, are also considered plant parts. Furthermore, any plant cell, regardless of its tissue origin, is considered a plant part. Similarly, plant parts such as certain tissues and cells isolated to produce polypeptides disclosed herein are also considered plant parts, such as embryo sacs, endosperm, alleurons, and seed coats.

[0154] The scope of this disclosure also includes such plants, plant parts, and offspring of plant cells.

[0155] Transgenic plants or plant cells expressing the polypeptides disclosed herein can be constructed by methods known in the art. In short, the plants or plant cells are constructed by incorporating one or more expression constructs encoding hybrid polypeptides into a plant host genome or chloroplast genome, and then growing the resulting modified plants or plant cells into transgenic plants or plant cells.

[0156] Useful methods for measuring the expression of polypeptides described herein are well known in the art (e.g., Southern blotting (DNA detection), dot or slot blotting (DNA, RNA), Northern blotting (RNA), and RT-PCR (RNA) analysis). Other useful methods include processes well known in the art (e.g., Western blotting (protein detection) to determine the translational state of a cell by measuring the abundance of constituent protein species present in the cell).

[0157] Composition: In another aspect, the present disclosure envisions a composition comprising one or more polynucleotide sequences described herein and a carrier. The compositions described herein may be prepared by acceptable pharmaceutical procedures as described in Remington's Pharmaceutical Sciences, 17th edition, ed. Alfonoso R. Gennaro, Mack Publishing Company, Easton, Pa. (1985), etc.

[0158] In one embodiment, this disclosure, (a) one or more of the polynucleotide constructs described herein, (b) Describe a composition comprising a pharmaceutically acceptable carrier.

[0159] In another embodiment, this disclosure is, (a) one or more of the polynucleotide constructs described herein, (b) One or more polynucleotides described herein (for example, one polynucleotide, two polynucleotides, three polynucleotides, four polynucleotides, five polynucleotides, six polynucleotides, seven polynucleotides, eight polynucleotides, nine polynucleotides, ten polynucleotides, etc.) (c) A composition comprising a pharmaceutically acceptable carrier is described.

[0160] In another embodiment, the present disclosure relates to a composition comprising one or more polynucleotides (for example, one polynucleotide, two polynucleotides, three polynucleotides, four polynucleotides, five polynucleotides, six polynucleotides, seven polynucleotides, eight polynucleotides, nine polynucleotides, ten polynucleotides, etc.), (a) A first polynucleotide encoding a polypeptide that can have NK cell inhibitory activity, (b) A second polynucleotide encoding a polypeptide that can induce an immune response, or a protein that is normally produced endogenously, (c) A pharmaceutically acceptable carrier, The composition comprising the above is described.

[0161] It should be understood that the ratio of the first polynucleotide to the second polynucleotide can be any ratio that is effective in achieving the desired effect (e.g., enhancing the persistence of one or more exogenously delivered polynucleotides, or reducing the undesirable side effects of one or more exogenously delivered polynucleotides). For example, the molar ratio of the first polynucleotide to the second polynucleotide can be approximately 100:1 to 1:100 (for example, the molar ratio of the first polynucleotide to the second polynucleotide can be 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, 1:5, 1:5.5, 1:6, 1:6.5, 1:7, 1:7.5, 1:8, 1:8.5, 1:9, 1:9.5, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1:75, 1:80, 1:85, 1:9). The molar ratio of the second polynucleotide to the first polynucleotide may be 0, 1:95, or 1:100, or the molar ratio of the second polynucleotide to the first polynucleotide may be 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, 1:5, 1:5.5, 1:6, 1:6.5, 1:7, 1:7.5, 1:8, 1:8.5, 1:9, 1:9.5, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1:75, 1:80, 1:85, 1:90, 1:95, or 1:100). Additionally or alternatively, the weight ratio of the first polynucleotide to the second polynucleotide may be approximately 100:1 to 1:100.And approximately 1:100 (for example, the weight ratio of the first polynucleotide to the second polynucleotide is 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, 1:5, 1:5.5, 1:6, 1:6.5, 1:7, 1:7.5, 1:8, 1:8.5, 1:9, 1:9.5, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1:75, 1:80, 1:85, 1:90, 1:95, or from 1:100) (The weight ratio of the second polynucleotide to the first polynucleotide may be 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, 1:5, 1:5.5, 1:6, 1:6.5, 1:7, 1:7.5, 1:8, 1:8.5, 1:9, 1:9.5, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1:75, 1:80, 1:85, 1:90, 1:95, or 1:100.)

[0162] In certain embodiments, the molar ratio of the first polypeptide to the second polypeptide is 1:1 or approximately 1:1.

[0163] In certain embodiments, the molar ratio of the first polypeptide to the second polypeptide is 1:100 or approximately 1:100.

[0164] In certain embodiments, the molar ratio of a first polypeptide capable of having NK cell inhibitory activity to a second polypeptide encoding an antigen is 1:1 or approximately 1:1.

[0165] In certain embodiments, the molar ratio of a first polypeptide capable of having NK cell inhibitory activity to a second polypeptide encoding an antigen is 1:100 or approximately 1:100.

[0166] In certain embodiments, the molar ratio of a first polypeptide capable of having NK cell inhibitory activity to a second polypeptide encoding a normally endogenously produced protein is 1:1 or approximately 1:1.

[0167] In certain embodiments, the molar ratio of a first polypeptide capable of having NK cell inhibitory activity to a second polypeptide encoding a normally endogenously produced protein is 1:100 or approximately 1:100.

[0168] Carrier: A carrier (i.e., a pharmaceutically acceptable carrier) typically has properties (e.g., viscosity, yield value, shear stress, shear rate, etc.) that, once formulated, allow the composition disclosed herein to remain effective (e.g., enhance the persistence of the vaccine, reduce undesirable side effects of the vaccine, etc.). The carrier is typically compatible with the polynucleotides described herein and, optionally, may stabilize them. One or more solubilizers may be used as a suitable carrier for the polynucleotides described herein, or may be present in the carrier. Examples of acceptable carriers include, but are not limited to, biocompatible vehicles, adjuvants, additives, and diluents for achieving compositions usable as dosage forms for the intended route of administration. Other examples of carriers include, but are not limited to, colloidal silicon dioxide, magnesium stearate, cellulose, and sodium lauryl sulfate. Additional suitable pharmaceutically acceptable carriers and diluents, and their pharmaceutically necessary use, are described in Remington's Pharmaceutical Sciences.

[0169] In specific embodiments, the carrier is a solid carrier. In more specific embodiments, suitable solid carriers include, but are not limited to, calcium phosphate, magnesium stearate, talc, sugars, lactose, dextrin, starch, gelatin, cellulose, methylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidine, low-melting-point waxes, ion exchange resins, and combinations thereof.

[0170] The solid carriers described herein may further contain, but are not limited to, one or more substances including, fragrances, lubricants, solubilizers, suspending agents, fillers, flow promoters, compression aids, binders or tablet disintegrants, or encapsulating materials.

[0171] In specific embodiments, the carrier is a liquid carrier. In more specific embodiments, the liquid carrier is water, an organic solvent, an oil or fat, and a combination thereof. In even more specific embodiments, preferred liquid carriers include water, alcohols (e.g., monohydric alcohols and polyhydric alcohols, e.g., glycols) and their derivatives, sugar alcohols (e.g., erythritol, arabitol, ribitol, isomalt, polyglycitol, maltitol, dulciitol, iditol, mannitol, xylitol, lactitol, sorbitol, etc.), and oils and fats (e.g., short-chain triglycerides, medium-chain triglycerides). Glycerides, long-chain triglycerides, saturated, monounsaturated, and / or polyunsaturated oils, soybean oil, coconut oil, canola oil, safflower oil, olive oil, corn oil, cottonseed oil, linseed oil, safflower oil, coconut oil, peanut oil, linseed oil, sunflower oil, rice bran oil, sesame oil, rapeseed oil, cocoa butter, almond oil, cashew oil, hazelnut oil, macadamia oil, mongongo nut oil, pecan oil, pine nut oil, pistachio oil, sacha inchi oil, walnut oil, gourd oil Buffalo pumpkin oil, butternut squash seed oil, pumpkin seed oil, watermelon seed oil, acai oil, blackcurrant seed oil, borage seed oil, evening primrose oil, carob pod oil, amaranth oil, apricot oil, apricot kernel oil, apple seed oil, argan oil, artichoke oil, avocado oil, babassu oil, bean oil, borneo butter, cape chestnut oil, cassia oil, cocoa butter, cockle oil, burdock oil, coriander seed oil, dika oil, grape seed oil Oils, hemp oil, kapok seed oil, kenaf seed oil, larle manzia oil, marula oil, meadowfoam seed oil, mustard oil, nutmeg butter, okra seed oil, papaya seed oil, perilla seed oil, pequi oil, poppy oil, prune kernel oil, quinoa oil, rum till oil, royle oil, tea seed oil, thistle oil, tiger nut oil, tomato seed oil, wheat germ oil, radish oil, salicorn oil, kiri oil, algae oil, copaiba oil, honge oil, jatropha oil, petroleum nut oil, WL1349 Oil, Silicone Oil, Mineral Oil, Lauroyl Macrogol-6 Glyceride, Lauroyl Polyoxyl-6 Glyceride, Oleoyl Macrogol-6 Glyceride, Oleoyl Polyoxyl-6 Glyceride, Linoleyl Macrogol-6 Glyceride, Linoleyl Polyoxyl-6 Glyceride, Propylene Glycol Monocaprylate, Propylene Glycol Monolaurate, Propylene Glycol Monolaurate, Polyglyceryl-3 Dioleate, Propylene Glycol Dicaprylocate, Diethylene Glycol Monotyl Ether, Caprylocaproyl Macrogol-8 Glyceride, Caprylocaproyl Polyoxyl-8 Glyceride, Bergamot, Caddisi, Chamomile, Caraway, Carnauba, Castor This includes, but is not limited to, cinnamon, cod liver, coffee, emu, eucalyptus, fish, geraniol, hyssop, jojoba, kukui nut, lavandin, lavender, lemon, litsea cubeba, mallow, mango seed, mink, orange, orange roughy, palm kernel, peach kernel, rosemary, sandalwood, camellia, mint, sea buckthorn, rare butter, tea tree, camellia, vetiver, butyl stearate, caprylic triglyceride, capric triglyceride, cyclomethicone, diethyl sebacate, dimethicone 360, isopropyl myristate, octyldodecanol, oleyl alcohol, etc., oily esters (e.g., ethyl oleate, etc.), and combinations thereof.

[0172] The liquid carriers described herein may further contain one or more suitable pharmaceutically acceptable excipients, such as solubilizers, emulsifiers, buffers, preservatives, sweeteners, flavorings, suspending agents, thickeners, pigments, viscosity modifiers, stabilizers, osmotic pressure modifiers, surfactants, and combinations thereof.

[0173] In certain embodiments, the carrier comprises lipid particles. In certain specific embodiments, the polynucleotide constructs and / or polynucleotides described herein are assumed to be formulated into lipid particles. In more specific embodiments, the polynucleotides and / or polynucleotide constructs described herein may be completely encapsulated within a lipid particle carrier. Those skilled in the field of formulation will understand how to formulate lipid particles for compositions such as those disclosed herein (Musielak et al., Synthesis and Potential Applications of Lipid Nanoparticles in Medicine. Materials (Basel). 2022 Jan 17;15(2):682. doi:10.3390 / ma15020682).

[0174] In certain embodiments, the lipid particles used in the compositions described herein are formed from lipids selected from the group consisting of cationic lipids, non-cationic lipids, and combinations thereof.

[0175] In one embodiment, lipid particles are formed from cationic lipids. Suitable cationic lipids include NN-dioleyl-N,N-dimethylammonium chloride ("DODAC"), N-(2,3-dioleyloxy)propyl-N,NN-triethylammonium chloride ("DOTMA"), N,N-distearyl-N,N-dimethylammonium bromide ("DDAB"), N-(2,3-dioleyloxy)propyl-N,N,N-trimethylammonium chloride ("DOTAP"), 1,2-dioleyloxy-3-trimethylaminopropane chloride salt ("DOTAP.C1"), 313-(N-(N',N'-dimethylaminoethane)-carbamoyl)cholesterol ("DC-Chol"), and N-(1-(2,3-dioleyl) This includes, but is not limited to, xy)propioni-1)-N-2-(sperminecarboxamide)ethyl)-N,N-dimethylammonium trifluoroacetic acid ("DOSPA"), dioctadecylamide glycyl carboxyspermine ("DOGS"), 1,2-dioleoyl-sn-3-phosphoethanolamine ("DOPE"), 1,2-dioleoyl-1-3-dimethylammonium propane ("DODAP"), N,N-dimethi-1-2,3-dioleyloxy)propylamine ("DODMA"), and N-(1,2-dimyristyloxypropane-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide ("DMRIE"), and combinations thereof. In addition, several commercially available preparations of cationic lipids may be used. Non-exclusive examples include LIPOFECTIN (including DOTMA and DOPE, available from GIBCO / BRL) and LIPOFECTAMINE (including DOSPA and DOPE, available from GIBCO / BRL).

[0176] In one embodiment, lipid particles are formed from noncationic lipids. Suitable noncationic lipids include lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, cephalin, cardiolipin, phosphatidic acid, cerebroside, dicetyl phosphate, distearoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylcholine (DOPC), dipalmitoyl phosphatidylcholine (DPPC), dioleoyl phosphatidylglycerol (DOPG), dipalmitoyl phosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyl oleoyl This includes, but is not limited to, phosphophosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), and dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), 16-O-monomethylPE, 16-O-dimethylPE, 18-1-transPE, and 1-stearoyl-2-oleoyl-phosphatidiethanolamine (SOPE).

[0177] The amount of carrier present in a composition as disclosed herein, as well as the formulation, the amount and type of polynucleotide being formulated, the intended route of administration, etc., may vary. In some embodiments, the compositions disclosed herein may be in amounts of about 0.01% to 99.99% by mass (e.g., 0.01% to 99.99%, 0.1% to 99.99%, 1.0% to 99.99%, 5.0% to 99.99%, 10.0% to 99.99%, 15.0% to 99.99%, 20.0% to 99.99%, 25.0% to 99.99%, 30.0% to 99.99%, 35.0% to 99.99%, 40.0%) Amount % ~ 99.99 mass %, 45.0 mass % ~ 99.99 mass %, 50.0 mass % ~ 99.99 mass %, 55.0 mass % ~ 99.99 mass %, 60.0 mass % ~ 99.99 mass %, 65.0 mass % ~ 99.99 mass %, 70.0 mass % ~ 99.9 9 mass%, 75.0 mass% to 99.99 mass%, 80.0 mass% to 99.99 mass%, 85.0 mass% to 99.99 mass%, 90.0 mass% to 99.99 mass%, 91.0 mass% to 99.99 mass%, 92.0 mass% to 99.99 mass%, 93 .0 mass% to 99.99 mass%, 94.0 mass% to 99.99 mass%, 95.0 mass% to 99.99 mass%, 96.0 mass% to 99.99 mass%, 97.0 mass% to 99.99 mass%, 98.0 mass% to 99.99 mass%, 99.0 mass% to 9 9.99 mass%, 99.10 mass% to 99.99 mass%, 99.20 mass% to 99.99 mass%, 99.30 mass% to 99.99 mass%, 99.40 mass% to 99.99 mass%, 99.50 mass% to 99.99 mass%, 99.60 mass% to 99 .99 mass%, 99.70 mass% to 99.99 mass%, 99.80 mass% to 99.99 mass%, 99.90 mass% to 99.99 mass%, 99.91 mass% to 99.99 mass%, 99.92 mass% to 99.99 mass%, 99.93 mass% to 99. 99% by mass, 99.94% by mass to 99.99% by mass, 99.95% by mass to 99.99% by mass, 99.96% by mass to 99.99% by mass, 99.97% by mass to 99.99% by mass, 99.98% by mass to 99.99% by mass).

[0178] method: This disclosure also relates to methods for enhancing the persistence of one or more exogenously delivered polynucleotides, and to methods for reducing one or more undesirable side effects associated with the administration of exogenously delivered polynucleotides (e.g., polynucleotides encoding antigens or proteins normally produced endogenously). This disclosure also relates to methods for enhancing one or both of the magnitude and persistence of an immune response induced by a vaccine, e.g., a vaccine comprising one or more exogenously delivered polynucleotides (e.g., an mRNA vaccine), e.g., one or both of the magnitude and / or persistence of an antibody response and T-cell response to a vaccine antigen that is exogenously present in or encoded in the vaccine polynucleotide (e.g., an antibody response and T-cell response to an antigen encoded by the mRNA of an mRNA vaccine). Additionally or alternatively, the methods disclosed herein may reduce one or more undesirable side effects associated with the administration of a vaccine, such as a vaccine comprising one or more exogenously delivered polynucleotides (e.g., an mRNA vaccine).

[0179] As used herein, “enhancing the persistence” of an exogenously delivered polynucleotide includes increasing the duration of time the polynucleotide is expressed and / or the duration of time the encoded polypeptide exhibits its intended biological effect, which includes enhancing the magnitude and / or duration of the immune response induced by a vaccine antigen encoded by the polynucleotide, or enhancing the magnitude and / or duration of the intended biological effect of a normally endogenously produced protein encoded by the polynucleotide. As used herein, “enhancing the persistence of a vaccine” or “enhancing the persistence of a vaccine-induced immune response” includes increasing the duration of time the vaccine exhibits its intended biological effect, which includes the magnitude and duration of the intended immune response to the vaccine.

[0180] The methods disclosed herein can be applied to any animal subject. In certain embodiments, the subject is a mammal. In more specific embodiments, the subject is a human. In other specific embodiments, the subject is a non-human subject. Non-human subjects include, but are not limited to, animals found in zoos and in captivity for research purposes, non-human primates (e.g., prosimians (e.g., lemurs), monkeys (e.g., Old World monkeys, e.g., baboons, macaques, and rhesus monkeys; New World monkeys, e.g., marmosets, capuchin monkeys, howler monkeys, and squirrel monkeys; and great apes, e.g., gibbons, orangutans, chimpanzees, and gorillas)), mice, rats, ferrets, hamsters, gerbils, guinea pigs, rabbits, pigs, raccoons, possums, opossums, skunks, bovine animals (e.g., cattle, bulls, buffalo), horses, zebras, deer, sheep, goats, birds, cats, dogs, foxes, coyotes, wolves, bats, etc.).

[0181] Certain embodiments include a method for enhancing the persistence of one or more exogenously delivered polynucleotides (e.g., polynucleotides encoding antigens that can induce an immune response, or polynucleotides encoding normally endogenously produced proteins administered to subjects having genetic mutations resulting in protein deficiency and / or insufficient quantity or function), comprising administering one or more polynucleotides encoding polypeptides that can have NK cell inhibitory activity in combination with a vaccine or normally endogenously produced protein, in combination with a polynucleotide encoding a vaccine antigen or a normally endogenously produced protein. In particular embodiments relating to vaccines, the methods described herein may be effective in increasing one or both of the magnitude and persistence of the immune response induced by one or more exogenous polynucleotides of a vaccine (e.g., an mRNA vaccine), for example, the magnitude and / or persistence of one or both of the antibody response and T cell response to one or more vaccine antigens encoded by one or more exogenous polynucleotides of the vaccine (e.g., vaccine antigens encoded by mRNA in an mRNA vaccine), for example, the magnitude and / or persistence of one or both of the antibody response and T cell response to vaccine antigens. Another specific embodiment envisions a method for reducing one or more undesirable side effects associated with the administration of one or more exogenously delivered polynucleotides, comprising administering one or more polynucleotides encoding polypeptides that can have NK cell inhibitory activity in combination with a vaccine or a protein that is normally produced endogenously, including in combination with a polynucleotide encoding a vaccine antigen or a protein that is normally produced endogenously.

[0182] Other aspects of the present disclosure also include methods for enhancing the persistence of one or more exogenously delivered polynucleotides, comprising administering to a subject (e.g., a patient) at least one of the compositions described herein (e.g., a composition comprising one or more NK cell inhibitors that may have the NK cell inhibitory activity described herein and a polynucleotide construct and / or polynucleotide encoding a vaccine antigen or a protein normally produced endogenously). In particular aspects, such methods may be effective in increasing the magnitude and / or persistence of one or both of the immune response induced by a vaccine antigen, for example, the magnitude and / or persistence of one or both of the antibody response and the T cell response to the vaccine antigen.

[0183] therefore, (a) A step of administering a pharmaceutically effective amount of the first polynucleotide described herein, which may have NK cell inhibitory activity, (b) The step of administering a pharmaceutically effective amount of one or more vaccines to a target, Inhibition of NK cell activity enhances the magnitude and / or duration of the immune response induced by one or more vaccines administered to a subject, and / or reduces one or more undesirable side effects associated with vaccine administration. Methods are provided for enhancing either the magnitude or duration of the immune response induced by the vaccine, or for reducing one or more undesirable side effects associated with vaccine administration.

[0184] In specific embodiments, the method enhances one or both of the antibody response and the T-cell response of the vaccine to the vaccine antigen.

[0185] The vaccine may be a polynucleotide vaccine comprising a polynucleotide encoding a vaccine antigen as described herein, and optionally, the polynucleotide is an mRNA molecule. The first polynucleotide and the polynucleotide encoding the vaccine antigen may be present in a single polynucleotide construct as described herein. The first polynucleotide and the polynucleotide encoding the vaccine antigen may be present in separate polynucleotide constructs formulated in a single composition. The first polynucleotide and the polynucleotide encoding the vaccine antigen may be present in separate polynucleotide constructs formulated in separate compositions.

[0186] In another specific embodiment, a method for enhancing the persistence of one or more exogenously delivered polynucleotides, (a) A step of administering a pharmaceutically effective amount of a pharmaceutical composition to a subject, comprising a first polynucleotide encoding one or more natural killer (NK cell) inhibitors capable of having NK cell inhibitory activity, (b) The step of administering a pharmaceutically effective amount of a pharmaceutical composition comprising one or more second exogenous polynucleotides (e.g., a polynucleotide encoding an antigen that can induce an immune response or a polynucleotide encoding a protein that is normally endogenously produced) to a subject, The present invention provides a method in which inhibition of NK cell activity enhances the persistence of one or more exogenous polynucleotides administered to a target.

[0187] The first polynucleotide may be administered in the same composition as the second exogenous polynucleotide, or the first polynucleotide and the second exogenous polynucleotide may be administered simultaneously or sequentially in separate compositions, as will be discussed in more detail below.

[0188] Further aspects of the present disclosure include a method for reducing one or more undesirable adverse events associated with the administration of one or more exogenously delivered polynucleotides to a subject, the method further comprising administering to a subject at least one of the compositions described herein (e.g., a composition comprising polynucleotide constructs and / or polynucleotides encoding one or more NK cell inhibitors that may have the NK cell inhibitory activity described herein) in combination with a vaccine antigen or a protein that is normally produced endogenously, the combination with a vaccine antigen or a protein that is normally produced endogenously.

[0189] In another specific embodiment, a method for reducing one or more undesirable side effects associated with the administration of one or more exogenously delivered polynucleotides, (a) A step of administering a pharmaceutically effective amount of a pharmaceutical composition comprising one or more polynucleotides encoding natural killer (NK cell) inhibitors capable of having NK cell inhibitory activity to a subject, (b) The step of administering a pharmaceutically effective amount of a pharmaceutical composition to a subject, comprising one or more exogenous polynucleotides (for example, a polynucleotide encoding an antigen that can induce an immune response or a polynucleotide encoding a protein that is normally produced endogenously), The method is provided, wherein inhibition of NK cell activity reduces one or more undesirable side effects associated with the administration of one or more exogenous polynucleotides.

[0190] The first polynucleotide may be administered in the same composition as the second exogenous polynucleotide, or the first polynucleotide and the second exogenous polynucleotide may be administered simultaneously or sequentially in separate compositions, as will be discussed in more detail below.

[0191] The compositions described herein, comprising polynucleotides encoding one or more NK cell inhibitors capable of having NK cell inhibitory activity, are intended to be administered before, after, or concurrently with the administration of one or more exogenous polynucleotides.

[0192] In a more specific embodiment, the method for enhancing one or both of the magnitude and duration of the immune response induced by a vaccine is the method, which involves administering a pharmaceutically effective amount of any of the compositions described herein to a subject in conjunction with the administration of the vaccine.

[0193] In a specific embodiment, the method for enhancing the persistence of a vaccine is as follows: (a) A step of administering a pharmaceutically effective amount of a pharmaceutical composition comprising one or more polynucleotides encoding natural killer (NK cell) inhibitors capable of having NK cell inhibitory activity to a subject, (b) The step of administering a pharmaceutically effective amount of one or more vaccines to a target, The method involves inhibiting NK cell activity to enhance one or both of the magnitude and duration of the immune response induced by one or more vaccines administered to a target.

[0194] In specific embodiments, this method enhances one or both of the antibody response and the T-cell response to the vaccine antigen of the vaccine.

[0195] In particular, in specific embodiments, the vaccine is a polynucleotide vaccine (for example, an RNA vaccine such as an mRNA vaccine).

[0196] In another, more specific embodiment, the method for reducing one or more side effects associated with the administration of a vaccine to a subject comprises administering a pharmaceutically effective amount of any of the compositions described herein to the subject.

[0197] In another specific embodiment, a method for reducing one or more undesirable side effects associated with vaccine administration, (a) A step of administering a pharmaceutically effective amount of a pharmaceutical composition comprising one or more polynucleotides encoding natural killer (NK cell) inhibitors capable of having NK cell inhibitory activity to a subject, (b) The step of administering a pharmaceutically effective amount of one or more vaccines to a target, The method involves inhibiting NK cell activity to reduce one or more undesirable side effects associated with vaccine administration.

[0198] In particular, in specific embodiments, the vaccine is a polynucleotide vaccine (for example, an RNA vaccine such as an mRNA vaccine).

[0199] The compositions described herein, comprising polynucleotides encoding one or more NK cell inhibitors capable of having NK cell inhibitory activity, are intended to be administered before, after, or concurrently with the administration of one or more vaccines.

[0200] In certain embodiments, compositions described herein comprising polynucleotide constructs and / or polynucleotides encoding one or more NK cell inhibitors capable of having the NK cell inhibitory activity described herein are administered to a subject prior to the administration of one or more vaccines (or one or more polynucleotides encoding polypeptides capable of inducing an immune response, or one or more polynucleotides encoding polypeptides normally produced endogenously). Such embodiments may be advantageous for depleting NK cells or NK cell activity before vaccination (or before administration of one or more polynucleotides encoding polypeptides capable of inducing an immune response, or one or more polynucleotides encoding polypeptides normally produced endogenously).

[0201] In another embodiment, the compositions described herein are administered to a subject simultaneously with (i.e., “concurrently administered)) one or more vaccines. Concurrent administration means that the compositions described herein, comprising one or more NK cell inhibitors capable of having NK cell inhibitory activity and one or more polynucleotides encoding vaccines, are administered at substantially the same time (e.g., simultaneously, as practically as possible) or at exactly the same time.

[0202] In another specific embodiment, a composition described herein comprising a polynucleotide encoding one or more NK cell inhibitors that may have NK cell inhibitory activity is administered to a subject after the subject has been administered one or more vaccines.

[0203] It should be understood that any of the method steps disclosed herein (i.e., administering one or more polynucleotide constructs and / or compositions comprising one or more polynucleotides that can have NK cell inhibitory activity as described herein, before, after, or concurrently with the administration of one or more vaccines) may be repeated as many times as necessary to achieve the desired effect of enhancing the persistence of one or more vaccines and / or reducing one or more undesirable side effects associated with the administration of the vaccines.

[0204] Furthermore, it should be understood that all methods described herein are intended to allow the delivery of one or more polynucleotide constructs and / or compositions comprising one or more polynucleotides, which may have NK cell inhibitory activity as described herein, to be administered to a subject by any of the administration methods generally known in the art. Non-limiting examples include delivery of the compositions herein by oral (PO), intravenous (IV), intramuscular (IM), intraarterial, intramedullary, subarachnoid, subcutaneous (SQ), intraventricular, percutaneous, intradermal, endothelial, rectal (PR), vaginal, intraperitoneal (IP), intragastric (IG), topical and / or percutaneous (e.g., by lotion, cream, powder, ointment, rub, gel, infusion, etc.), mucous membrane, intranasal cavity, oral cavity, enteral, vitreous humor, and / or sublingual administration, by tracheal drip, bronchial drip, and / or inhalation, as oral spray, nasal spray, and / or aerosol, and / or by portal vein catheter, and / or combinations thereof.

[0205] In certain embodiments, one or more polynucleotide constructs and / or compositions comprising one or more polynucleotides that may have the NK cell inhibitory activity described herein are administered to a subject via an intravenous method.

[0206] It is further understood that any of the method steps disclosed herein (i.e., administering one or more polynucleotide constructs and / or compositions comprising one or more polynucleotides that may have the NK cell inhibitory activity described herein) is administered to a subject in an amount effective to achieve a particular outcome (i.e., a therapeutic outcome).

[0207] In certain embodiments, an effective amount of polynucleotide constructs and / or polynucleotides (polynucleotides encoding polypeptides that may have NK cell inhibitory activity, and / or polynucleotides encoding polypeptides that may induce an immune response, or proteins that are normally endogenously produced) contained in the compositions described herein or administered to a subject as described herein is in the range of 0.5 μg to 300 μg and may be administered as a single dose or as two or more divided doses. In some embodiments, an effective amount of a single polynucleotide (encoding either a polypeptide that may have NK cell inhibitory activity, or a polypeptide that may induce an immune response, or a protein that is normally endogenously produced) described herein is 0.5 μg to 150 μg, or about 0.5 μg to 150 μg, or any value in between. In some embodiments, an effective amount of a polynucleotide construct or composition containing two polynucleotides described herein is 0.5 μg to 300 μg, or about 0.5 μg to 300 μg, or any value in between. For example, an effective dose of mRNA vaccine polynucleotide may be approximately 3 μg for children or 30 μg for adults, or 10-25 μg for children or 100 μg for adults.Therefore, the total doses of polynucleotides or polynucleotide constructs described herein are 0.5 μg, 1 μg, 2 μg, 3 μg, 4 μg, 5 μg, 6 μg, 7 μg, 8 μg, 9 μg, 10 μg, 11 μg, 12 μg, 13 μg, 14 μg, 15 μg, 16 μg, 17 μg, 18 μg, 19 μg, 20 μg, 25 μg, 3 μg 0μg, 35μg, 40μg, 45μg, 50μg, 55μg, 60μg, 65μg, 70μg, 75μg, 80μg, 85μg, 90μg, 95μg, 100μg, 110μg, 120μg, 130μg, 140μg, 150μg, 160μg, 170μg, 180μg, 190μg, 200μg, 250μg, or 3 It may be 00 μg, or approximately 0.5 μg, 1 μg, 2 μg, 3 μg, 4 μg, 5 μg, 6 μg, 7 μg, 8 μg, 9 μg, 10 μg, 11 μg, 12 μg, 13 μg, 14 μg, 15 μg, 16 μg, 17 μg, 18 μg, 19 μg, 20 μg, 25 μg, 30 μg, 35 μg, 40 μg, 45 μg, 50 μg, 55 μg, The effective amount may be 60 μg, 65 μg, 70 μg, 75 μg, 80 μg, 85 μg, 90 μg, 95 μg, 100 μg, 110 μg, 120 μg, 130 μg, 140 μg, 150 μg, 160 μg, 170 μg, 180 μg, 190 μg, 200 μg, 250 μg, or 300 μg, or any value in between. If additional polynucleotides, such as three, four, or more polynucleotides, are present (either individually or as part of a construct), the effective amount may be greater than these values, such as proportionally larger based on the number of polynucleotides.

[0208] Embodiment The following embodiments are non-limiting and illustrative of the subject matter of this disclosure. Embodiment 1. An isolated polynucleotide construct comprising (a) a first polynucleotide encoding a polypeptide capable of having natural killer (NK) cell inhibitory activity, and (b) a second polynucleotide encoding a polypeptide capable of inducing an immune response or a protein naturally endogenously produced. Embodiment 2. A composition comprising (a) one or more polynucleotide constructs described in Embodiment (Embodinet) 1, and (b) a pharmaceutically acceptable carrier. Embodiment 3. A composition comprising (a) a first polynucleotide encoding a polypeptide that can have natural killer (NK) cell inhibitory activity; (b) a second polynucleotide encoding a polypeptide that can induce an immune response or a protein that is naturally endogenously produced; and (c) a pharmaceutically acceptable carrier. Embodiment 4. The construct or composition according to any one of Embodiments 1 to 3, wherein the polynucleotide construct or the first and second polynucleotides are mRNA molecules. Embodiment 5. A construct or composition according to any one of the prior embodiments, wherein the first polynucleotide encodes a serine protease inhibitor (serpine) polypeptide or fragment thereof that can have natural killer (NK) cell inhibitory activity. Embodiment 6. The construct or composition according to Embodiment 5, wherein the serpine polypeptide is a serpine polypeptide selected from the group consisting of SERPINA1, SERPINA2, SERPINA3, SERPINA4, SERPINA5, SERPINA6, SERPINA7, SERPINA8, SERPINA9, SERPINA10, SERPINA11, SERPINA12, SERPINA13, SERPINB1, SERPINB2, SERPINB3, SERPINB4, SERPINB5, SERPINB6, SERPINB7, SERPINB8, SERPINB9, SERPINB10, SERPINB11, SERPINB12, SERPINB13, SERPINC1, SERPIND1, SERPINE1, SERPINE2, SERPINE3, SERPINF1, SERPINF2, SERPING1, SERPINH1, SERPINI1, SERPINI2, and combinations thereof. Embodiment 7. The construct or composition according to Embodiment 5 or 6, wherein the serpine polypeptide is SERPINB9. Embodiment 8. The first polynucleotide encodes a serpine polypeptide or a fragment thereof that can have NK cell inhibitory activity, and (a) the first polynucleotide has sequence identity with SEQ ID NO: 1, or at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or at least 99.99% of SEQ ID NO: 1 The construct or composition according to Embodiment 7, comprising a sequence, or (b) encoding a serpine polypeptide comprising a sequence having at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or at least 99.99% sequence identity with respect to Sequence ID No. 5. Embodiment 9. A construct or composition according to any one of Embodiments 1 to 4, wherein the first polynucleotide encodes a polypeptide of the major histocompatibility complex (MHC) class having NK cell inhibitory activity or a fragment thereof. Embodiment 10. The construct or composition according to Embodiment 9, wherein the MHC class polypeptide having NK cell inhibitory activity is selected from the group consisting of MHC class I polypeptides, MHC class II polypeptides, MHC class III polypeptides, and combinations thereof. Embodiment 11. A construct or composition according to Embodiment 9 or 10, wherein the first polynucleotide encodes an MHC class I polypeptide or fragment thereof that can have NK cell inhibitory activity, and optionally the MHC class I polypeptide is selected from the group consisting of HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, HLA-K, HLA-L, and combinations thereof. Embodiment 12. The construct or composition according to Embodiment 11, wherein the MHC class 1 polypeptide is an HLA-E polypeptide. Embodiment 13. The construct or composition according to Embodiment 12, wherein the HLA-E polypeptide is HLA-E*0103. Embodiment 14. The first polynucleotide encodes an HLA-E*0103 polypeptide or a fragment thereof that can have NK cell inhibitory activity, and (a) the first polynucleotide has sequence identity with SEQ ID NO: 2, or at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or at least 99.99% of SEQ ID NO: 2 The construct or composition according to Embodiment 13, comprising a sequence, or (b) encoding an HLA-E*0103 polypeptide comprising the sequence of SEQ ID NO: 6, or a sequence having at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or at least 99.99% sequence identity with respect to SEQ ID NO: 6. Embodiment 15. The construct or composition according to Embodiment 12, wherein the HLA-E polypeptide is HLA E*0101. Embodiment 16. The first polynucleotide encodes an HLA-E*0101 polypeptide or a fragment thereof that can have NK cell inhibitory activity, and (a) the first polynucleotide has sequence identity with SEQ ID NO: 3, or at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or at least 99.99% of SEQ ID NO: 3 The construct or composition according to Embodiment 15, comprising a sequence, or (b) encoding an HLA-E*0101 polypeptide comprising the sequence of SEQ ID NO: 7, or a sequence having at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or at least 99.99% sequence identity with respect to SEQ ID NO: 7. Embodiment 17. The construct or composition according to Embodiment 9 or 10, wherein the first polynucleotide encodes an MHC class II polypeptide or fragment thereof that can have NK cell inhibitory activity, and the MHC class II polypeptide is selected from the group consisting of HLA-DM, HLA-DO, HLA-DP, HLA-DQ, HLA-DR, and combinations thereof. Embodiment 18. A construct or composition according to any one of Embodiments 1 to 4, wherein the first polynucleotide encodes a lectin polypeptide or a fragment thereof that can have NK cell inhibitory activity. Embodiment 19. The construct or composition according to Embodiment 18, wherein the lectin polypeptide is selected from the group consisting of Clec2d8 (mouse), Clec2d11 (rat), CLEC2D (human), and combinations thereof. Embodiment 20. The construct or composition according to Embodiment 18 or 19, wherein the lectin polypeptide is CLEC2D. Embodiment 21. The first polynucleotide encodes CLEC2D or a fragment thereof that can have NK cell inhibitory activity, and (a) the first polynucleotide is the sequence of SEQ ID NO: 4, or a sequence having at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or at least 99.99% sequence identity to SEQ ID NO: 4. The construct or composition according to Embodiment 20, comprising, or (b) encoding a CLEC2D polypeptide comprising the sequence of SEQ ID NO: 8, or a sequence having at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or at least 99.99% sequence identity with respect to SEQ ID NO: 8. Embodiment 22. A construct or composition according to any one of Embodiments 1 to 4, wherein the first polynucleotide encodes a cadherin polypeptide or a fragment thereof that can have NK cell inhibitory activity. Embodiment 23. The construct or composition according to Embodiment 22, wherein the cadherin polypeptide is selected from the group consisting of classical cadherins, desmosome cadherins, protocadherins, ungrouped cadherins, and combinations thereof. Embodiment 24. The construct or composition according to Embodiment 22 or 23, wherein the first polynucleotide encodes a classical cadherin polypeptide or a fragment thereof that can have NK cell inhibitory activity, and the classical cadherin polypeptide is selected from the group consisting of CDH1(e-cadherin), CDH2, CDH3, CDH12, and combinations thereof. Embodiment 25. The construct or composition according to Embodiment 22 or 23, wherein the first polynucleotide encodes a desmosome cadherin polypeptide or a fragment thereof that can have NK cell inhibitory activity, and the desmosome cadherin polypeptide is selected from the group consisting of desmogleins, desmocolins, and combinations thereof. Embodiment 26. The construct or composition according to Embodiment 25, wherein the first polynucleotide encodes a desmoglein polypeptide or a fragment thereof that can have NK cell inhibitory activity, and the desmoglein polypeptide is selected from the group consisting of DSG1, DSG2, DSG3, DSG4, and combinations thereof. Embodiment 27. The construct or composition according to Embodiment 25, wherein the first polynucleotide encodes a desmocolin polypeptide or fragment thereof that can have NK cell inhibitory activity, and the desmocolin polypeptide is selected from the group consisting of DSC1, DSC2, DSC3, DSC4, and combinations thereof. Embodiment 28. The first polynucleotide encodes a protocadherin polypeptide or a fragment thereof that can have NK cell inhibitory activity, and the protocadherin polypeptide is PCDH1, PCDH7, PCDH8, PCDH9, PCDH10, PCDH11X / 11Y, PCDH12, PCDH15, PCDH17, PCDH18, PCDH19, PCDH20, PCDHA1, PCDHA2, PCDHA3, PCDHA4, PCDHA5, PCDHA6, PCDHA7, PCDHA8, PCDHA9, PCDHA10, PCDHA11, PCDHA12, PCDHA13, PCDHAC1, PCDHAC2, PCDHB1, PCDHB2, PCDHB3, PCDHB4, PCDHB5, PCDHB6, PCDHB7, PCDH A structure or composition according to Embodiment 22 or 23, selected from the group consisting of B8, PCDHB9, PCDHB10, PCDHB11, PCDHB12, PCDHB13, PCDHB14, PCDHB15, PCDHB16, PCDHB17, PCDHB18, PCDHGA1, PCDHGA2, PCDHGA3, PCDHGA4, PCDHGA5, PCDHGA6, PCDHGA7, PCDHGA8, PCDHGA9, PCDHGA10, PCDHGA11, PCDHGA12, PCDHGB1, PCDHGB2, PCDHGB3, PCDHGB4, PCDHGB5, PCDHGB6, PCDHGB7, PCDHGC3, PCDHGC4, PCDHGC5, FAT, FAT2, FAT4, and combinations thereof. Embodiment 29. The construct or composition according to Embodiment 22 or 23, wherein the first polynucleotide encodes a non-grouped cadherin polypeptide or fragment thereof that can have NK cell inhibitory activity, and the non-grouped cadherin polypeptide is selected from the group consisting of CDH4, CDH5, CDH6, CDH7, CDH8, CDH9, CDH10, CDH11, CDH13, CDH15, CDH16, CDH17, CDH18, CDH19, CDH20, CDH23, CDH22, CDH24, CDH26, CDH28, CELSR1, CELSR2, CELSR3, CLSTN1, CLSTN2, CLSTN3, DCHS1, DCHS2, LOC389118, PCLKC, RESDA1, RET, and combinations thereof. Embodiment 30. The first polynucleotide encodes a cadherin polypeptide variant or fragment thereof that can have NK cell inhibitory activity, and the cadherin polypeptide is CDH1(e-cadherin), CDH2, CDH3, CDH12, DSG1, DSG2, DSG3, DSG4, DSC1, DSC2, DSC3, DSC4, PCDH1, PCDH7, PCDH8, PCDH9, PCDH10, PCDH11X / 11Y, PCDH12, PCDH15, PCDH17, PCDH18, PCDH19, PCDH20 , PCDHA1, PCDHA2, PCDHA3, PCDHA4, PCDHA5, PCDHA6, PCDHA7, PCDHA8, PCDHA9, PCDHA10, PCDHA11, PCDHA12, PCDHA13, PCDHAC1, PCDHAC2, P CDHB1, PCDHB2, PCDHB3, PCDHB4, PCDHB5, PCDHB6, PCDHB7, PCDHB8, PCDHB9, PCDHB10, PCDHB11, PCDHB12, PCDHB13, PCDHB14, PCDHB15, PCD HB16, PCDHB17, PCDHB18, PCDHGA1, PCDHGA2, PCDHGA3, PCDHGA4, PCDHGA5, PCDHGA6, PCDHGA7, PCDHGA8, PCDHGA9, PCDHGA10, PCDHGA11, PC DHGA12, PCDHGB1, PCDHGB2, PCDHGB3, PCDHGB4, PCDHGB5, PCDHGB6, PCDHGB7, PCDHGC3, PCDHGC4, PCDHGC5, FAT, FAT2, FAT4, CDH4, CDH5, CD A structure or composition according to Embodiment 22, selected from the group consisting of H6, CDH7, CDH8, CDH9, CDH10, CDH11, CDH13, CDH15, CDH16, CDH17, CDH18, CDH19, CDH20, CDH23, CDH22, CDH24, CDH26, CDH28, CELSR1, CELSR2, CELSR3, CLSTN1, CLSTN2, CLSTN3, DCHS1, DCHS2, LOC389118, PCLKC, RESDA1, RET, and combinations thereof. Embodiment 31. A construct or composition according to any one of Embodiments 1 to 4, wherein the first polynucleotide encodes CDH1(e-cadherin) or a fragment thereof that can have NK cell inhibitory activity, or a variant or fragment thereof of CDH1(e-cadherin) that can have NK cell inhibitory activity. Embodiment 32. A construct or composition according to any one of Embodiments 1 to 4, wherein the first polynucleotide encodes CD155 (poliovirus receptor) or a fragment thereof that can have NK cell inhibitory activity, or a variant or fragment thereof of CD155 that can have NK cell inhibitory activity. Embodiment 33. A construct or composition according to any one of Embodiments 1 to 4, wherein the first polynucleotide encodes RTN4 or a fragment thereof that can have NK cell inhibitory activity, or a variant or fragment thereof of RTN4 that can have NK cell inhibitory activity. Embodiment 34. A construct or composition according to any one of the prior embodiments, wherein the second polynucleotide encodes an antigen capable of inducing an immune response. Embodiment 35. The construct or composition according to Embodiment 34, wherein the antigen is a COVID-19 antigen capable of inducing an immune response to COVID-19. Embodiment 36. A construct or composition according to any one of Embodiments 1 to 33, wherein the second polynucleotide encodes a protein that is normally produced endogenously. Embodiment 37. The construct or composition according to Embodiment 36, wherein the protein normally produced endogenously is factor VIII or a biologically active fragment thereof. Embodiment 38. A method for enhancing the persistence of a vaccine or for reducing one or more undesirable side effects associated with vaccine administration, comprising the steps of (a) administering a pharmaceutically effective amount of a first polynucleotide encoding a polypeptide capable of having NK cell inhibitory activity to a subject, and (b) administering a pharmaceutically effective amount of one or more vaccines to a subject, wherein inhibition of NK cell activity enhances the persistence of one or more vaccines administered to a subject and / or reduces one or more undesirable side effects associated with vaccine administration. Embodiment 39. The method according to Embodiment 38, wherein the vaccine is a polynucleotide vaccine comprising a polynucleotide encoding a vaccine antigen, and optionally the polynucleotide is an mRNA molecule. Embodiment 40. The method according to Embodiment 39, wherein the polynucleotide encoding the first polynucleotide and the vaccine antigen is present in a single polynucleotide construct. Embodiment 41. The method according to Embodiment 39, wherein the polynucleotide encoding the first polynucleotide and the vaccine antigen are present in separate polynucleotide constructs formulated in a single composition. Embodiment 42. The method according to Embodiment 40 or 41, wherein a single polynucleotide construct or single composition is a construct or composition according to any one of Embodiments 1 to 35. Embodiment 43. The method according to Embodiment 39, wherein the polynucleotide encoding the first polynucleotide and the vaccine antigen are present in separate polynucleotide constructs formulated in separate compositions. Embodiment 44. A method for enhancing the persistence of an exogenously administered polynucleotide or for reducing one or more undesirable side effects associated with the administration of an exogenously administered polynucleotide, comprising the steps of (a) administering a pharmaceutically effective amount of a first polynucleotide encoding a polypeptide capable of having NK cell inhibitory activity to a target, and (b) administering a second exogenously administered polynucleotide to a target, wherein inhibition of NK cell activity enhances the persistence of the exogenously administered polynucleotide and / or reduces one or more undesirable side effects associated with the administration of an exogenously administered polynucleotide. Embodiment 45. The method according to Embodiment 44, wherein a second exogenously administered polynucleotide encodes an antigen capable of inducing an immune response. Embodiment 46. The method according to Embodiment 45, wherein the antigen is a COVID-19 antigen capable of inducing an immune response to COVID-19. Embodiment 47. The method according to Embodiment 44, wherein the second exogenously administered polynucleotide encodes a protein that is normally produced endogenously. Embodiment 48. The method according to Embodiment 47, wherein the protein normally produced endogenously is factor VIII or a biologically active fragment thereof. Embodiment 49. The method according to any one of Embodiments 38 or 39 or 43-48, wherein step (a) is performed before step (b). Embodiment 50. The method according to any one of Embodiments 38 or 39 or 43-48, wherein step (a) is performed after step (b). Embodiment 51. The method according to any one of Embodiments 38 or 39 or 43-48, wherein step (a) is performed simultaneously with step (b). Embodiment 52. A construct or composition according to any one of Embodiments 1 to 35 for enhancing the persistence of a polynucleotide vaccine or for reducing one or more undesirable side effects associated with the administration of a polynucleotide vaccine. Embodiment 53. A construct or composition according to Embodiment 36 or 37 for enhancing the persistence of an exogenously administered polynucleotide, or for reducing one or more undesirable side effects associated with the administration of an exogenously administered polynucleotide. Embodiment 54. A cell expressing a polynucleotide construct according to any one of Embodiments 1 to 37. [Examples]

[0209] The following embodiments are provided for illustrative purposes only and are not intended to limit the scope of the disclosure as provided herein. Any modifications of the exemplary embodiments that can be imagined by those skilled in the art are intended to fall within the scope of the disclosure.

[0210] Example 1 Materials and methods Study participants: Participants were enrolled in the PASS study, an observational, longitudinal cohort study of healthcare workers (HCWs) evaluating clinical and immunological responses to SARS-CoV-2 infection and vaccination. The PASS study commenced in August 2020, with participants observed monthly during the first year of the study at either the Naval Medical Research Center (NMRC) Clinical Trials Center or the Uniformed Services University (USU) Translational Medicine Unit, and then quarterly during the second year. The study protocol was approved by the USU Institutional Review Board. The cohort consisted of generally healthy adults who were ≥18 years old, working at a WRNMMC, not severely immunocompromised, and seronegative for SARS-CoV-2 at the time of study enrollment (Jackson-Thompson et al., 2021. Prospective Assessment of SARS-CoV-2 Seroconversion (PASS) study: an observational cohort study of SARS-CoV-2 infection and vaccination in healthcare workers. BMC Infect Dis 21:544). The subset of PASS participants included in this study for analysis also met the following criteria (see Figure 1A). 1. No prior medical history of COVID-19 diagnosis before vaccination; 2. Remained seronegative for SARS-CoV-2 spike-specific IgG during monthly testing prior to vaccination; 3. Received two doses of the Pfizer / BioNTech BNT162b2 vaccine; 4. Completed two vaccine-related symptom questionnaires by March 30, 2021; and 5. Serum samples were provided 20-50 days and 150-200 days after the second vaccination.

[0211] Post-vaccination symptom assessment: As shown in Figure 1B, participants completed a structured vaccine-related symptom questionnaire at the first monthly visit after each vaccination dose. The questionnaire asked about the presence and severity of 12 symptoms (8 classified as systemic, 3 classified as localized to the vaccination site, and 1 classified as neither local nor systemic). The severity of each symptom was defined as the intensity of the symptom, measured on a scale of 0 to 4 (0 = "none at all", 1 = "slight", 2 = "moderate", 3 = "considerable", 4 = "severe"), and scores for each symptom were summed to obtain a total symptom severity score ranging from 0 to 48.

[0212] Antibody testing: Binding IgG antibodies against the vaccine strain (D614G) SARS-CoV-2 spike protein and receptor binding domain (RBD) were measured using a microsphere-based multiplex immunoassay (MMIA) constructed using Luminex xMAP-based technology as previously described (Laing et al., 2022).

[0213] Isolation and purification of PBMCs: PBMCs were isolated from PASS participants at baseline and at various time points after COVID-19 vaccination and stored frozen as previously described (Jackson-Thompson et al., 2021).

[0214] Cell preparation and flow cytometry for NK cell phenotyping: Frozen PBMC samples were thawed and then washed with pre-warmed complete RPMI medium (RPMI, 10% FBS, and 1% penicillin / streptomycin). Cells were resuspended in pre-warmed complete RPMI containing 50 U / ml DNase (Invitrogen). Cells were then counted and transferred at 1×10 per tube in 100 μl of cold 1×PBS to 6 FACS tubes with cells. A viability control sample (5×10 5 viable cells and 5×10 5A sample of heat-killed cells was prepared. Then, all cell samples except for the unstained control sample were incubated with 1 μL of LIVE / DEAD® Fixable Blue (Invitrogen) viability dye on ice in the dark for 30 minutes. The cells were then washed, resuspended in PBS / 0.5% BSA, and incubated with BD Horizon® Brilliant Stain Buffer for 5 minutes. Next, the cells were incubated on ice in the dark for 30 minutes with fluorescent dye conjugated antibodies against CD16 (BUV496), CD3 (BUV737), NKG2D (Super Bright 436), CD14 (BV510), CD19 (BV510), KIR3DL1 (BV711), CD57 (BV785), CD56 (FITC), KIR2DL2 / L3 / S2 (PE-Cy5.5), NKG2A (PE-Vio770), NKG2C (APC), and KIR2DL1 (APC-Vio770). The cells were washed, resuspended in fixation buffer (BD Biosciences), and incubated at room temperature in the dark for 15 minutes. After fixation, the cells were washed, resuspended in PBS / 0.5% bovine serum albumin (BSA), and stored at 4°C until flow cytometry analysis. Monochromatic reference controls were prepared using UltraComp eBeads™ correction beads (Invitrogen). All antibodies were titrated before use to determine the optimal staining concentration. Positive cutoffs for CD56, CD16, CD57, NKG2A, NKG2C, NKG2D, KIR2DL1, KIR2DL2 / L3 / S2, and KIR3DL1 were established using a fluorescence minus one (FMO) control. All samples were analyzed using a Cytek Aurora spectral cytometer (Cytek Biosciences) at the USU Biomedical Instrumentation Center Flow Cytometry Facility, and the generated data were analyzed using FlowJo software v10 (BD Biosciences).

[0215] The gating strategies for identifying NK cells and NK cell subsets are shown in Figure 2. The NK cell percentage was calculated by dividing the number of CD56+ NK cells ("total NK cells" gate) by the number of PBMCs ("total cells" gate) and multiplying by 100. The absolute NK cell frequency was calculated by multiplying the number of PBMCs / 1 μl of blood by the previously calculated NK cell percentage. The frequency of NK subsets was calculated by dividing the number of cells from a particular subset by the number of NK cells ("total NK cells" gate) and multiplying by 100.

[0216] NK cell function assay: PBMC samples were thawed and incubated overnight at 37°C in 5% CO2 in pre-warmed complete RPMI-1640 medium supplemented with 10% FBS, 100 U / mL penicillin, 100 ug / mL streptomycin, and 2 mM L-glutamine (assay medium). The following morning, PBMCs were collected from the wells of a 6-well plate and counted. 50,000 PBMCs were stained, and the percentage of live CD3- / CD56+ / CD16+ NK cells was assessed using LIVE / DEAD Fixable Near-IR Dead Cell Stain (Invitrogen) for 30 minutes at room temperature in the dark. The cells were washed twice and then incubated with CD56 (PE), CD16 (PE), and CD3 (FITC) at 4°C for 30 minutes in the dark. All samples were analyzed using BD FACSCanto II, and the generated data were analyzed using FCS Express (v6) (De Novo Software). Total 1 x 10 6 K562 target cells were labeled with 1 μL of human TVA® dye containing calcein-AM (Immunospot, CTL Inc.) in 1 mL of PBS for 20 minutes at 37°C. After this incubation and two washes in PBS, 1 × 10¹ K562 cells were added to assay medium. 5The cells were resuspended in 1 / mL. PBMCs and K562 were then plated into U-bottom 96-well plates in 200 μL of assay medium at NK cell:K562 (E:T) ratios of 10:1, 5:1, 2.5:1, and 1.25:1. Labeled K562 alone was also plated. The plates were centrifuged at 200 × g for 1 minute, and then incubated in 5% CO2 at 37°C for 4 hours. After 4 hours of incubation, each well was mixed, and 50 μL of cell suspension was transferred in three portions to flat-bottom 96-well plates. Plate images were acquired using the S6 Universal Analyzer (Immunospot, CTL Inc.), available at Frederick National Laboratory for Cancer Research. Live cell counts and target cell death percentages were determined using NK-TVA software (Welter et al, 2018. High-Throughput GLP-Capable Target Cell Visualization Assay for Measuring Cell-Mediated Cytotoxicity. Cells 7(5):35. doi:10.3390 / cells7050035). To obtain a relative measurement of the size of highly cytotoxic NK cells for each individual, the cytotoxicity index of NK cells was calculated by multiplying the percentage of target cells killed at a specific E:T ratio by the absolute number of NK cells / 1 μl of blood.

[0217] Statistical analysis: Normality tests were performed on each dataset. If the data were normally distributed, an unpaired t-test was used to compare the two groups. If the data were not normally distributed, the Mann-Whitney U test was performed for unpaired group comparisons. Spearman correlation was performed to assess the relationships between factors, and repeated one-way ANOVA followed by Dunnett's multiple comparison test was used for comparisons between multiple time points. Statistical analyses were performed using GraphPad9. For data showing mortality percentage or NK cytotoxicity index at multiple effector-versus-target cell ratios, the area under the curve (AUC) was calculated. AUC was calculated in GraphPad9 by calculating the area under each data point; these areas were then compared between groups and analyzed using either a t-test or the Mann-Whitney U test depending on normality. Exploratory analyses of the association between NK cell receptor expression and symptom scores after vaccination 1 and 2, as well as IgG levels at 1 and 6 months post-vaccination, were performed using Spearman correlation with Bonferroni adjustment for multiple comparisons.

[0218] Analysis 1: Participant Selection and Demographics By March 30, 2021, the PASS study had enrolled 271 individuals. Of these participants, 15 had evidence of SARS-CoV-2 infection prior to vaccination, and the purpose of this analysis was to evaluate the impact of NK cells on the vaccine response in COVID-naive individuals, so they were excluded from the study. Of the remaining 256 participants, 188 completed the symptom questionnaire after both the first and second vaccinations and provided serum samples after the second vaccination at 20–50 days and 150–200 days after the second vaccination (Figure 1A).

[0219] Participants self-reported demographic characteristics, including sex, race, and ethnicity. Of the 188 individuals included in this study, 67.5% were identified as female and 32.5% as male (Table 1). 171 individuals were non-Hispanic, 12 were Hispanic, and 5 did not specify their ethnicity. Participants identified their race as 72.3% White, 10.6% Black, 9.0% Asian, 2.1% Other Race, 5.3% Multiracial, and 0.7% did not specify their race. The mean age was 42.4 years (range 20–69). [Table 1]

[0220] Analysis 2: Evaluation of the relationship between NK cell characteristics and sex or age. Flow cytometry was performed on PBMCs collected from individuals at baseline and mean 71.2 (SD 31.9) days prior to vaccination with BNT162b2. Given the relatively large size of our cohort, we initially assessed whether NK cell frequency, absolute number, or functionality differed among study participants based on either sex or age. As seen in Figure 3 (Panels a and b), no statistically significant differences were observed between NK cell frequency or absolute number in women compared to men (frequency as % of total cells: 5.9 [SD 2.9] vs. 6.9 [SD 3.7]; absolute NK cell count / 1 μl of blood: 101.6 [SD 65.2] vs. 106.9 [SD 63.0]). Functional cytotoxicity of NK cells was assessed on PBMCs from a subset of participants. No differences were found between men and women in terms of AUC of mortality percentage (Figure 3, Panel c) or NK cytotoxicity index (Figure 3, Panel d). Individual results for each effector-to-target (E:T) ratio for NK cell killing and cytotoxicity index are shown in Figure 4 (Panels a and b). There was no significant relationship between age and the percentage of PBMCs (NK cells) (Figure 5, Panel a), nor between age and absolute NK cell count / 1 μl of blood (Figure 5, Panel b). Furthermore, there was no significant correlation between age and NK cell functionality or AUC of the NK cell cytotoxicity index (Figure 5, Panels c and d). The results indicate a relationship between NK cell characteristics and sex or age.

[0221] Analysis 3: Frequency of NK cells at baseline, within 1 week after the first vaccine dose, and within 1 month after the second vaccine dose. A subset of participants (n=18) had PBMC samples collected within one week of their first BNT162b2 vaccine dose. For these participants, NK cell flow cytometry analysis was performed at baseline, 4.2 (range 1–7) days after the first BNT162b2 vaccine dose, and 25 (range 14–41) days after the second BNT162b2 vaccine dose to assess any potential short-term changes in circulating NK cells after mRNA vaccination (Figure 6, panels a–e). The frequency of total NK cells as a percentage of all PBMCs (Figure 6, panel a) and absolute NK cell count / 1 μl of blood (Figure 6, panel b) did not differ significantly from baseline levels at the two post-vaccination time points we analyzed. Immature CD56 bright CD16 - The frequency of NK cells remained virtually unchanged after vaccination (Figure 6, panel c), while CD56 dim CD16 + The frequency of mature NK cells decreased in some individuals during the first week after vaccination, but this difference was not statistically significant (Figure 6, panel d). Interestingly, CD56 dim CD16 - The percentage of mature NK cells was significantly higher during the first week after vaccination compared to baseline (11.51% [SD3.8] vs. 20.65% [SD17]; p=0.02) (Figure 6, panel e). Since mature NK cells are known to lose CD16 expression upon activation (Romee et al., 2013. NK cell CD16 surface expression and function is regulated by a disintegrin and metalloprotease-17 (ADAM17). Blood 121:3599-3608), CD16 + Decreased mature NK cells, and CD16 - A balanced increase in mature NK cells suggests early NK cell activation after vaccination. The results show NK cell frequencies at baseline, within one week after the first vaccination, and within one month after the second vaccination.

[0222] Analysis 4: Relationship between NK cell characteristics and symptoms after COVID-19 vaccination Because mRNA vaccination-induced NK cell activation may be a potentially significant contributor to vaccine-related inflammatory symptoms, we assessed baseline NK cell frequency using symptom severity scores measured by a structured questionnaire collected after both BNT162b2 vaccinations. After the first vaccination, participants reported a mean symptom score of 7.45 (SD 6.19), and after the second vaccination, they reported a mean symptom score of 11.0 (SD 9.22). The breakdown of various local and systemic symptoms shown in the cohort is summarized in Figures 7A-7B. As seen in Figure 8 (panels a and b), no significant association was observed between absolute NK cell count / 1 μl of blood and post-vaccination symptoms.

[0223] Due to limitations in conducting NK cell functionality studies on all samples, baseline NK cell functionality based on target cell death was examined in a subset of individuals who reported high (≥9) or low (≤3) scores after the first vaccine dose. After the first vaccination, individuals in the low symptom score group had a mean symptom score of 1.2 (range 0-3), while individuals with high symptom scores had a mean symptom score of 16.8 (range 9-33) (data not shown). After the second vaccination, all individuals who underwent baseline NK functionality testing were regrouped into low and high symptom score groups for the second dose. Individuals in the low symptom score group after the second dose had a mean symptom score of 3.7 (range 0-9), while individuals in the high symptom score group had a mean symptom score of 19.4 (range 11-43) (data not shown). There was no significant difference in AUC for the percentage of cell death between the low and high symptom score groups after the first vaccination (Figure 8, panel c). However, there was a significant difference in the AUC of the NK cytotoxicity index measured between the low symptom score group and the high symptom score group after vaccination 1 (118.5 [SD77.5] vs. 195.7 [SD170.1], p=0.04) (Figure 8, panel d). Higher NK cytotoxicity index values ​​in highly symptomatic individuals suggest that NK cells may contribute to vaccine-related inflammatory symptoms. Baseline NK death percentage (Figure 8, panel e) and NK cytotoxicity index (Figure 8, panel f) did not show statistically significant differences between the low symptom group and the high symptom group after the second vaccine dose. Individual results for each E:T ratio for NK death and cytotoxicity index after vaccination 1 and 2 are shown in Figure 9 (panels a-d). The results show the association between NK cell characteristics and symptoms after COVID-19 vaccination.

[0224] Analysis 5: Relationship between NK cell frequency and functionality and spike-specific IgG levels after two doses of BNT162b2 vaccination. Because NK cells have the ability to suppress or enhance the development of adaptive immune responses, we next evaluated whether baseline NK cell frequency and functionality were associated with higher or lower antibody levels after vaccination. SARS-CoV-2 spike-specific IgG levels were measured from serum samples collected approximately 1 month and 6 months after the second BNT162b2 vaccination. No statistically significant correlation was observed between the absolute NK cell count before vaccination and the spike-specific IgG level at 1 month (Rho=-0.019, p=0.79) (Figure 10, panel a), but a statistically significant negative correlation was observed between the absolute NK cell count before vaccination and the spike-specific IgG level at 6 months (Rho=-0.14, p=0.04) (Figure 10, panel b). This finding suggests that a large number of NK cells before vaccination may interfere with the persistence of the mRNA vaccine-induced antibody response. This negative correlation was also observed between the number of mature NK cells at 6 months and the spike-specific IgG level (data not shown). There was no significant correlation between NK cell killing or NK cytotoxicity index and IgG levels at 1 and 6 months post-vaccination (Figure 10, panels c-d; Figure 11, panels a-h). There was no significant correlation between spike-specific IgG levels and NK cytotoxicity at 6 months post-vaccination at various E:T ratios, although there was a tendency for a negative correlation between these two factors (Figure 11, panels e-h). The results show the relationship between NK cell frequency and functionality and spike-specific IgG levels after two BNT162b2 vaccinations.

[0225] Analysis 6: Immature NK cells expressing NKG2A are positively associated with IgG levels at 1 month and 6 months post-vaccination. The association between NK cell receptor expression and symptom scores after vaccination 1 and 2, as well as IgG levels at 1 and 6 months post-vaccination, was investigated in a series of exploratory secondary analyses (Figure 12). Surface receptors examined included NK-activating receptors NKG2C and NKG2D, and NK-inhibiting receptors NKG2A, KIR2DL1, KIR2DL2 / L3, and KIR3DL1. Receptor expression was analyzed for total NK cells (CD3 -CD56 + ), immature NK cells (CD56 bright CD16 - ) and mature (CD56 dim CD16 + ) NK cells. A negative correlation was found between NKG2D expression on NK cells and spike-specific IgG levels at 6 months after vaccination (rho=-0.15, p=0.04). A positive correlation was found between NKG2A expression on immature NK cells and spike-specific IgG levels at 1 month (rho=0.26, p=0.0003) and 6 months (rho=0.15, p=0.03) after vaccination. NKG2A expression was also positively correlated with higher spike-specific IgG levels in total NK cells and mature NK cells at 1 and 6 months after vaccination, but the correlation was not significant. Even after applying Bonferroni correction for 76 comparisons, the positive association between NKG2A expression on immature NK cells and spike-specific IgG levels at 1 month after vaccination remained statistically significant.

[0226] Example 2 Protection of Leukocytes from Killing by NK Cells Natural killer (NK) cells inject abnormal cell types with packets containing a set of proteins (cytolytic granules) to kill them. Granzyme B, one of the key proteins involved in killing target cells, is a proteolytic enzyme. A naturally occurring inhibitor of this enzyme (SERPINB9) is expressed by several immune cell types and protects them from being killed by NK cells. Recognition and triggering of NK cells to kill a target can also be prevented by a protein on the surface of NK cells (NKG2A) that recognizes a protein expressed on the surface of healthy cells (HLA-E).

[0227] Generally, to evaluate the ability of SERPINB9 and / or HLA-E protein to protect the leukocyte cell line (721.221), which is normally a target for killing by NK cells, the killing of unmodified cell lines was compared with the killing of cell lines engineered to contain SERPINB9 protein, HLA-E protein, or both.

[0228] Specifically, to obtain NK cells, PBMCs were obtained by density gradient centrifugation of whole blood obtained from 6 different human donors. After isolation, cells were counted using the Muse® Count and Viability Kit (Cytek®, MCH600103) on a Muse® Cell Analyzer (Cytek®), and seeded at 10-15×10 per well in 6-well plates in assay medium RPMI 1640 medium (Gibco, 21870-084), 10% heat-inactivated fetal bovine serum (Sigma, 12306C), and 1% Pen Strep Glutamine (Gibco, 10378-016) 6 and incubated overnight. After overnight incubation, NK cells were isolated with a human NK cell isolation kit (Miltenyi Biotec, 130-092-657) according to the manufacturer's instructions.

[0229] To prepare test cells, 721.221 cells (parental cell line or cells expressing SERPINB, HLA-E, both, or GFP) were labeled with calcein violet dye, and adjusted to 2×10 in assay medium 5The cells were resuspended at 100 cells / mL. 100 microliters of 721.221 cells were cultured with NK cells in assay medium in U-bottom 96-well plates at effector:target ratios of 10:1, 5:1, 2.5:1, and 1.25:1, in a final volume of 200 μL / well. Labeled 721.221 cells alone were also plated as a negative control. The plates were centrifuged at 200 × g for 1 minute and then incubated at 37°C in 5% CO2 for 4 hours. After 4 hours of incubation, each well was mixed, and 50 μL of the cell suspension was transferred to the 96-well plate in three portions for imaging. The percentage of target cell death was determined using the following formula: (control count - experimental count) / control count * 100.

[0230] Results for samples with a 5:1 effector:target ratio are reported in Figure 13 for each of the six donors, with parent cell line death set to 1 for each donor. The black line represents the average level of lysis for all six donors combined. As seen in Figure 13, cells containing the SERPINB9 protein were protected from death to a greater extent than cells with the HLA-E protein on their surface. Cells with both SERPINB9 and HLA-E were protected to the same extent as cells with only SERPINB9, demonstrating the superior protective effect of the SERPINB9 protein.

[0231] Example 3 Immunization by the Covid spike protein and SERPINB9 enhances the immune response. In this animal model study, 8-week-old female C57BL / 6 mice were intramuscularly vaccinated in the right hind limb with a lipid nanoparticle (LNP) vaccine formulation (total 1 μg) in one of three groups (5 mice per group). Each group contained 0.5 μg of LNPs containing mRNA encoding the SARS-CoV-2 spike protein, one group contained an additional 0.5 μg of empty LNPs, one group contained an additional 0.5 μg of LNPs containing mRNA encoding the SERPINB9 protein, and one group contained 0.5 μg of LNPs containing mRNA encoding Qa1 (the mouse equivalent of HLA-E) (see Sequence IDs 9 and 10): 0.5 μg spike LNP + 0.5 μg empty LNP 0.5 μg of spike LNP + 0.5 μg of serpine LNP 0.5 μg of spike LNP + 0.5 μg of Qa1 LNP

[0232] The LNP was SM-102 LNP (used in the Moderna COVID vaccine).

[0233] Three weeks after vaccination, mice were euthanized, and single-cell suspensions of spleen and absorptive lymph nodes were prepared. Lymph node cells were pooled from five mice, and the experiment was repeated once. Splenocytes and lymph node cells were divided into 5 × 10⁶ cells. 6Cells were cultured at 100 cells / ml and then stimulated with either two pools of spike protein peptides in DMSO (1.1 mcg / ml S1 peptide pool and 1.1 mcg / ml S2 peptide pool) or DMSO alone, with a final DMSO concentration of 0.2 vol% of the medium. Each peptide pool (obtained from Stemcell Technologies) contained 158 15mer peptides with 11 amino acid duplications covering the entire SARS-CoV-2 spike protein between the two peptide pools. The following day, cells were stained and fixed for flow cytometry, and the percentages of all viable cells, CD3+, and CD8+ T cells, which were also CD25+, were evaluated in relation to determining the percentage of activated (CD25+) CD8 cells. The results are shown in Figure 14 (panels a and b). As shown in the figure, in splenocytes of mice vaccinated with LNPs containing spike protein-encoding mRNA and SERPINB9 or Qa1 mRNA, the percentage of spike protein-specific CD8+ T cells activated by incubation with a spike S1 peptide pool was significantly higher than in mice vaccinated only with LNPs containing spike mRNA-encoding mRNA (Panel A). Similar results were observed in lymph node cells (Panel B), but statistical significance was not observed in lymph node cells because it is likely that there are only two data points for each condition, as lymph node cells need to be pooled from five mice for each experiment. Therefore, these results indicate that mice immunized with mRNA LNP vaccines by this disclosure showed a greater CD25+ response to the vaccine antigen (spike protein in this experiment), demonstrating that the approach of this disclosure enhances the immune response to the vaccine antigen (spike protein in this experiment).

[0234] Desktop Example 1 To test whether NK cell depletion alters the levels of clinical symptoms, acute inflammation, and / or antigen-specific responses induced by initial BNT162b2 vaccination.

[0235] To determine the role of NK cells in eliciting mRNA-induced inflammatory side effects and pathogen-specific adaptive immune responses, we evaluate acute inflammatory cytokines, signs of inflammatory side effects, spike protein-specific antibody levels, and spike protein-specific T cell responses in C57BL / 6 mice, in the presence or absence of NK cell depletion.

[0236] mRNA vaccine: For these studies, we plan to use either bivalent BNT162b2+BNT162b2 Omi(BA.1), a bivalent mRNA COVID vaccine encoding the pre-fusion SARS-CoV-2 spike protein of the D614G and BA.1 SARS-CoV-2 variants, or monovalent BNT162b2 (simply encoding the ancestral D614G spike protein). The vaccine dose will be obtained from a multipurpose vial with the remaining / residual dose, which is to be discarded at the end of the day from the Walter Reed National Military Medical Center (WRNMMC) COVID Vaccine Center.

[0237] Vaccination consists of an intramuscular (IM) injection of 50 μl of mRNA vaccine (0.2 or 5 μg per mouse). These doses have been shown to induce robust spike-specific antibody levels and moderate levels of spike-specific T cells in the spleen and lungs of C57BL / 6 mice three weeks after vaccination. Mice receive a single dose of mRNA vaccine administered by intramuscular injection into the thigh muscle of the hind limb. NK cell depletion was initiated one day prior to vaccination by intraperitoneal injection of 200 μg of anti-NK1.1 antibody (clone PK136; Bio X Cell) in a total of five doses every three days. This approach resulted in sustained depletion of over 90% of NK cells in C57BL / 6 mice over a two-week period, as confirmed in Aim 1 (Schmitt, DM, et al., Role of NK cells in host defense against pulmonary type AFrancisella tularensis infection. Microbes Infect, 2013.15(3):p.201-11). We selected this duration of NK cell depletion because most adverse reactions in response to mRNA vaccination occur in the first few days, and antigen expression by somatic cells that take up mRNA from the mRNA vaccine expresses the vaccine-coding protein for about 10 days (Cagigi, A. and K. Lore, Immune Responses Induced by mRNA Vaccination in Mice, Monkeys and Humans. Vaccines (Basel), 2021.9(1)). Control mice with undepleted NK cells are administered the same dose and frequency of isotyped antibody (clone C1.18.4; Bio X Cell).

[0238] To evaluate the effect of NK cell depletion on vaccine-induced production of inflammatory cytokines, 50 μl of blood was obtained from mice 6 and 24 hours after vaccination via tail vein or submandibular hemorrhage (depending on the technical proficiency of the laboratory staff), and serum was then measured for inflammatory cytokines, including IFN-γ, TNF-α, IL-1, IL-6, and IFN-α, using microsphere (Luminex-based) multiplex assay (R&D systems).

[0239] To assess the clinical signs of inflammatory side effects, mice will be monitored daily for 5 days after vaccination by a blinded observer in the treatment group for the presence or absence of hair bristles, hunched posture, and lethargy. In addition, rectal temperature will be taken twice daily for the first 5 days after vaccination.

[0240] On day 21, mice are euthanized according to V.4.6. Following in vitro stimulation with a pool of peptides from one or both SARS-CoV-2 vaccine strains, influencing inguinal and iliac lymph nodes, as well as splenic cells, are isolated by flow cytometry and / or ELISPOT to enumerate SARS-CoV-2 spike-specific CD4 and CD8 T cells. Serum obtained from terminal hemorrhage is measured for spike-specific antibody levels using a highly sensitive and specific microsphere immunoassay developed by the Broder / Laing Laboratory in the Department of Microbiology.

[0241] Results and number of mice used: The primary outcome is spike-specific antibody levels. We hypothesize that NK cell depletion allows for a 30% increase in spike-specific antibody levels. A sample size of 13 mice in each group has 80% power to detect a 30% difference in antibody levels, with a two-sided significance level of 0.05, assuming a standard deviation of 0.25 in each group (25% from the mean). Therefore, we use 15 animals per group. 15 animals per group allows for the loss of up to 2 animals per group due to reasons such as inability to obtain blood samples or removal of animals from the study due to illness. Since there are two main experimental groups (vaccinated and NK depleted vs. vaccinated and unvaccinated), this requires a total of 30 mice. Additionally, to ensure that mice do not have a baseline response that is cross-reactive with the spike protein, we include a third set of 15 unvaccinated mice to allow for comparison between vaccinated and unvaccinated mice. Total mice = 45.

[0242] Desktop Example 2 This study investigates whether NK cell depletion alters the levels of acute inflammation and / or antigen-specific antibody and T cell responses induced by booster BNT162b2 vaccination.

[0243] For this experiment, mice received an initial dose of vaccine on day 0 and a booster dose on day 21, initiating NK cell depletion one day before the booster. The prime / boost regimen with vaccine doses given on day 0 and day 21 resulted in higher levels of spike-specific antibodies, as well as a high frequency of spike-specific CD4 and CD8 T cells in the spleen, three weeks after booster vaccination.

[0244] As in Working Example 1 on the machine, vaccination consists of intramuscular injection of 50 μl of mRNA vaccine (0.2 or 5 μg per mouse). Anti-NK1.1 antibody injection (or isotype control injection for control group mice) is continued for 5 doses every 3 days.

[0245] To evaluate the effect of NK cell depletion on booster vaccine-induced production of inflammatory cytokines, 50 μl of blood is obtained from mice 6 hours and 24 hours after booster vaccination via tail vein or submandibular bleeding, then serum is measured for inflammatory cytokines including IFN-g, TNF-a, IL-1, IL-6, and IFN-a using a microsphere (Luminex-based) multiplex assay (R&D systems).

[0246] To evaluate clinical signs of inflammatory side effects after booster vaccination, mice are monitored daily for 5 days after booster vaccination by an observer blinded to the treatment group for the presence or absence of piloerection, hunched posture, and lethargy. Additionally, rectal temperature is obtained twice a day for the first 5 days after booster vaccination.

[0247] Mice are euthanized on day 42 (day 21 after booster vaccination). To enumerate SARS-CoV-2 spike-specific CD4 and CD8 T cells by flow cytometry and / or ELISPOT after in vitro stimulation with a pool of peptides from one or both vaccine strains of SARS-CoV-2, draining inguinal and iliac lymph nodes, and spleen cells are isolated. Serum obtained from terminal bleeding is measured for spike-specific antibody levels using a sensitive and specific microsphere immunoassay developed by the Broder / Laing Institute at the Department of Microbiology.

[0248] Results and number of mice used: The primary outcome is spike-specific antibody levels. We hypothesize that NK cell depletion during the mRNA-boosted vaccination period allows for a 30% increase in spike-specific antibody levels. A sample size of 13 mice in each group has 80% power to detect a 30% difference in antibody levels, with a two-sided significance level of 0.05, assuming a standard deviation of 0.25 in each group (25% from the mean). Therefore, we use 15 animals per group. 15 animals per group allows for the loss of up to 2 animals per group due to reasons such as inability to obtain blood samples or removal of animals from the study due to illness. Since there are two main experimental groups (vaccinated and NK depleted vs. vaccinated and without NK depletion), this requires a total of 30 mice.

[0249] Desktop Example 3 In primary or booster vaccination settings, we will test whether NK cell depletion prolongs the duration of spike-specific mRNA expression after COVID mRNA vaccination.

[0250] Our central hypothesis is that NK cells deplete cells expressing the vaccine-delivered spike mRNA. To directly test this, we evaluate the duration of spike mRNA expression in muscle and lymph node cells after primary or booster vaccination, in the presence or absence of NK cell depletion.

[0251] The group includes the following: A. Single-dose COVID mRNA vaccination and NK cell depletion accompanied by euthanasia on days 1, 3, and 7 after vaccination. B. Single-dose COVID mRNA vaccination and isotype injection accompanied by euthanasia on days 1, 3, and 7. C. Euthanasia on days 1, 3, and 7, COVID mRNA vaccination on days 0 and 21, and NK depletion. Euthanasia on days D1, 3, and 7, along with COVID mRNA vaccination and isotype injection on days 0 and 21.

[0252] Mice are vaccinated by intramuscular injection of monovalent or bivalent BNT162b2 at a dose of 0.5 or 5 μg / mouse, as detailed.

[0253] For group A, NK cell depletion is initiated one day before vaccination by intraperitoneal injection of 200 μg of anti-NK1.1 antibody (clone PK136; Bio X Cell) in a total of five doses every three days. For group C, NK cell depletion is initiated on day 20, one day before the booster vaccination on day 21. Control mice in groups B and D, whose NK cells are not depleted, are given the same dose and frequency of isotype antibody (clone C1.18.4; Bio X Cell) as groups A and C receive Ab, which depletes NK cells.

[0254] Five mice from each mouse group will be euthanized on days 1, 3, and 7 after single mRNA vaccination for groups A and B, and on days 22, 23, 25, and 28 (i.e., days 1, 3, and 7 after booster vaccination) for the booster group. After euthanasia, lymph nodes and muscle tissue will be evaluated by RT-PCR for the presence and relative amount of spike mRNA expression, and the cells expressing spike mRNA will be determined by single-cell RNA sequencing. Blood collected from terminal bleeding will be evaluated by ELISA for the circulating level of spike protein. Flow cytometry may also be performed on inflow lymph nodes to identify the frequency of NK cells in lymph nodes at various time points and to assess the activation status of these NK cells (this information will also be evaluated using transcriptomics data obtained from single-cell sequencing studies performed).

[0255] Results and number of mice used: The primary outcome is the duration of spike mRNA expression in lymph nodes. We assume that spike-specific mRNA in NK-depleted mice is present at a level 55% or higher than in non-depleted mice 7 days after vaccination or booster (previous studies have shown that most mice do not have detectable mRNA 7 days after vaccination with 5 μg of BNT162b2). The sample size of 5 mice in each group has 80% power to detect a 55% difference in quantitative spike mRNA levels by Mann-Whitney rank-sum test, with a two-sided significance level of 0.05, assuming a standard deviation of 0.3 within each group. Since there are 4 groups, each of which has 3 time points for euthanasia, this requires a total of 5 mice / group × 4 groups × 3 time points per group = 60 mice.

[0256] Desktop Example 4 This study will test whether vaccination with an mRNA vaccine containing both mRNA for the SARS-CoV-2 spike protein and mRNA for the inhibitory ligand of natural killer (NK) cells results in a greater immune response and / or fewer side effects than vaccination with an mRNA vaccine containing only mRNA for the spike protein.

[0257] Since NK cell function can be inhibited by inhibitory ligands expressed on the host cell surface, we hypothesize that a bivalent mRNA vaccine in which lipid nanoparticles (LNPs) contain mRNA sequences encoding both the SARS-CoV-2 spike protein and proteins that inhibit NK cell function will induce a higher antibody and T cell response than a vaccine containing only the mRNA encoding the spike protein. The encoded NK cell inhibitory proteins may include one or more selected from HLA-E, CD155, PI-9 (also known as protease inhibitor 9 or SERPINB9), and CEACAM-1, all of which have an inhibitory effect on NK cell function or on the effects of NK cells on target cells.

[0258] The age-matched C57BL / 6 mouse group includes: A. COVID mRNA vaccination accompanied by euthanasia on days 3, 7, and 21. B. COVID+NK inhibitor ligand vaccination accompanied by euthanasia on days 3, 7, and 21. C. Euthanasia on days 3, 7, and 21 after booster, with COVID mRNA vaccination and booster dose on day 21. COVID+NK inhibitor ligand vaccination with euthanasia on days 3, 7, and 21.

[0259] Mice will be evaluated for the symptoms detailed in Experiment 2 (all groups), the release of acute inflammatory cytokines into the serum by obtaining blood 6 and 24 hours after vaccination or booster dose, the duration of spike protein expression in the serum and spike mRNA expression in the lymph nodes measured after euthanasia (groups on days 3 and 7), and antibody and T cell responses (group on day 21). Blood will be obtained by either the tail vein or submandibular hemorrhage method, depending on the technical expertise and experience of the laboratory staff conducting the experiment.

[0260] Results and number of mice used: The primary outcome is spike-specific antibody levels. We hypothesize that an mRNA vaccine containing mRNA expressing an NK inhibitory ligand will enable a 30% increase in spike-specific antibody levels. A sample size of 13 mice in each group has 80% power to detect a 30% difference in antibody levels, with a two-sided significance level of 0.05, assuming a standard deviation of 0.25 in each group (25% from the mean). Therefore, we use 15 animals per group. 15 animals per group allows for the loss of up to 2 animals per group due to reasons such as inability to obtain blood samples or removal of animals from the study due to illness. There are four main experimental groups (single-dose BNT162b2 vs. single-dose BNT162b2 + NK inhibitory ligand, booster dose BNT162b2 vs. booster dose BNT162b2 + NK inhibitory ligand), and a control unvaccinated group. The unvaccinated control group, present solely to confirm baseline levels of cross-reactive immune responses to spike proteins, is the same as that used in bench examples 2 and 5. Therefore, this experiment requires 60 mice.

[0261] Desktop Example 5 This study investigates whether NK cell depletion in age-matched mice alters the levels of acute inflammation and / or antigen-specific antibody and T cell responses induced by mRNA vaccines against intestinal filarial antigens.

[0262] This experiment is an exact reproduction of bench examples 2 and 3, except that instead of using the bivalent BNT162b2 vaccine, we utilize an mRNA vaccine expressing mRNA for BMA-LAD-2 and / or BmUDP-GT. The objective of this study is to determine whether the effect observed by NK cell depletion against COVID mRNA vaccines also occurs with mRNA vaccines expressing different antigens. BMA-LAD-2 is an essential adhesion molecule for Brugia malayi, functioning to maintain tight junctions between intestinal epithelial cells in adult B. malayi. BMA-UDP-GT is a transmembrane protein with a large extracellular portion present in the lumen of the intestinal tract of adult B. malayi. BmUDP-GT functions as a UDP-glucoronosyltransferase and has been demonstrated to be an essential protein for adult B. malayi.

[0263] Results and number of mice used: The primary outcome is the level of filarial antigen-specific antibody. We hypothesize that NK cell depletion during the period of first-line mRNA vaccination or boost vaccination allows for a 30% increase in spike-specific antibody levels. A sample size of 13 mice in each group has 80% power to detect a 30% difference in antibody levels, with a two-sided significance level of 0.05, assuming a standard deviation of 0.25 in each group (25% from the mean). Therefore, we use 15 animals per group. In addition to the four main experimental groups (vaccinated and NK depleted vs. vaccinated and without NK depletion, boosted and NK depletion vs. boosted and without NK depletion), there is also an unvaccinated group. Therefore, this experiment requires 60 mice.

[0264] Desktop Example 6 This study will test whether vaccination with an mRNA vaccine containing both mRNA for the vaccine antigen and mRNA for a natural killer (NK) cell inhibitory ligand extends the expression time of the vaccine antigen protein.

[0265] To demonstrate that the addition of an NK inhibitor to an mRNA vaccine increases the expression time of the vaccine antigen protein, mice were vaccinated with one of the following mRNA vaccines: Each mRNA vaccine contained an equal amount of LNP loaded with mRNA encoding the SARS-CoV-2 spike protein (as the vaccine antigen), one additionally containing 0.5 μg of LNP loaded with mRNA encoding the green fluorescent protein (as a control), one additionally containing 0.5 μg of LNP loaded with mRNA encoding the serpin protein (e.g., SERPIN B9 NK inhibitor), and one additionally containing 0.5 μg of LNP loaded with mRNA encoding Qa1 (an NK inhibitor): 0.5 μg of spike LNP + 0.5 μg of GFP LNP (S+GFP) 0.5 μg of spike LNP + 0.5 μg of serpine LNP (S + serpine) 0.5 μg of spike LNP + 0.5 μg of Qa1 LNP (S + Qa1).

[0266] Mice were euthanized on days 1, 3, and 7 after vaccination, and spike protein mRNA levels in the lymph nodes and spleen were quantified by qPCR. Additionally, serum spike protein levels were measured by ELISA on days 1, 3, and 7 after vaccination. The results indicate whether vaccination with (S+serpine) or (S+Qa1)mRNA LNP vaccine formulations prolongs the expression time of the vaccine antigen (spike protein).

[0267] Desktop Example 7 This study will test whether vaccination with an mRNA vaccine containing both mRNA for the vaccine antigen and mRNA for a natural killer (NK) cell inhibitory ligand enhances the persistence of the antibody response.

[0268] One of the main limitations of current mRNA vaccines is the low persistence of the antibody response. To demonstrate that the addition of an NK inhibitor to mRNA vaccines enhances the persistence of circulating antibodies, three groups of mice are vaccinated as described above. Serum is obtained 4, 8, 12, and 16 weeks after vaccination and tested for levels of anti-spike IgG antibodies. The results indicate whether vaccination with (S+serpine) or (S+Qa1) mRNA LNP vaccine formulations enhances the persistence of the antibody response.

[0269] This specification and its embodiments are illustrative of this embodiment, and it will be understood that other embodiments within the spirit and scope of the embodiments described will themselves be suggested to those skilled in the art. While this disclosure is described in relation to its particular form and embodiments, it should be understood that various modifications other than those discussed above can be relied upon without departing from the spirit or scope of this disclosure. For example, equivalents may be replaced with those specifically described, and in certain particular cases, the specific application of the steps may all be reversed or intervened without departing from the spirit or scope of this disclosure.

[0270] array Serpin B9 (Sequence ID 1) (Natural human sequence from GenBank RefSeq file NM_004155) 120 auggaaaccuu uucuaaugca agugguacuu uugccauacg ccuuuuaaag auacuguguc 181 aagauaaccc uucgcacaac guguucuguu cuccugugag caucuccucu gcccuggcca 241 ugguucuccu aggggcaaag ggaaacaccg caacccagau ggcccaggca cugucuuuaa 301 acacagagga agacauucau cgggcuuucc agucgcuucu cacugaagug aacaaggcug 361 gcacacagua ccugcugaga acggccaaca ggcucuuugg agaaaaacu ugucaguucc 421 ucucaacguu uaaggaaucc ugucuucaau ucucaccaugc ugagcugaag gagcuuucc 481 uuaucagagc ugcagaagag uccaggaaac acaucaacac cugggucuca aaaaagaccg 541 aagguaaaau ugagaguug uugccgggua gcuaauuga ugcagaaacc aggcugguug 601 uugucaaugc caucuacuuc aaaggaaagu ggaaugaacc guuugacgaa acauacacaa 661 gggaaaugcc cuuuaaaaua aaccaggagg agcaaagcc agugcagaug auguaucagg 721 781 841 cgguggaaaa aagucucacu uuugagaaac ucacagccug gaccaagcca gacuguauga 901 961 aaucugugcu ucggcauuuug ggaauuguug augccuucca acagggcaag gcugacuugu 1021 cggcaauguc agcggagaga gaccuguguc uguccaaguu cguccaaag aguuuuggug 1081 aggugaauga agaaggcacc gaggcagcgg cagcgucgag cugcuuugua guugcagagu 1141 gcugcaugga aucuggcccc agguucugug cugaccaccc uuuccuuuuc uucaucaggc 1201 acaacagagc caacagcauu cuguucugug gcagguucuc aucgccauaa HLA-E*0103 (SEQ ID NO: 2) (native human sequence from GenBank RefSeq file AY216681) 1 augguagaug gaacccuccu uuuacuccuc ucggaggccc uggcccuuac ccagaccugg 61 gcgggcuccc acuccuugaa guauuuccac acuuccgugu cccggcccgg ccgcggggag 121 ccccgcuuca ucucuguggg cuacguggac gacacccagu ucgugcgcuu cgacaacgac 181 gccgcgaguc cgaggauggu gccgcgggcg ccguggaugg agcaggaggg gucagaguau 241 ugggaccggg agacacggag cgccagggac accgcacaga uuuuccgagu gaaccugcgg 301 acgcugcgcg gcuacuacaa ucagagcgag gccgggucuc acacccugca guggaugcau 361 ggcugcgagc uggggcccga cgggcgcuuc cuccgcgggu augaacaguu cgccuacgac 421 ggcaaggauu aucucacccu gaaugaggac cugcgcuccu ggaccgcggu ggacacggcg 481 gcucagaucu ccgagcaaaa gcucagaucu gccucugagg cggagcacca gagagccuac 541 cuggagaca caugcgugga guggcuccac aaauaccugg agagggaa ggagacgcug 601 cucacugg agccccaaa gacacgug cucucaccacc cucucacugg ccaugaggcc 661 acccugaggu gcugggcccu gggcuucuac ccugcggaga acccugac cuggcagcag 721 gaugggagg gccauaccca ggacacggag cucguggaga ccaggcugc agggaugga 781 accuccaga agugggcagc uguggugc ccucuccag agggaggag auacacgugc 841 caugugcagc augaggggcu acccgagccc caugucccuga gauggaagcc ggcuucccag 901 squirrel squirrel squirrel squirrel squirrel squirrel squirrel 961 ggagcugugg ugcugcugu gauauggagg agagagcu cagguggaaa aggaggc 1021 uacucuaagg cugaguggag cgacagugcc cagggucug agcucacag cuuguaa HLA-E*0101(XoL-E*0101) 1 augguagaug gaacccuccu uuuacuccuc ucggaggccc uggcccuuac scagaccugg 61 gcggcucccc acuccuugaa guauuuccac acuuccgugu cccggccgg ccgcgggg 121 ccccgcuuca ucucugugggg cuacguggac cavacccagu ucgugcgcuu cgacaacgac 181 gccgcgaguc cgaggauggu gccgcggcg ccguggaugg agcaggagg aggaguau 241 ugggaccggg agacacggag cgccagggac accgcacaga uuuuccgagu gaaccugcgg 301 acgcugcgcg gcuacuacaa ucagagcgag gccgggucuc acacccugca guggaugcau 361 ggcugcgagc uggggcccga caggcgcuuc cuccgcgggu augacaguu cgccuacgac 421 ggcaggaou aucucacccu gaugaggac cugcgcuccu ggaccgcggu ggacacggcg 481 gcucagaucu ccgagcaaaa gcucagaucu gccucugagg cggagcacca gagagccuac 541 cuggagaca caugcgugga guggcuccac aaauaccugg agagggaa ggagacgcug 601 cucacugg agccccaaa gacacgug cucucaccacc cucucacugg ccaugaggcc 661 acccugaggu gcugggcccu gggcuucuac ccugcggaga acccugac cuggcagcag 721 gaugggagg gccauaccca ggacacggag cucguggaga ccaggcugc agggaugga 781 accuccaga agugggcagc uguggugc ccucuccag agggaggag auacacgugc 841 caugugcagc augaggggcu acccgagccc gucacccuga gauggaagcc ggcuucccag 901 cccaccaucc ccaucguggg caucauugcu ggccugguuc uccuuggauc uguggucucu 961 ggagcugugg uugcugcugu gauauggagg aagaagagcu cagguggaaa aggagggagc 1021 uacucuaagg cugaguggag cgacagugcc caggggucug agucucacag cuuguaa CLEC2D (SEQ ID NO: 4) 1 augcaugaca guaacaaugu ggagaaagac auuacaccau cugaauugcc ugcaaaccca 61 gguugucugc auucaaaaga gcauucuauu aaagcuaccu uaauuuggcg cuuauuuuuc 121 uuaaucaugu uucugacaau cauagugugu ggaaugguug cugcuuuaag cgcaauaaga 181 gcuaacugcc aucaagagcc aucaguaugu cuucaagcug caugcccaga aagcuggauu 241 gguuuucaaa gaaaguguuu cuauuuuucu gaugacacca agaacuggac aucaagucag 301 agguuuugug acucacaaga ugcugaucuu gcucagguug aaagcuucca ggaacugaau 361 uuccuguuga gauauaaagg cccaucugau cacuggauug ggcugagcag agaacaaggc 421 caaccaugga aauggauaaa ugguacugaa uggacaagac aguuuccuau ccugggagca 481 ggagagugug ccuauuugaa ugacaaaggu gccagugug ccaggcacua cacagagagg 541 aaguggauuu guuccaaauc agauauacau gucuag Serpin B9 (Sequence ID 5) METLSNASGTFAIRLLKILCQDNPSHNVFCSPVSISSALAMVLLGAKGNTATQMAQALSLNTEEDIHRAFQSLLTEVNKAGTQYLLRTANRLFGEKTCQFLSTFKESCLQFYHAELKELSFIRAAEESRKHINTWVSKKTEGKIEELLPGSSIDAETRLVLVNAIYFKGKWNEPFDETYTREMPFKIN QEEQRPVQMMYQEATFKLAHVGEVRAQLLELPYARKELSLLVLLPDDGVELSTVEKSLTFEKLTAWTKPDCMKSTEVEVLLPKFKLQEDYDMESVLRHLGIVDAFQQGKADLSAMSAERDLCLSKFVHKSFVEVNEEGTEAAAASSCFVVAECCMESGPRFCADHPFLFFIRHNRANSILFCGRFSSP HLA-E*0103 (Sequence ID 6) MVDGTLLLLLSEALALTQTWAGSHSLKYFHTSVSRPGRGEPRFISVGYVDDTQFVRDFDNDAASPRMVPRAPWMEQEGSEYWDRETRSARDTAQIFRVNLRTLRGYYYNQSEAGSHTLQWMHGCELGPDGRFLRGYEQFAYDGKDYLTLNEDLRSWTAVDTAAQISEQKSNDASEAEHQRA YLEDTCVEWLHKYLEKGKETLLHLEPPKTHVTHHPISDHEATLRCWALGFYPAEITLTWQQDGEGHTQDTELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPVTLRWKPASQPTIPIVGIIAGLVLLGSVVSGAVVAAVIWRKKSSGGKGGSYSKAEWSDSAQGSESHSL HLA-E*0101 (Sequence ID 7) MVDGTLLLLLSEALALTQTWAGSHSLKYFHTSVSRPGRGEPRFISVGYVDDTQFVRDFDNDAASPRMVPRAPWMEQEGSEYWDRETRSARDTAQIFRVNLRTLRGYYYNQSEAGSHTLQWMHGCELGPDRRFLRGYEQFAYDGKDYLTLNEDLRSWTAVDTAAQISEQKSNDASEAEHQRA YLEDTCVEWLHKYLEKGKETLLHLEPPKTHVTHHPISDHEATLRCWALGFYPAEITLTWQQDGEGHTQDTELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPVTLRWKPASQPTIPIVGIIAGLVLLGSVVSGAVVAAVIWRKKSSGGKGGSYSKAEWSDSAQGSESHSL CLEC2D (Sequence ID 8) MHDSNNVEKDITPSELPANPGCLHSKEHSIKATLIWRLFFLIMFLTIIVCGMVAALSAIRANCHQEPSVCLQAACPESWIGFQRKCFYFSDDTKNWTSSQRFCDSQDADLAQVESFQELNFLLRYKGPSDHWIGLSREQGQPWKWINGTEWTRQFPILGAGECAYLNDKGASSARHYTERKWICSKSDIHV

Claims

1. (a) A first polynucleotide encoding a polypeptide that can have natural killer (NK) cell inhibitory activity, (b) A second polynucleotide encoding a polypeptide that can induce an immune response, or a protein that is naturally endogenously produced, Isolated polynucleotide constructs containing [the specified element].

2. (a) One or more polynucleotide constructs according to claim 1, (b) A pharmaceutically acceptable carrier, A composition containing the following:

3. (a) A first polynucleotide encoding a polypeptide that can have natural killer (NK) cell inhibitory activity, (b) A second polynucleotide encoding a polypeptide that can induce an immune response, or a protein that is naturally endogenously produced, (c) A pharmaceutically acceptable carrier, A composition containing the following:

4. The construct or composition according to any one of claims 1 to 3, wherein the polynucleotide construct or the first and second polynucleotides are mRNA molecules.

5. The construct or composition according to any one of the prior claims 1 to 4, wherein the first polynucleotide encodes a serine protease inhibitor (serpine) polypeptide or a fragment thereof that can have natural killer (NK) cell inhibitory activity.

6. Serpine polypeptides include SERPINA1, SERPINA2, SERPINA3, SERPINA4, SERPINA5, SERPINA6, SERPINA7, SERPINA8, SERPINA9, SERPINA10, SERPINA11, SERPINA12, SERPINA13, SERPINB1, SERPINB2, SERPINB3, SERPINB4, SERPINB5, SERPINB6, SERPINB7, and SERPINB 8. A construct or composition according to claim 5, which is a serpine polypeptide selected from the group consisting of SERPINB9, SERPINB10, SERPINB11, SERPINB12, SERPINB13, SERPINC1, SERPIND1, SERPINE1, SERPINE2, SERPINE3, SERPINF1, SERPINF2, SERPING1, SERPINH1, SERPINI1, SERPINI2, and combinations thereof.

7. The construct or composition according to claim 5 or 6, wherein the serpine polypeptide is SERPINB9.

8. The first polynucleotide encodes a serpine polypeptide or a fragment thereof that can have NK cell inhibitory activity. (a) The first polynucleotide contains the sequence of SEQ ID NO: 1, or a sequence having at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99%, at least 99.5%, at least 99.9%, or at least 99.99% sequence identity with respect to the sequence of SEQ ID NO: 1, or (b) The construct or composition according to claim 7, wherein the first polynucleotide encodes a serpine polypeptide comprising the sequence of SEQ ID NO: 5, or a sequence having at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or at least 99.99% sequence identity with respect to the sequence of SEQ ID NO:

5.

9. The first polynucleotide is Polypeptides of the major histocompatibility complex (MHC) class having NK cell inhibitory activity, or fragments thereof, Lectin polypeptides or fragments thereof that can have NK cell inhibitory activity, Cadherin polypeptides or fragments thereof that can have NK cell inhibitory activity, CDH1 (e-cadherin) or a fragment thereof that can have NK cell inhibitory activity, or a variant or fragment thereof of CDH1 (e-cadherin) that can have NK cell inhibitory activity, CD155 (poliovirus receptor) or a fragment thereof that can have NK cell inhibitory activity, or a variant of CD155 or a fragment thereof that can have NK cell inhibitory activity, A construct or composition according to any one of claims 1 to 4, encoding RTN4 or a fragment thereof that can have NK cell inhibitory activity, or a variant of RTN4 or a fragment thereof that can have NK cell inhibitory activity.

10. The construct or composition according to any one of claims 1 to 9, wherein the second polynucleotide encodes an antigen capable of inducing an immune response, and optionally the antigen is a COVID-19 antigen capable of inducing an immune response to COVID-19.

11. The construct or composition according to any one of claims 1 to 9, wherein the second polynucleotide encodes a protein that is normally endogenously produced, and optionally the normally endogenously produced protein is factor VIII or a biologically active fragment thereof.

12. A method for enhancing one or both of the magnitude and duration of the immune response induced by a vaccine, or for reducing one or more undesirable side effects associated with the administration of a vaccine, (a) A step of administering a pharmaceutically effective amount of a first polynucleotide encoding a polypeptide capable of having NK cell inhibitory activity to the target, (b) The step of administering a pharmaceutically effective amount of one or more vaccines to a target, Inhibition of NK cell activity enhances the magnitude and / or duration of the immune response induced by the one or more vaccines administered to the subject, and / or reduces one or more undesirable side effects associated with the administration of the vaccine. Optionally, the method enhances one or both of the antibody response and T cell response to the vaccine antigen of the vaccine. The method wherein, optionally, the vaccine is a polynucleotide vaccine containing a polynucleotide encoding a vaccine antigen, and optionally, the polynucleotide is an mRNA molecule.

13. The method according to claim 12, wherein a polynucleotide encoding a first polynucleotide and a vaccine antigen is present in a single polynucleotide construct, and optionally the single polynucleotide construct is a construct according to any one of claims 1 to 11.

14. The method according to claim 12, wherein the polynucleotide encoding the first polynucleotide and the vaccine antigen are present in separate polynucleotide constructs formulated in a single composition, and optionally the single composition is the composition according to any one of claims 1 to 11.

15. The method according to claim 12, wherein the polynucleotide encoding the first polynucleotide and the vaccine antigen are present in separate polynucleotide constructs formulated in separate compositions.

16. A method for enhancing the persistence of an exogenously administered polynucleotide, or for reducing one or more undesirable side effects associated with the administration of an exogenously administered polynucleotide, (a) A step of administering a pharmaceutically effective amount of a first polynucleotide encoding a polypeptide capable of having NK cell inhibitory activity to the target, (b) A step of administering a second exogenously administered polynucleotide to the subject, wherein optionally, the second exogenously administered polynucleotide encodes a protein normally produced endogenously, and optionally, the protein normally produced endogenously is factor VIII or a biologically active fragment thereof, The method wherein inhibition of NK cell activity enhances the persistence of the exogenously administered polynucleotide and / or reduces one or more undesirable side effects associated with the administration of the exogenously administered polynucleotide.

17. The method according to claim 16, wherein a second exogenously administered polynucleotide encodes an antigen capable of inducing an immune response, and optionally, the antigen is a COVID-19 antigen capable of inducing an immune response to COVID-19.

18. A construct or composition according to any one of claims 1 to 11, for enhancing one or both of the magnitude and duration of the immune response induced by the vaccine, or for reducing one or more undesirable side effects associated with the administration of the vaccine, optionally the vaccine being a polynucleotide vaccine comprising a polynucleotide encoding a vaccine antigen, and optionally the polynucleotide being an mRNA molecule.

19. The construct or composition according to claim 11 for enhancing the persistence of an exogenously administered polynucleotide, or for reducing one or more undesirable side effects associated with the administration of an exogenously administered polynucleotide.

20. A cell expressing a polynucleotide construct according to any one of claims 1 to 11.

Images