Method for enhancing antiviral activity of NK cells

Abstract

The present invention relates to a method for enhancing antiviral activity of NK cells in vitro, and in particular to the step of: enabling the NK cells to be in contact with NUCB2 protein or mRNA or an expression vector expressing the NUCB2 protein, thereby enhancing antiviral activity of the NK cells.

Description

Methods to promote the antiviral activity of NK cells Technical Field

[0001] This invention relates to the field of medicine, and more specifically to a method for promoting the antiviral activity of NK cells. Background Technology

[0002] Cytomegalovirus (CMV) is a DNA virus belonging to the herpesvirus group. Also known as a cell inclusion body virus, it causes cells to swell and possess large intranuclear inclusion bodies. CMV is widely distributed and can infect other animals, causing infections in various systems, primarily the reproductive and urinary systems, central nervous system, and liver, ranging from mild asymptomatic infection to severe defects or death. Mouse CMV infection in rats and mice is similar to HCMV infection in humans, making it a common animal model.

[0003] MCMV infection is divided into three phases: (a) acute infection, where viral titers in visceral organs peak on days 3 to 5 post-infection (pi), CD8 + T cell responses peak on day 7 post-infection; (b) persistent infection, viral replication is usually controlled in visceral organs, but titers peak in salivary glands; (c) latent infection, limited viral gene expression can be detected by RT-PCR, but infectious virus is only produced sporadically. In NK cells, Ly49H is the primary cell responsible for resisting MCMV infection. + NK cell population, Ly49H after MCMV infection + NK cells increased significantly, and Ly49H + NK cell cytotoxicity was also significantly increased.

[0004] Therefore, it is necessary to further promote the function of NK cells so that they can play an efficient role in fighting viral infections. Summary of the Invention

[0005] The purpose of this invention is to provide a method for enhancing the antiviral activity of NK cells.

[0006] A first aspect of the present invention provides a method for promoting the antiviral activity of NK cells in vitro, comprising the steps of:

[0007] (a) Contacting NK cells with NUCB2 protein or mRNA or expression vector expressing said NUCB2 protein to enhance the antiviral activity of said NK cells.

[0008] In another preferred embodiment, step (a) further includes contacting NK cells with the m157 protein or mRNA or expression vector expressing the m157 protein.

[0009] In another preferred embodiment, the contact between NK cells and the NUCB2 protein, and the contact between NK cells and the m157 protein, occur sequentially or simultaneously.

[0010] In another preferred embodiment, the method further includes step (b): isolating LY49H-positive NK cells therein.

[0011] In another preferred embodiment, in step (a), the NK cells do not come into contact with the NUCB1 protein.

[0012] In another preferred embodiment, the virus includes: cytomegalovirus.

[0013] In another preferred embodiment, the expression vector includes a viral vector.

[0014] In another preferred embodiment, the viral vector is selected from the group consisting of adeno-associated virus vectors, lentiviral vectors, or combinations thereof.

[0015] In another preferred embodiment, the protein comprises a full-length protein or a protein fragment.

[0016] In another preferred embodiment, the amino acid sequence of m157 is shown in SEQ ID NO.:4.

[0017] In another preferred embodiment, the amino acid sequence of NUCB2 is shown in SEQ ID NO.:2.

[0018] A second aspect of the invention provides the use of NUCB2 protein or its mRNA or expression vector for the preparation of compositions or formulations selected from the group consisting of: (A) enhancing the ability of NK cells to fight viral infections; and (B) treating a subject with a viral infection.

[0019] In another preferred embodiment, the composition or formulation does not contain the NUCB1 protein.

[0020] In another preferred embodiment, the subject does not have cancer or tumors.

[0021] A third aspect of the invention provides the use of a protein binding promoter for preparing a formulation or composition for use selected from the group consisting of: (a) promoting the binding of nucleobinding protein 2 (NUCB2) to the LY49H receptor; (b) promoting the proportion of LY49H-positive NK cells; (c) downregulating the cytomegalovirus (CMV) load in a subject; and (d) treating a subject with a viral infection.

[0022] The protein binding promoter is selected from m157 or fragments thereof or similar substances.

[0023] In another preferred embodiment, the analogue has a homology or sequence identity with m157 of ≥85%, preferably ≥90%, more preferably ≥95%, and most preferably ≥98%.

[0024] In another preferred embodiment, the protein binding promoter does not significantly promote the binding of nucleobinding protein 1 (NUCB1) to the LY49H receptor.

[0025] In another preferred embodiment, the virus includes cytomegalovirus.

[0026] In another preferred embodiment, the cytomegalovirus is mouse cytomegalovirus (MCMV) or human cytomegalovirus (HCMV).

[0027] In another preferred embodiment, the subject has a normal or elevated level of NUCB2 expression.

[0028] A fourth aspect of the present invention provides an active ingredient combination comprising:

[0029] (i) a first active ingredient, wherein the first active ingredient is selected from nucleobinding protein 2 (NUCB2); and

[0030] (ii) A second active ingredient, wherein the second active ingredient is selected from m157 or a fragment thereof or the like.

[0031] In another preferred embodiment, the NUCB2 is of non-viral origin.

[0032] In another preferred embodiment, the NUCB2 is derived from mammals, preferably from rodents (rats, mice) and primates (e.g., humans), and more preferably from humans.

[0033] In another preferred embodiment, the analogue has a homology or sequence identity with m157 of ≥85%, preferably ≥90%, more preferably ≥95%, and most preferably ≥98%.

[0034] In another preferred embodiment, the weight ratio of the first active ingredient to the second active ingredient is 1:1000 to 1000:1, more preferably 1:100 to 100:1, and even more preferably 1:10 to 10:1.

[0035] The fifth aspect of the invention provides the use of the combination of active ingredients as described in the fourth aspect of the invention for the preparation of a medicament or formulation for use selected from the group consisting of: (a) promoting the binding of nucleobinding protein 2 (NUCB2) to the LY49H receptor; (b) promoting the proportion of LY49H-positive NK cells; (c) downregulating the cytomegalovirus (CMV) load in a subject; and (d) treating a subject with a viral infection.

[0036] A sixth aspect of the present invention provides a pharmaceutical composition comprising:

[0037] (1) The combination of active ingredients as described in the fourth aspect of the present invention; and

[0038] (2) Pharmaceutically acceptable carrier.

[0039] In another preferred embodiment, the content of the active ingredient combination in the pharmaceutical composition is 0.01-99 wt%, more preferably 0.1-90 wt%, based on the total weight of the pharmaceutical composition.

[0040] In another preferred embodiment, the dosage form of the pharmaceutical composition is selected from the group consisting of: injections, oral preparations, patches, and transdermal preparations.

[0041] In another preferred embodiment, the injectable is a liquid formulation or a lyophilized formulation.

[0042] In another preferred embodiment, the method of administration of the pharmaceutical composition is selected from the group consisting of intraperitoneal, intravenous, intramuscular, parenteral, transdermal, or combinations thereof.

[0043] In another preferred embodiment, the preparation is a laboratory preparation.

[0044] A seventh aspect of the present invention provides a method for treating a subject with a viral infection, comprising the steps of administering to the subject a therapeutically effective amount of NUCB2 protein, m157, or NK cells activated by the method described in the first aspect of the present invention.

[0045] In another preferred embodiment, the virus includes: cytomegalovirus.

[0046] In another preferred embodiment, the object is a human or a non-human mammal.

[0047] In another preferred embodiment, the non-human mammals include rodents (such as mice, rats, and rabbits) and primates (such as monkeys).

[0048] In another preferred embodiment, the NK cells are LY49H-positive NK cells.

[0049] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0050] Figure 1 shows that m157 promotes the binding of NUCB2 and Ly49H. Figure A is a schematic diagram of the co-secretion expression system. The extracellular segment of Ly49H with a secretion signal peptide and its derived protein are tagged with an FC tag. This protein, along with NUCB cells carrying their own secretion signal and a modified m157 cell carrying an interleukin-2 secretion signal, is co-transfected into HEK293T cells, thus concentrating them all in the culture medium. 24 hours after transfection, the FC tag can be directly captured by Protein A / G, resulting in co-precipitation with the binding protein. Figures B and C show the co-precipitation of HEK293T cells 24 hours after transfection with the corresponding plasmid, followed by Western blot analysis. All results were independently replicated at least twice.

[0051] Figure 2 shows the establishment of a mouse model of acute MCMV infection. Six-week-old C57 male mice were intraperitoneally injected with purified third-generation salivary MCMV virus diluted with PBS, 2 x 10⁶ mmol / L per mouse. 4 The relative content of the viral gene IE1 in the genomic DNA of spleen (Figure A) and liver (Figure B) of uninfected and infected mice 1 to 4 days after infection was detected by PFU and qPCR. Four mice were used in each group, and unpaired t-test (two-tailed) statistical analysis was performed. P values ​​were set as 0.05 (*), 0.01 (**), 0.001 (***), and 0.0001 (****). A P value greater than 0.05 was expressed as not statistically significant (ns). Data are expressed as mean ± standard error (SEM).

[0052] Figure 3 shows that NUCB2 KO mice, but not NUCB1 KO mice, had higher MCMV viral loads than WT mice. Eight-week-old C57 male mice were intraperitoneally injected with purified third-generation salivary gland MCMV diluted in PBS, at a dose of 2 x 10⁻⁶ per mouse for the NUCB1 / 2 groups. 4 PFU, 5 x 10 g per Ly49H KO group mouse 3 The relative viral genome content in the genomic DNA of mouse spleen (Figure AC) or liver (Figure DF) tissues 3 days after infection was detected by PFU and qPCR. Unpaired t-tests (two-tailed) were performed on NUCB1 (n=5), NUCB2 WT (n=6), NUCB2 KO (n=5), and Ly49H KO (n=5) mice. P-values ​​were set at 0.05 (*), 0.01 (**), and 0.001 (***), respectively. A P-value greater than 0.05 was considered statistically insignificant (ns). Data are expressed as mean ± standard error (SEM).

[0053] Figure 4 shows that the virus was not completely cleared and liver damage persisted in NUCB2 KO mice on day 7 after infection. Eight-week-old C57 male mice were intraperitoneally injected with purified third-generation salivary gland MCMV virus diluted with PBS, 2 x 10⁸ mmol / L per mouse. 4The content of viral genome IE1 in genomic DNA of spleen (Figure A) or liver (Figure B) tissues of mice 7 days after infection was detected by PFU and qPCR. Five mice were used in each group, and an unpaired t-test (two-tailed) was performed. P-values ​​were set at 0.05 (*), 0.01 (**), and 0.001 (***), respectively. A P-value greater than 0.05 was expressed as not statistically significant (ns). Data are expressed as mean ± standard error (SEM). Figure C shows a representative HE-stained image of the liver of NUCB2 WT or KO mice on day 7 after infection; black arrows indicate lymphocyte infiltration and hepatocyte ballooning degeneration. All experimental results were independently replicated at least twice.

[0054] Figure 5 shows the NK cells and Ly49H levels in NUCB2 KO mice during acute infection. + The proportion of NK cells decreased. Figure A shows purified third-generation salivary gland MCMV virus diluted with PBS, administered intraperitoneally to 8-week-old C57 male mice (2 x 10⁻⁶ cells per mouse). 4 PFU, flow cytometry representation of spleens in infected mice 3 days post-infection; Figures B and C show the percentage of positive cells, with 5 mice per group; Figures D and E show the percentage of positive cells in uninfected mice, with 3 mice per group, using unpaired t-tests (two-tailed). P-values ​​were set at 0.05 (*), 0.01 (**), and 0.001 (***), respectively. A P-value greater than 0.05 was considered statistically insignificant (ns). Data are expressed as mean ± standard error (SEM).

[0055] Figure 6 shows that overexpression of NUCB2, but not NUCB1, promotes the expression of hepatic perforin, IL15, and Ly49H. Seven-week-old C57 WT male mice were intraperitoneally injected with purified AAV virus diluted in PBS, 2 x 10⁻⁶ per mouse. 10 Seven days after infection, qPCR was used to detect the expression of NUCB2 (Figure A), perforin (Figures B, F), IL15 (Figure C, G), Ly49H (Figure D, H), and NUCB1 (Figure E). Unpaired t-tests (two-tailed) were performed on AAV-NUCB2 (n=4 per group) and AAV-NUCB1 (n=5 in the control group and n=6 in the experimental group). P-values ​​were set at 0.05 (*), 0.01 (**), and 0.001 (***), respectively. A P-value greater than 0.05 was considered not statistically significant (ns). Data are expressed as mean ± standard error (SEM).

[0056] Figure 7 shows the NUCB2 overexpression treatment regimen and expression efficiency assay. Figure A, NUCB2 overexpression treatment regimen for MCMV infection, seven-week-old C57 WT male mice, intraperitoneally injected with purified AAV virus diluted with PBS, 2 x 10⁻⁶ per mouse. 10VG, seven days after infection, were intraperitoneally injected with purified third-generation salivary gland MCMV virus diluted with PBS, 2 x 10^6 viruses per animal. 4 PFU was used to measure viral load on day 10 after infection. Figure B shows AAV in seven-week-old C57 male mice, with purified AAV virus diluted in PBS administered intraperitoneally, 2 x 10⁻⁶ ppm per mouse. 10 VG was performed, and liver and spleen homogenates were collected one week later. Western blot was used to detect HA tag expression, and Image J analysis was performed. All experimental results were independently repeated more than twice.

[0057] Figure 8 shows the reduced MCMV viral load in NUCB2-overexpressing mice. Seven-week-old C57 WT male mice were intraperitoneally injected with purified AAV virus diluted in PBS, 2 x 10⁻⁶ per mouse. 10 VG, seven days after infection, were intraperitoneally injected with purified third-generation salivary gland MCMV virus diluted with PBS, 2 x 10^6 viruses per animal. 4 PFU. qPCR was used to detect the relative viral genome content in NUCB2 mRNA in mouse livers (Fig. A, D) and in genomic DNA from spleen (Fig. B, E) or liver (Fig. C, F) tissues 3 days after infection. Unpaired t-tests (two-tailed) were performed on WT mice (n=5) and Ly49H KO mice (n=4), with P values ​​set at 0.05 (*), 0.01 (**), and 0.001 (***), respectively. A P value greater than 0.05 was considered statistically insignificant (ns). Data are expressed as mean ± standard error (SEM). Detailed Implementation

[0058] Through extensive and in-depth research, the inventors unexpectedly discovered for the first time that the protein binding enhancer m157, rather than NUCB1, enhances the binding of NUCB2 to the surface receptor LY49H, thereby promoting the antiviral function of NK cells and effectively inhibiting MCMV infection. This invention was completed based on this discovery.

[0059] Cytomegalovirus

[0060] Cytomegalovirus (CMV) is a DNA virus belonging to the herpesvirus group. Also known as a cell inclusion body virus, it infects cells with enlarged cytoplasmic inclusions. Human cytomegalovirus (HCMV) belongs to the Herpesviridae family and is a subfamily of human herpesvirus type 5 (HHV-5). HCMV is a widespread virus, with infection rates ranging from 40% to 100% in different countries and regions. The process of mouse cytomegalovirus (MCMV) infection in rats and mice is similar to that of HCMV infection in humans.

[0061] Nucleus-binding proteins (NUCBs)

[0062] Nucleobindins (NUCBs) are a family of calcium-binding secreted proteins, including NUCB1 and NUCB2. The function of NUCB2 was first reported in 2006 as suppressing appetite and reducing weight, but subsequent studies showed that NUCB2 does not have appetite or weight regulation functions under physiological conditions. The biological function of NUCB1 remains unclear. Therefore, the biological functions and molecular mechanisms of the NUCB family are still poorly understood. NUCB1 and NUCB2 proteins are evolutionarily conserved and share high homology; for example, the similarity between human and rat NUCB2 proteins is 87.4%, and the similarity between rat and mouse NUCB2 proteins is 95.7%. The DNA similarity between humans and mice is as follows: mouse NUCB1 is 84% ​​similarity, and mouse NUCB2 is 83% similarity. The protein sequence similarity is as follows: mouse NUCB1 is 89% similarity, and mouse NUCB2 is 87% similarity. NUCB proteins are expressed in a variety of tissues and cells. Under conditions such as stress or inflammation, NUCBs proteins can be induced to express.

[0063] m157

[0064] m157 is an MHC-like glycoprotein encoded by mouse cytomegalovirus (MCMV) and expressed on the surface of infected cell membranes. m157 belongs to the m145 family, which consists of related glycoproteins (approximately 20% amino acid identity), including the immune escape protein m152. These proteins are typically expressed early in infection and are essential for viral growth and replication in vitro. In vitro stimulation of m157 promotes IFN-γ secretion and ERK-phosphorylation levels in Ly49H+ NK cells and enhances NK cell killing ability; in vivo experiments show that m157 is deeply involved in NK cell proliferation and secondary immunity during viral infection via the Ly49H signaling pathway. The m145 family and m157 are also present in the genome of rat CMV. The sequence of m157 is shown in SEQ ID NO:4.

[0065] Combination of active ingredients, pharmaceutical composition

[0066] The present invention provides an active ingredient comprising NUCB2 and m157 or similar substances thereof.

[0067] Based on the above-mentioned components, the present invention provides a pharmaceutical composition that can be used for: (a) promoting the binding of nucleobinding protein 2 (NUCB2) to the LY49H receptor, (b) downregulating the viral load in a subject, and (c) treating a subject with a viral infection.

[0068] The present invention also provides a pharmaceutical composition comprising a combination of active ingredients within a safe and effective range, and a pharmaceutically acceptable carrier.

[0069] "Pharmaceutically acceptable carriers" refer to one or more compatible solid or liquid fillers or gel substances that are suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatibility" here refers to the ability of the components in the composition to interact with and incorporate the active ingredient of the invention without significantly reducing the efficacy of the active ingredient. Examples of pharmaceutically acceptable carriers include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerin, mannitol, sorbitol, etc.), and emulsifiers. Wetting agents (such as sodium dodecyl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, saline, buffer solutions, glucose, water, glycerol, polysorbate, ethanol, etc.

[0070] There are no particular limitations on the administration of the active ingredient combination or pharmaceutical composition of the present invention. Representative administration methods include (but are not limited to): oral, rectal, parenteral (intravenous, intramuscular or subcutaneous), etc.

[0071] As used herein, the term “effective amount” or “effective dose” means an amount that is functional or active in humans and / or animals and / or cells and is acceptable to humans and / or animals.

[0072] When using pharmaceutical preparations, safe and effective amounts of active ingredients are combined and administered to mammals.

[0073] It should be understood that the effective amount of the active ingredient described in this invention may vary depending on the administration method and the severity of the disease. The preferred effective amount can be determined by those skilled in the art based on various factors (e.g., through clinical trials). These factors include, but are not limited to: pharmacokinetic parameters such as bioavailability, metabolism, and half-life; disease severity; patient weight; patient immune status; and route of administration.

[0074] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules.

[0075] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, or tinctures. In addition to the active ingredient, liquid dosage forms may contain inert diluents conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, e.g., ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butanediol, dimethylformamide, and oils, particularly cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil, and sesame oil, or mixtures thereof. Besides these inert diluents, the composition may also contain adjuvants such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents, and fragrances.

[0076] In addition to the active ingredient, the suspension may contain suspending agents, such as ethoxylated isooctadecyl alcohol, polyoxyethylene sorbitol and dehydrated sorbitol esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances.

[0077] Compositions for parenteral injection may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions, or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents, or excipients include water, ethanol, polyols, and suitable mixtures thereof.

[0078] Experimental methods

[0079] 1. Plasmids and Antibodies

[0080] AAV 2 / 8, pHelper, and pAAV-TBG-PI-eGFP-WPRE-bGH were purchased from Addgene. pCMV-NUCB1, pCMV-NUCB2, pFUSE-hIgG1-Fc2-Ly49H, as well as L196P, H216D, and truncated mutant forms of Ly49H, and px330-Cas9 were pre-existing laboratory plasmids. All other plasmids were constructed in-house using homologous recombination; specific methods are detailed in the Vazyme ClonExpress™ II One Step Cloning Kit manual. pCMV-m157 used the pCMV vector. AAV-TBG-NUCB1 / 2 used the pAAV vector. The N143D, H144P, and S152A mutants of pCMV-NUCB2 were derived from pCMV-NUCB2.

[0081] NUCB2 N-segment antibody (N9414) was purchased from Sigma Aldrich; HRP-conjugated goat anti-human IgG antibody (D110150-0100) was purchased from Sangon Biotech; GAPDH antibody (AP0063) was purchased from Bioworld; HA tag antibody (901515) was purchased from Biolegend; Phospho-p44 / 42MAPK (Erk1 / 2) (Thr202 / Tyr204, D13.14.4E) was also purchased. Rabbit antibody (#8544) and p44 / 42MAPK (Erk1 / 2) antibody (#4695) were purchased from CST Biotechnology. HRP-conjugated goat anti-mouse and goat anti-rabbit antibodies (catalog numbers 31430 and 31460) were purchased from Invitrogen. TM Company, Brilliant Violet 605 TM Anti-mouse CD3 antibody, Brilliant Violet 421 TM Anti-mouse NK-1.1 antibody, Ly49H monoclonal antibody (3D10) FITC, and Alexa Flow cytometry antibodies such as 700 anti-mouse IFN-γ antibody and CD16 / 32 (MAb) were purchased from Biolegend.

[0082] 2. Cell lines and mice

[0083] HEK293T cells were purchased from ATCC, mouse hepatocellular carcinoma Hepa1-6 cells (catalog number: SCSP-512) were from the Chinese Academy of Sciences Cell Bank, and MEF10.1 cells were from the Shanghai Public Health Clinical Center of Fudan University. Cell passages were limited to no more than 8 times. HEK293T and Hepa1-6 cells were cultured in DMEM (Life Technologies) supplemented with 10% fetal bovine serum (GIBCO), while MEF10.1 cells were cultured in DMEM supplemented with 10% FBS and additionally containing 100X NEAA, 100X sodium pyruvate, and 1% penicillin antibiotic (GIBCO). All cell lines were regularly checked for mycoplasma contamination. Cells were cultured in a 37°C, 5% CO2 incubator.

[0084] NUCB1 / NUCB2 KO mice were constructed in our previous laboratory using CRISPER-Cas9 technology, and the background DNA was purified for at least 5 generations. Ly49H KO mice were constructed in-house; candidate sgRNAs were generated using inDelphi prediction and determined after excluding off-target effects. The design strategy is shown in Figure 2-1. The construction method is as described previously (Yang et al., 2014), where the ssDNA template was synthesized by PlatinumSmithKline. After the template plasmid was constructed, mMESSAGE and mMACHINE were used. TM T7 ULTRA Transcription Kit (Invitrogen TM AM1345) and MEGA shortscript TM Kit (Invitrogen) TM The AM1354 kit was used for in vitro transcription of Cas9 mRNA and sgRNA, respectively, using MEGA clear reagents. TM Kit (Invitrogen) TM After purification (AM1908), store at -80℃. When using, use UltraPure... TM DNase / RNase-free distilled water (Invitrogen) TM A mixture of Cas9 mRNA (100 ng / μl) and sgRNA (100 ng / μl) was prepared and injected into the cytoplasm via an animal platform. After backcrossing with WT C57 mice and background purification for 5 generations, it was used in the experiments described in this paper.

[0085] All mice were C57BL / 6J background mice and held in a dedicated pathogen-free (SPF) animal facility. Infectivity experiments were conducted in the biosafety laboratory of the Shanghai Institute of Immunology and Infection, Chinese Academy of Sciences. Sex- and age-matched mice aged 7 to 16 weeks were used in paired experiments, with littermates used whenever possible. Experiments consisted of male and female mice, with donors and recipients of the same sex used in each individual experiment.

[0086] 3. Immunoprecipitation and in vitro binding

[0087] HEK293T cells were seeded and cultured in 6-well cell culture plates using Lipofectamine. TM 3000 (Invitrogen) TMAfter transfecting the corresponding plasmid (L3000075), the culture medium was cultured for 24 h. The supernatant was collected and centrifuged at 12000 rpm, 4℃ for 5 min. The supernatant was transferred to a new EP tube. 100 μl of the supernatant was taken out, 20 μl of 6X SDS Loading Buffer was added, mixed, centrifuged, boiled at 100℃ for 10 min, centrifuged at low speed, and stored at -20℃ as the Input sample. 20 μl of Protein A / G beads (Santa Cruz, SC-2003) was added to the remaining supernatant and incubated at 4℃ for 2 h. The agarose beads were washed three times with 1X Lysis Buffer, the supernatant was aspirated, 40 μl of 2X SDS Loading Buffer was added, mixed, centrifuged, boiled at 100℃ for 10 min, centrifuged at low speed, and stored at -20℃ as the IP sample. The sample was detected by Western blot.

[0088] 4. In vitro stimulation and interferon secretion experiment

[0089] HEK293T cells were seeded and cultured in 10cm culture dishes using Lipofectamine. TM After transfecting plasmids Lac Z, NUCB1, NUCB2, and m157 with 3000, culture for 48 h, collect the culture supernatant, take 50 μl for detection, and aliquot the rest and freeze at -80℃.

[0090] Spleen tissue was aseptically harvested from WT / Ly49H L196P mice. After grinding with a syringe, the tissue was passed through a 40-micron sieve, lysed with erythrocyte lysis buffer, and resuspended in 40% Percoll. The tissue was then plated with a 50 / 47.5 / 45 / 42.5 / 40% Percoll gradient and centrifuged at 2000 rpm for 25 min, with the speed increasing and decreasing by 3 times. The 45% and 47.5% bilayers were collected and purified to obtain LGL cells, predominantly NK cells. After counting, the cells were seeded into 24-well plates at 2 x 10⁶ cells per well. CM stimulation with NUCB / m157 was added for 20 min. 100 μl of 2X SDS loading buffer was added to each well, mixed, centrifuged, boiled at 100°C for 10 min, mixed again, and centrifuged again. Samples were analyzed by Western blot.

[0091] For the interferon secretion assay, after red blood cell cleavage and washing once with PBS, cells were counted and seeded into low-absorption 24-well plates at 3 x 10⁶ cells per well. CM stimulation containing NUC B / m157 was added directly. One hour later, Monensin (1000X, Invitrogen) was added. TM Continue culturing for 4-6 hours. Fix with 2% PFA on ice for 30 minutes, and analyze the samples by flow cytometry.

[0092] 5. Preparation of AAV virus

[0093] Virus packaging and preparation: HEK293T cells were seeded and cultured in 15cm culture dishes. Plasmids were prepared at a molar ratio of AAV2 / 8, pHelper, and AAV-TBG-GFP / NUCB1 / NUCB2 of 1:1:1, with 420μg of plasmid per 20 cell trays. The plasmids were dissolved in 40mL PBS, mixed by inversion, and then 1.26mL of 1mg / mL PEI was added. After mixing by pipetting, the mixture was incubated at room temperature for 30min. 2mL of the above liquid mixture was slowly added to each cell tray, and the cells were cultured for approximately 72h.

[0094] Collect the culture supernatant into a 50 mL centrifuge tube, centrifuge at 4000 rpm for 5 min, and filter the supernatant through a 0.22 μm PES filter membrane into a sterile 500 mL container. Collect the remaining cells in PBS using a cell scraper, combine with the centrifuged precipitate, flash freeze in liquid nitrogen, and thaw in a 37°C water bath. Repeat this process four times to release virus particles. Centrifuge at 4°C, 3900 rpm for 10 min to remove cell debris, resulting in Virus Stock Solution I. Add 25 mL of PEG solution to every 100 mL of supernatant. Add a magnetic rotor, stir slowly at 4°C for 1 hour, and let stand for 3 hours to allow complete precipitation. Centrifuge at 4°C, 3900 rpm for 15 min, discard the supernatant, and resuspend in Virus Stock Solution I. Add 50 units of benzonase per mL, incubate at 37°C for 30 min, centrifuge at 4°C, 3900 rpm for 15 min, and collect the supernatant as Virus Stock Solution II. Store on ice overnight for subsequent purification.

[0095] Virus purification and titer: Prepare 15% Opti-prep using NaCl / PBS-MK buffer, and prepare 25%, 40%, and 60% Opti-prep solutions using PBS-MK buffer. Add phenol red-labeled 25% and 60% Opti-prep solutions. Lay a gradient according to the ratio of Virus Stock II: 8 mL of 15% Opti-prep, 5 mL of 25% Opti-prep, 5 mL of 40% Opti-prep, and 3 mL of 60% Opti-prep. Make up the remaining volume with PBS.

[0096] After centrifugation at 200,000g and 4℃ for 2 hours, 40% of the sample was transferred to Centrifugal filter units (MWCO 100kDa, Millipore). The sample was washed with PBS + 200mM NaCl + 0.001% Pluronic F68, and then centrifuged at 3500rpm at 4℃ until the liquid was concentrated to 500μL. The liquid was then aliquoted and stored at -80℃.

[0097] Prepare 2x 10⁻¹⁰ AAV-TBG-GFP original plasmid. 8 2 x 10 7 2 x 10 6 2 x 10 5 2 x 10 4 A concentration gradient of mol / L was used to plot a standard curve. The purified virus sample was serially diluted, and the ITR sequence was detected by qPCR analysis using the SYBR Green method. The titer of AAV virus, VG / mL (Vector Genomes per mL), was calculated: the number of genomes of the viral vector per unit volume.

[0098] 6. Preparation and titer determination of MCMV virus

[0099] MCMV (Smith strain ATCC VR-1399, batch number 1698918) 5x10 3 Balb / C mice were infected with plaque-forming unit (PFU) MCMV via intraperitoneal injection for 3-5 weeks. After 3 weeks, salivary gland tissue was aseptically isolated, weighed, ground on ice, and prepared into a 10% tissue homogenate. The homogenate was subjected to three freeze-thaw cycles at -80°C, centrifuged, and the supernatant was collected as P1 generation. The homogenate was aliquoted and stored at -80°C. After determining the titer, the above operation was repeated. Each infection lasted for two weeks. The virus after three generations of purification was used in the experiments described in this paper.

[0100] MCMV virus titer determination: MEF10.1 cells were seeded in 12-well plates and tested on the same day they reached 90% confluence. The virus was serially diluted 10-fold, with 0.2 mL of virus dilution added to each well, in duplicate for each dilution. The cells were incubated at 37°C for 1 hour, agitated every 10 minutes. The virus inoculum was discarded, and 2 mL of DMEM containing 1.5% agarose was quickly added to each well. The cells were incubated at room temperature for 30 minutes, then cultured at 37°C for approximately one week.

[0101] Add 1 mL of 4% PFA solution to each well and incubate at room temperature for 1 h. Discard the supernatant and rinse the plate with running water to remove the agarose coating. Wash with water to remove residual PFA, invert for 5 minutes to air dry, and stain with 1 mL / well of 0.03% methylene blue for 5 minutes. Wash with water and air dry. Count the corresponding wells with a gradient of 10 to 100 plaques under a microscope and calculate the corresponding viral titer (PFU) per mL.

[0102] 7. Analysis of MCMV In vivo infection and viral load

[0103] The aforementioned salivary gland MCMV was diluted with PBS according to experimental requirements, and C57 mice were intraperitoneally injected with 2x10⁻⁶ ppm. 4 / 5x10 3One PFU of MCMV was collected. Infection experiments were conducted in a biosafety level 2 animal facility. Liver and spleen tissues were aseptically isolated on the third and seventh days after infection, weighed, ground, and prepared into 10% tissue homogenates. Genomic DNA was extracted using a blood tissue genomic extraction kit (Tiangen, DP304). Samples were stored at -20°C, and the relative contents of β-actin and the early viral gene MCMV-IE1 were detected by q-PCR.

[0104] For flow cytometry samples: Spleen tissue was aseptically harvested from WT and KO mice infected with MCMV, ground, and passed through a 40 μm sieve. After red blood cell lysis, the cells were fixed on ice with 2% PFA for 30 minutes, followed by cell perforation and staining with 0.2% saponin buffer. Cells were incubated on ice with anti-CD16 / 32 (MAb) for 30 minutes to avoid nonspecific staining.

[0105] AAV-TBG-GFP / NUCB1 / NUCB2 viral suspension was diluted with PBS according to experimental requirements, and 2 x 10⁻⁶ doses were injected intraperitoneally into C57 mice. 10 For each VG animal, liver and spleen tissues were aseptically isolated 7 days after infection and stored in Trizol at -80℃. The relative mRNA levels of β-actin, NUCB2, perforin, IL15, and Ly49H were detected by qPCR.

[0106] 8. Flow cytometry

[0107] After the samples are fixed, count approximately 2 x 10^6 samples per tube. 6 Cells were perforated and stained with 0.2% saponin buffer, and incubated in the dark for 15 min. Subsequent antibody preparations were all done using saponin buffer. Cells were incubated on ice with anti-CD16 / 32 (MAb) for 30 min to avoid non-specific staining. After inhibition, a mixture of CD3, NK1.1, Ly49H, and IFN-γ monoclonal antibodies was prepared according to the sample number and incubated on ice in the dark for 1 h for staining. Cells were then washed with PBS containing 2% fetal bovine serum (FBS) and analyzed. Cell fluorescence signals were monitored using a Cytoflex flow cytometer (Beckman Coulter) and analyzed using the accompanying Cytexpert.

[0108] 9. Preparation of paraffin sections of mouse liver and hematoxylin-eosin staining

[0109] On day 7 after C57 mice were infected with MCMV, liver tissue was aseptically isolated, and the right lobe of the liver was fixed overnight in 4% PFA. It was washed three times with PBS for 10 min each time. Dehydration was performed using a gradient of 40-75-80-90-100% alcohol for 1 h each. The liver was then immersed in xylene for 10 min three times, followed by immersion in heated molten paraffin solution, then benzene paraffin for 30 min, and finally paraffin paraffin for 30 min. The mixture was then transferred to a 65℃ oven for overnight paraffin immersion. The next day, the paraffin solution was changed and immersed for 1 h before embedding. The paraffin blocks were appropriately trimmed, cut into 3-10 μm slides, spread in a 37℃ water bath, and then dried at 37℃ overnight.

[0110] Dewaxing was performed by washing with fresh xylene for 20 minutes each, followed by two immersions in anhydrous ethanol for 5 minutes each, and then washing with 75% ethanol for 5 minutes to rehydrate. The slides were then rinsed three times with tap water, stained with hematoxylin for approximately 5 minutes, rinsed three times with tap water, stained with eosin for 45 seconds, rinsed three times with tap water, and then immersed in anhydrous ethanol three times for 5 minutes each. Dehydration and clearing were then performed by immersion in fresh xylene for 5 minutes each. The slides were then mounted with neutral resin and air-dried overnight in a fume hood. Images were taken and analyzed using a Scan Z1 / BX53.

[0111] 10. Western blot for protein immunoblotting

[0112] Protein samples were separated using SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Polyacrylamide gels were prepared using 40% Acr-Bis (purchased from Sangon Biotech, B546013-0500). The stacking gel was 4% acrylamide, and the gel buffer was Tris-HCl at pH 6.8 with added SDS. The separating gel was prepared with an appropriate concentration based on the size of the target protein, and the gel buffer was Tris-HCl at pH 8.8 with added SDS. The electrode buffer was Tris-glycine buffer at pH 8.3 with added SDS. 500X PAGE blue dye (Beyotime Biotech, P0701) was added to the stacking gel for clear observation of the lanes. Electrophoresis was performed at a constant voltage of 80V for 30 min, then reduced to 120V until bromophenol blue reached the bottom of the gel, at which point electrophoresis was stopped. After electrophoresis, proteins were transferred to a nitrocellulose (NC) membrane using wet transfer. The membrane was then subjected to a constant current of 340mA for 60 min. Block with 5% skim milk for 1 hour, wash three times with TTBS, prepare antibody according to the instructions using primary antibody dilution buffer (Beyotime Biotech, P0701), incubate at room temperature for 2 hours or overnight at 4°C, wash three times with TTBS, prepare secondary antibody dilution buffer with 0.25% BSA at a working concentration of 0.25 μg / mL using TTBS, incubate at room temperature for 1 hour, wash three times with TTBS, and use chemiluminescent substrate (Thermo Scientific). TM (34580, 34095) Developed, photographed with Tanon 5200 scanning film scanner, and then processed and analyzed by Image J.

[0113] 11. Real-time quantitative PCR

[0114] RNA samples were extracted using the chloroform / isopropanol method to obtain total RNA from tissues. For every 500 μl Trizol, 100 μl chloroform was added, and the mixture was centrifuged at 13000 rpm for 15 min. The upper aqueous phase was aspirated, and an equal volume of isopropanol was added to precipitate the precipitate. The mixture was washed twice with 500 μl 75% ethanol, air-dried for 30 min, and then dissolved in a suitable amount of DEPC-treated water. Reverse transcription was performed using Hiscript II Reverse Transcriptase (Novizan, R201). The obtained cDNA was diluted tenfold, and based on the number of genes to be tested, 2 μL of diluent was required per well. This was then diluted twofold with DEPC-treated water and mixed 1:1 with SYBR Green premix (Takara, RR420A) containing the corresponding primers for assembly. Genomic DNA samples were directly mixed with SYBR Green premix containing the corresponding primers. Samples were added to Roche 96 / 384-well plates on ice, sealed, and briefly centrifuged to ensure homogenization. Fluorescent qPCR was performed using a Roche LightCycler R 384 / 96 with a pre-denaturation condition of 95℃ for 50 s. 45 amplification cycles were performed: 95℃ for 5 s, 60℃ for 20 s, and 72℃ for 35 s.

[0115] 12. Quantitative and Statistical Analysis

[0116] GraphPad Prism software (version 9) was used to perform t-tests (two-tailed), multiple t-tests, and the nonparametric statistical test Mann-Whitney test for unpaired and paired students. Statistical significance was determined by probability (P). P values ​​were indicated by asterisks and set to 0.05 (*), 0.01 (**), and 0.001 (***), respectively. A P value greater than 0.05 was considered statistically insignificant (ns). Data are presented as mean ± standard error (SEM). The number of mice used to generate the data is provided in the graph legend.

[0117] Example 1. The presence of m157 affects the binding of NUCB2 and Ly49H.

[0118] NUCB1 / 2 are secretory proteins containing a secretion signal peptide, while Ly49H and the viral ligand m157 are receptors on the cell membrane surface. Therefore, studying their interactions requires selecting a suitable medium. A co-secretion expression system was constructed, in which the extracellular domain of Ly49H (amino acid residues 67-266) (SEQ ID NO:9) is linked to an FC tag, and NUCB1 / 2 and m157 containing the interleukin IL2 secretion signal peptide are linked to an HA tag, and both are simultaneously overexpressed in HEK 293T (Figure 1A). Immunoprecipitation experiments verified that both the viral ligand m157 and the NUCB in this system can bind to the extracellular domain of Ly49H, and the binding affinity of m157 is stronger than that of NUCB (Figure 1B).

[0119] NUCB is a widely expressed protein under physiological conditions, while m157 is expressed only during MCMV infection and is not present on the cell surface under physiological conditions. To simulate the changes in the interaction between NUCB and Ly49H before and after infection, the co-expression of m157 was compared to determine whether it affected NUCB binding, while keeping the transfection levels of Ly49H and NUCB constant. The presence of m157 was found to enhance the binding efficiency of NUCB2, but had no significant effect on the binding of NUCB1 (Figure 1C). These results suggest that the binding of NUCB2 and Ly49H may play an important role during viral infection.

[0120] Example 2. The MCMV viral load in NUCB2 KO mice, instead of NUCB1 KO mice, was significantly higher than that in WT control mice.

[0121] Literature reports that NK cell activity and viral load reach a peak 3-4 days after MCMV infection (Allan and Shellam, 1984). The viral replication cycle is definite, and the order of infection in different tissues obviously affects the results at the time endpoint. Therefore, time-point analysis was first used to determine the optimal time for phenotypic analysis. C57B6 mice were infected with MCMV derived from salivary glands, purified three generations in BALB-c mice. Infected tissues were collected at different time points during the acute infection phase, and genomic DNA was extracted for relative quantification. Overall, the content of the early MCMV gene IE1 in genomic DNA at different time points showed a curve-like pattern, reaching a peak first and then decreasing. Furthermore, the time to reach the peak differed in different tissues: day 3 in the spleen (Figure 2A) and day 2 in the liver (Figure 2B), consistent with previous assumptions. On day 3 of MCMV infection, the viral load was higher in the main infected tissues, the liver and spleen, and the data dispersion was small; therefore, day 3 was determined as the time point for subsequent experimental detection.

[0122] To verify whether NUCB exerts an antiviral effect during the acute infection phase, previously constructed NUCB1 and NUCB2 KO mice, as well as a self-constructed Ly49H KO mouse, were used. The viral genome content in the main infected tissues of KO mice on day 3 of MCMV infection was compared with that of their littermates (WT mice). First, by comparing the viral load in the spleen and liver tissues of Ly49H KO mice and WT mice, the viral load in Ly49H KO mice was significantly higher than that in WT mice, confirming that the decreased antiviral function due to the loss of Ly49H function is well reflected in the system (Figure 3C, F). Comparing the viral load in the liver and spleen tissues of NUCB1 / 2 KO mice and WT mice on day 3 of infection, there was no significant difference in viral load between NUCB1 KO mice and WT mice (Figure 3A, D); however, the viral load in NUCB2 KO mice was significantly higher than that in WT mice (Figure 3B, E). These results suggest that NUCB2, rather than NUCB1, exerts an anti-MCMV effect during the acute infection phase.

[0123] Example 3. NUCB2 may still play a role at the end of the acute infection phase of MCMV.

[0124] According to literature reports, mice with impaired Ly49H function cannot achieve complete viral clearance (Alexandre et al., 2014). Comparing viral load and liver damage in NUCB2 KO mice and WT mice on day 7 after infection, the MCMV-IE1 content in the liver tissue of NUCB2 KO mice was significantly higher than that in WT mice, indicating that the virus was not completely cleared from the liver tissue; while there was no significant difference in viral content in the spleen tissue (Figure 4A). CD8+ T cell immunity was initiated on day 7 after infection, and the liver is the main site of latent MCMV virus infection, which may explain this. HE staining results showed that lymphocyte infiltration and hepatocyte ballooning degeneration were still present in the portal area of ​​NUCB2 KO mice (Figure 4B), while these had been repaired in normal mice. These results suggest that NUCB2 still plays a role in the liver at the end of the acute infection phase.

[0125] Example 4. NK and Ly49H in NUCB2 KO mice during acute infection + NK ratio decline

[0126] During the acute infection phase, the control of MCMV virus mainly relies on innate immunity, primarily NK cells. Is the phenotype of NUCB2 KO mice during the acute infection phase due to impaired NK cell function? To address this question, flow cytometry analysis was performed on the spleen lymphocytes of NUCB2 KO mice on day 3 of MCMV infection. The analysis revealed a significant decrease in the proportion of NK1.1+ cells compared to WT mice, and also showed an increase in NK1.1+ Ly49H cells. +The proportion of NK1.1+ cells was also reduced (Figure 5A-C). Furthermore, the proportion of NK1.1+ cells and NK1.1+ cells in uninfected NUCB2 KO mice was also lower. + Ly49H + The cell proportions were not significantly different from those in WT (Fig. 5D, E). In summary, the interaction among NUCB2, MCMV virus with m157, and Ly49H is crucial for the normal functioning of NK cells during the acute infection phase.

[0127] Example 5. Overexpression of NUCB2, instead of NUCB1, can promote the expression of liver perforin, IL15, and Ly49H.

[0128] The previous results showed that NUCB2 KO significantly affected the antiviral function of NK cells. Next, we investigated whether overexpression of NUCB2 would affect NK cell function. Recombinant adeno-associated virus (AAV) is a commonly used overexpression vector system in mouse models. Using AAV with the TBG promoter, NUCB2-specific overexpression in liver tissue was achieved. Compared to the control group overexpressing GFP, the mRNA expression levels of perforin, interleukin-15, and Ly49H in the liver tissue of mice overexpressing NUCB2 were significantly increased (Figures 6A-6D). Perforin-mediated cell killing is an important mechanism for NK cells to clear viruses, interleukin-15 is a cytokine that promotes NK cell proliferation, and the expression level of Ly49H is directly related to NK cell function; all of these genes can be expressed by NK cells. In contrast, overexpression of NUCB1 had no significant effect on perforin, IL15, and Ly49H in mice (Figures 6E-G).

[0129] Example 6. NUCB2 overexpressing mice had lower MCMV viral load than controls.

[0130] Overexpression of NUCB2 can prematurely activate NK cells. The next step was to investigate whether premature NUCB2 overexpression could achieve better control or even prevention of MCMV infection. In mice, the protein overexpressed via AAV virus maintains a stable expression level for approximately one week. To eliminate the influence of unstable NUCB2 overexpression on the experimental results, MCMV infection was initiated one week after AAV overexpression of NUCB2 (Figure 7A). Western blot analysis showed that NUCB2 produced in the liver could not be detected in the corresponding spleen homogenate, possibly due to the shorter half-life of NUCB2 in the spleen (Figure 7B). This phenomenon allows for comparison of different infection statuses in the liver and spleen of mice within the same group, thus better characterizing whether NUCB2 overexpression has an antiviral effect.

[0131] qPCR results showed that in WT mice overexpressing NUCB2, reinfection with MCMV on day 7 significantly increased the level of NUCB2 mRNA in the liver compared to the control group (Fig. 8A, D). On day 3 of infection, the viral load in the liver of WT mice was significantly lower than that in the control group (Fig. 8C), while the viral load in the spleen was not different from the control group (Fig. 8B). However, in Ly49H KO mice, this inhibitory activity of NUCB2 on viral load disappeared. Although the level of NUCB2 mRNA in the liver was significantly increased (Fig. 8D), the viral load in the liver and spleen was not different from that in the control group (Fig. 8E-8F). These results indicate that overexpression of NUCB2 has an inhibitory effect on MCMV infection, and this effect is mediated through Ly49H.

[0132] The sequences used in this invention are shown in Table 1 below:

[0133] Table 1

[0134] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A method of promoting NK cell antiviral activity in vitro, characterized in that, Including the following steps: (a) Contacting NK cells with NUCB2 protein or mRNA or expression vector expressing said NUCB2 protein to enhance the antiviral activity of said NK cells.

2. The method of claim 1, wherein, Step (a) further includes: contacting NK cells with the m157 protein or mRNA or expression vector expressing the m157 protein.

3. The method of claim 1, wherein, The contact between NK cells and the NUCB2 protein, and the contact between NK cells and the m157 protein, may occur sequentially or simultaneously.

4. The method of claim 1, wherein, The method further includes step (b): isolating LY49H-positive NK cells therein.

5. Use of NUCB2 protein or mRNA or expression vector thereof, characterized in that, Used to prepare a composition or formulation for use selected from the group consisting of: (A) enhancing the ability of NK cells to fight viral infections; (B) treating a subject with a viral infection.

6. Use of a protein binding promoter, characterized in that For the preparation of a formulation or composition, said formulation or composition for use selected from the group consisting of: (a) promoting the binding of nucleobinding protein 2 (NUCB2) to the LY49H receptor; (b) promoting the proportion of LY49H-positive NK cells; (c) downregulating the cytomegalovirus (CMV) load in a subject; (d) treating a subject with a viral infection; The protein binding promoter is selected from m157 or fragments thereof or similar substances.

7. Use according to claim 6, characterized in that, The protein binding promoters did not significantly promote the binding of nucleobinding protein 1 (NUCB1) to the LY49H receptor.

8. An active ingredient combination, characterized in that The combination of active ingredients includes: (i) a first active ingredient, wherein the first active ingredient is selected from nucleobinding protein 2 (NUCB2); and (ii) A second active ingredient, wherein the second active ingredient is selected from m157 or a fragment thereof or the like.

9. Use of an active ingredient combination according to claim 7, characterized in that Used to prepare a drug or formulation for use selected from the group consisting of: (a) promoting the binding of nucleobinding protein 2 (NUCB2) to the LY49H receptor; (b) promoting the proportion of LY49H-positive NK cells; (c) downregulating the cytomegalovirus (CMV) load in a subject; and (d) treating a subject with a viral infection.

10. A pharmaceutical composition, characterized by, The pharmaceutical composition comprises: (1) The combination of active ingredients as described in claim 8; and (2) Pharmaceutically acceptable carrier.

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