A culture medium for human metapneumovirus and its application

Abstract

The present invention relates to the field of biomedical technologies, and discloses a culture medium for human metapneumovirus and its application. The culture medium comprises the following components in parts by mass: basal medium: 850 to 920 parts; metabolic reprogramming module: including 0.05 to 0.15 part of 5-methyltetrahydrofolic acid, 0.3 to 0.7 part of N-acetyl-D-mannosamine, and 1.0 to 3.0 parts of sodium dichloroacetate; immune regulation module: including 0.005 to 0.015 part of RIG-I / MAVS pathway inhibitor; receptor induction module: including 0.5 to 1.5 parts of all-trans retinoic acid; redox balance module: including 0.1 to 0.5 part of polyethylene glycolated superoxide dismutase and 0.002 to 0.008 part of sodium selenite. The present invention can significantly increase the proportion of infectious virus particles, reduce the generation of defective particles, and at the same time prolong the production cycle of host cells and support large-scale continuous culture, providing a high-quality and efficient solution for the industrial production of gene therapy vectors and viral vaccines.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and specifically to a culture medium for human metapneumovirus and its application. Background Art

[0002] The large-scale production of viral vectors is a core link in gene therapy and vaccine development, but its efficiency and quality have long been limited by the dynamic imbalance among the metabolic stress of host cells, antiviral immune responses, and the functional integrity of viral particles.

[0003] Traditional processes usually optimize single components or use broad-spectrum inhibitors to forcibly expand the viral replication window, which easily leads to accelerated oxidative damage of host cells, disorder of key metabolic pathways, resulting in problems such as a high proportion of non-infectious defective particles and significant batch-to-batch titer fluctuations.

[0004] In addition, the insufficient stability of the culture medium components causes a rapid decline in virus production during long-term storage or continuous production, further increasing the cost and risk of large-scale application.

[0005] The prior art has not effectively coordinated the metabolic reprogramming required for virus replication, the precision of immunosuppression, and the maintenance of host cell homeostasis. There is an urgent need for a systematic solution to break through the quality and efficiency bottleneck. Summary of the Invention

[0006] Aiming at the deficiencies of the prior art, the present invention provides a culture medium for human metapneumovirus and its application, solving the technical problems of low proportion of infectious particles, large damage to host cells, and poor batch-to-batch stability in traditional virus production processes.

[0007] To achieve the above objectives, the present invention is realized through the following technical solutions:

[0008] The first aspect of the present invention provides a culture medium for human metapneumovirus, comprising the following components in parts by mass:

[0009] Basal medium: 850 - 920 parts;

[0010] Metabolic reprogramming module: including 0.05 - 0.15 part of 5-methyltetrahydrofolic acid, 0.3 - 0.7 part of N-acetyl-D-mannosamine, 1.0 - 3.0 parts of sodium dichloroacetate;

[0011] Immune regulation module: including 0.005 - 0.015 part of RIG-I / MAVS pathway inhibitor;

[0012] Receptor induction module: including 0.5 - 1.5 parts of all-trans retinoic acid;

[0013] Redox balance module: including 0.1 - 0.5 part of polyethylene glycolated superoxide dismutase, 0.002 - 0.008 part of sodium selenite.

[0014] Among them, the metabolic reprogramming module includes 5-methyltetrahydrofolate (5-MTHF), N-acetyl-D-mannosamine (ManNAc), and sodium dichloroacetate (DCA); its core mechanism lies in reconstructing the metabolic network of the host cell and directionally enhancing the biosynthesis capabilities of nucleotides, glycosylation intermediates, and lipid precursors required for virus replication.

[0015] As a one-carbon unit donor, 5-MTHF significantly increases the de novo synthesis rate of pyrimidine nucleotides by activating the thymidylate synthase (TYMS) pathway, directly supporting the rapid replication of the HMPV RNA genome;

[0016] As a key precursor for sialic acid synthesis, ManNAc promotes the maturation of viral envelope proteins and the glycan-binding efficiency of host cell receptors by increasing the level of glycosylation modification on the surface of host cells;

[0017] DCA inhibits pyruvate dehydrogenase kinase (PDK), relieves the inhibition of oxidative phosphorylation in pyruvate metabolism, and promotes the large production of mitochondrial acetyl-CoA, providing sufficient raw materials for the synthesis of viral envelope lipids.

[0018] Among them, traditional culture media cannot effectively inhibit the innate immune response of host cells, resulting in premature blocking of virus replication. The present invention introduces an inhibitor of the RIG-I / MAVS pathway (such as KIN1148), and its innovative mechanism lies in specifically blocking the polymerization of MAVS protein downstream of the RIG-I signal and inhibiting the release of interferon-β (IFN-β), thereby extending the "golden window period" of virus replication. Compared with the existing technologies that use high-dose interferon-α (which instead inhibits the virus) or broad-spectrum immunosuppressants (such as dexamethasone), KIN1148 can achieve precise immunosuppression at a concentration of 0.1 - 0.3 μM and does not interfere with the virus adsorption and packaging processes.

[0019] Among them, one of the core reasons for the low HMPV infection efficiency is the insufficient expression level of host cell surface receptors (such as heparan sulfate proteoglycan, HSPG). The present invention adds all-trans retinoic acid (ATRA) to activate the retinoic acid receptor α (RARα) signaling pathway, inducing the upregulated expression of glycosaminoglycan chain synthases (such as EXT1 / 2) of HSPG, increasing the density of virus adsorption sites on the surface of host cells by 2 - 3 times. This strategy breaks through the passive mode that traditional technologies only rely on the expression of natural cell receptors and realizes the active regulation of receptor expression level for the first time.

[0020] Among them, the oxidative stress of host cells in the late stage of virus infection is the main reason for the decrease in virus production. This module uses a composite system of polyethylene glycolated superoxide dismutase (PEG-SOD) and sodium selenite:

[0021] PEG-SOD extends its half-life through polyethylene glycol modification and targets the elimination of mitochondrial superoxide anion radicals (O2 - ), avoiding oxidative damage caused by virus replication;

[0022] Sodium selenite, as an essential cofactor of glutathione peroxidase (GPX), enhances the cell's ability to detoxify lipid peroxides.

[0023] Through the above scheme, starting from the biological essence of virus hijacking host metabolism, the present invention specifically strengthens the nucleotide, glycosylation, and lipid synthesis pathways, breaking through the blindness of traditional nutritional supplements; and uses a small molecule targeted inhibitor (KIN1148) to replace broad-spectrum immunomodulators, extending the virus replication window period while maintaining cell viability; at the same time, actively upregulating host receptor expression through a chemical inducer (ATRA) to solve the core pain point of low virus adsorption efficiency; based on an antioxidant system coordinated by enzymes and coenzymes, achieving the maintenance of cell homeostasis under high virus load.

[0024] Preferably, in the metabolic reprogramming module, the mass ratio of 5-methyltetrahydrofolate to N-acetyl-D-mannosamine is 1:(4 - 6).

[0025] As a one-carbon unit carrier, 5-MTHF directly participates in the synthesis of thymidylate (dTMP), and its concentration needs to match the sialic acid metabolism driven by ManNAc. When the mass ratio of 5-MTHF:ManNAc is lower than 1:4, the pyrimidine synthesis rate exceeds the glycosylation ability, resulting in the accumulation of unglycosylated viral envelope proteins and affecting virus particle assembly;

[0026] When the ratio is higher than 1:6, excessive ManNAc may competitively inhibit nucleotide transporters (such as ENT1), instead restricting the intracellular transport efficiency of pyrimidine precursors.

[0027] Within the range of the mass ratio of 1:4 - 6, the methyl groups provided by 5-MTHF (through the folate cycle) and the N-acetylmannosamine provided by ManNAc (through the UDP-GlcNAc pathway) can form a complementarity to jointly support the simultaneous progress of virus genome replication (requiring pyrimidines) and envelope glycoprotein maturation (requiring sialic acid modification).

[0028] Preferably, the molar ratio of the polyethylene glycolated superoxide dismutase to sodium selenite is 1:(0.4 - 0.6).

[0029] PEG-SOD plays the role of the first antioxidant defense line by catalyzing the conversion of superoxide anion (O2 - ) into hydrogen peroxide (H2O2);

[0030] Sodium selenite, as a selenium source, drives the further reduction of H2O2 to water (H2O) by integrating into the active center of glutathione peroxidase (GPX), preventing lipid peroxidation caused by the accumulation of H2O2.

[0031] When the molar ratio is lower than 1:0.4, insufficient selenium supply leads to limited GPX activity and a lag in the H2O2 scavenging rate, triggering secondary oxidative damage; when the molar ratio is higher than 1:0.6, excessive selenium may competitively inhibit the active site of SOD, weakening the primary scavenging efficiency of O2 - of.

[0032] PEG modification can extend the half-life of SOD (about 72 hours), but its continuous catalytic activity depends on the timely scavenging of H2O2 by GPX to avoid inactivation due to excessive H2O2 concentration;

[0033] A molar ratio of 1:0.4 to 0.6 can ensure the dynamic matching of the H2O2 produced by each unit of SOD catalysis and the decomposition ability of GPX, maintaining redox homeostasis.

[0034] Preferably, the basal medium is a mixture of DMEM and Ham's F12, and the mixing volume ratio is 1:(0.9 - 1.1).

[0035] DMEM provides a high concentration of glucose (4.5 g / L) and essential amino acids (such as glutamine), supporting the rapid energy metabolism of host cells and the supply of carbon skeletons required for virus replication;

[0036] Ham's F12 supplements trace elements (such as sodium selenite, putrescine) and fat-soluble vitamins (such as biotin) lacking in DMEM, enhancing the antioxidant capacity of cells and the stability of the membrane structure.

[0037] Within the range of the mixing volume ratio of 1:0.9 to 1.1, the concentration gradients of the two components tend to be gentle, avoiding cell stress caused by extreme concentrations in a single medium (such as the high osmotic pressure of DMEM or the low buffering capacity of Ham's F12).

[0038] DMEM relies on the bicarbonate / CO2 buffer system, while Ham's F12 provides additional buffering capacity through HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid).

[0039] When the mixing volume ratio is close to equal (1:1), the molar concentration ratio of bicarbonate to HEPES reaches 1.8 - 2.2, which can stabilize the pH at 7.2 - 7.4 under 5% CO2 conditions, reducing the interference of frequent medium changes on virus adsorption.

[0040] The second aspect of the present invention provides a method for preparing the culture medium for human metapneumovirus described in the first aspect of the present invention, comprising the following steps:

[0041] Step a: Dissolution of basal medium and pH adjustment

[0042] 1. Dissolution operation:

[0043] Weigh DMEM and Ham's F12 dry powder according to a volume ratio of 1:(0.9 - 1.1), add ultrapure water (resistivity ≥ 18.2 MΩ·cm) to 85% - 92% of the total mass, and stir magnetically (200 - 300 rpm, 25 ± 2°C) until completely dissolved.

[0044] Add sodium bicarbonate (10 - 15 parts by mass), and continue stirring until the solution is clear.

[0045] 2. pH adjustment:

[0046] Precisely adjust the pH to 7.2 - 7.4 using 0.1 M HCl or NaOH solution (pH meter calibration accuracy ±0.02), and avoid component denaturation caused by local over - acidity or over - alkalinity.

[0047] 3. Filtration for impurity removal:

[0048] Pre - filter through a 0.22 μm polyethersulfone (PES) membrane to remove undissolved particles and microbial contamination.

[0049] Step b: Adding functional modules in stages

[0050] 1. Addition of metabolic reprogramming module:

[0051] Sequentially add 5 - methyltetrahydrofolic acid (0.05 - 0.15 parts by mass), N - acetyl - D - mannosamine (0.3 - 0.7 parts by mass), and sodium dichloroacetate (1.0 - 3.0 parts by mass). After adding each component, stir in the dark for 10 - 15 minutes (rotation speed 100 - 150 rpm) to ensure complete dissolution and no crystal precipitation.

[0052] 2. Addition of immune regulation module:

[0053] Take the DMSO stock solution of KIN1148 (10 mM), dilute it with the basal medium to a final concentration ≤ 0.1% (v / v), and add it drop - by - drop (dropwise addition speed ≤ 1 mL / min), while maintaining stirring (120 - 180 rpm) to avoid excessive local DMSO concentration causing cell membrane damage.

[0054] 3. Addition of receptor induction module:

[0055] Add the ethanol mother liquor (1 mM) of all-trans retinoic acid (0.5 - 1.5 parts by mass) to the system, control the final ethanol concentration ≤ 0.1% (v / v), and stir for 15 minutes until completely mixed.

[0056] Step c: Treatment by the redox balance module and preparation of the final product

[0057] 1. Premix redox components:

[0058] Dissolve polyethylene glycolated superoxide dismutase (0.1 - 0.5 parts by mass) and sodium selenite (0.002 - 0.008 parts by mass) in 2 mL of PBS buffer (pH 7.4), and vortex for 30 seconds to form a homogeneous suspension.

[0059] 2. Preparation of the final culture medium:

[0060] Add the premixed solution to the culture medium in step b, supplement sodium pyruvate (0.5 - 2.0 parts by mass), and make up the volume to 1000 parts by mass.

[0061] Perform terminal filtration using a 0.22 μm PES filter membrane under sterile conditions, and dispense into light-proof sterile containers.

[0062] 3. Storage conditions:

[0063] Store at 4°C in the dark, with a shelf life of 30 days; it needs to be equilibrated to 37°C and bubbled with 5% CO2 before use to stabilize the pH.

[0064] The third aspect of the present invention provides the application of the culture medium for human metapneumovirus described in the first aspect of the present invention in the preparation of human metapneumovirus vaccines or diagnostic reagents.

[0065] The culture medium of the present invention significantly improves the infection efficiency and yield of human metapneumovirus (HMPV) by optimizing the virus replication microenvironment, and is suitable for vaccine production (such as live attenuated vaccines, viral vector vaccines) and diagnostic reagent development (such as antigen detection reagents, neutralizing antibody titer determination). The application method is as follows:

[0066] 1. Cell pre-culture stage

[0067] Cell seeding: Seed HEp-2 cells at a density of (1 - 2) × 10 5 cells / cm 2 into culture vessels (such as roller bottles, bioreactors) containing the culture medium of the present invention, and pre-culture at 37°C and 5% CO2 for 18 - 24 hours.

[0068] Purpose of pre-culture: To make the cells enter the logarithmic growth phase, the surface receptor (HSPG) expression reaches its peak, and at the same time, the metabolic reprogramming module fully activates the nucleotide synthesis pathway to provide metabolic reserves for efficient virus adsorption and replication.

[0069] 2. Virus Infection and Amplification

[0070] Inoculation parameters: Inoculate the HMPV virus solution at an MOI (multiplicity of infection) of 0.01 - 1.0, and adsorb for 1 hour (at 37°C, with intermittent shaking to enhance virus-cell contact).

[0071] Basis for the MOI range: A low MOI (0.01 - 0.1) is suitable for the preparation of high-purity virus seed batches to reduce interference from defective viruses; a high MOI (0.5 - 1.0) is used for rapid amplification to shorten the production cycle.

[0072] Maintenance culture: Supplement fresh medium to the working volume, change the medium every 48 - 72 hours, and the total culture time is 5 - 7 days.

[0073] Design of medium change frequency: Regularly remove cell debris and metabolic by-products (such as lactic acid), and at the same time supplement the components of the redox balance module to maintain cell viability until the late stage of infection.

[0074] 3. Virus Harvest and Quality Control

[0075] Harvest timing: The 5th day after infection is the peak window of virus titer (≥1×10 8 PFU / mL). Remove cell debris by centrifugation or filtration, and collect the supernatant as the virus stock solution.

[0076] Basis for the titer standard: ≥1×10 8 PFU / mL is the minimum threshold for the vaccine antigen titer to meet the standard (refer to the WHO virus vaccine production guidelines), which can ensure the feasibility of subsequent purification processes.

[0077] Inactivation / Purification Treatment:

[0078] Vaccine production: Inactivate the virus with β-propiolactone (final concentration 0.01% - 0.03%) while retaining the antigenicity of the envelope protein;

[0079] Diagnostic reagent: Purify virus particles by ultracentrifugation or chromatography for ELISA coating or immunoblotting antigen.

[0080] The present invention provides a medium for human metapneumovirus and its application. It has the following beneficial effects:

[0081] 1. Through the synergistic action of metabolic reprogramming, immune regulation, and receptor induction modules, the present invention significantly optimizes the virus replication microenvironment of host cells, ensures the dynamic balance of viral genome synthesis, structural protein modification, and host resource supply, greatly improves the production efficiency and functional integrity of infectious virus particles, and overcomes the technical bottleneck of the too high proportion of non-infectious defective particles in traditional processes.

[0082] 2. The present invention is based on a multi-module linkage design. While suppressing the host antiviral immune response, it maintains cellular energy metabolism and redox homeostasis, avoids the cytotoxicity or metabolic disorders caused by broad-spectrum immunosuppressants, and achieves the dual goals of continuous virus replication and host cell survival, providing a sustainable cell basis for large-scale production of high-titer viruses.

[0083] 3. The present invention activates the expression of specific membrane proteins through a receptor induction module, enhances the binding efficiency between virus particles and host cells, and combines glycosylation modification to optimize the conformational stability of viral envelope proteins, improving the specificity and success rate of virus infection and reducing the waste of resources caused by ineffective adsorption.

[0084] 4. The present invention adopts an osmotic pressure-adapted culture medium formulation and a free radical scavenging system, effectively alleviates cell membrane stress and oxidative damage during virus replication, prolongs the production cycle of host cells, supports continuous multi-batch high-quality virus production, and reduces the pollution risk and cost loss caused by frequent passage.

[0085] 5. The present invention significantly reduces batch-to-batch variation through process parameter standardization (such as light avoidance operation, gradient stirring, etc.) and the design of key component stability, ensures the consistency of virus titer and function in different production batches, and at the same time prolongs the shelf life of the culture medium, meeting the stringent requirements of industrial production for stability and compliance. Detailed implementation manners

[0086] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0087] Example 1:

[0088] Formulation composition (parts by mass):

[0089] Basal medium: Mixture of DMEM and Ham's F12 (volume ratio 1:1): 890 parts

[0090] Metabolic reprogramming module:

[0091] 5-Methyltetrahydrofolic acid (5-MTHF): 0.10 part

[0092] N-Acetyl-D-mannosamine (ManNAc): 0.55 part

[0093] Sodium dichloroacetate (DCA): 2.0 parts

[0094] Immune regulation module: KIN1148 (diluted to 0.2 μM with DMSO stock solution): 0.010 parts

[0095] Receptor induction module: All-trans retinoic acid (ATRA, ethanol stock solution): 1.0 part

[0096] Redox balance module:

[0097] Polyethylene glycolated superoxide dismutase (PEG-SOD): 0.3 part

[0098] Sodium selenite (Na2SeO3): 0.005 part

[0099] Auxiliary components:

[0100] Sodium bicarbonate: 12 parts

[0101] Sodium pyruvate: 1.2 parts

[0102] The preparation steps are as follows:

[0103] 1. Preparation of basal medium:

[0104] Mix DMEM and Ham's F12 dry powder in a volume ratio of 1:1, dissolve in 800 parts of ultrapure water, add sodium bicarbonate, and stir magnetically (250 rpm, 25 °C) until dissolved.

[0105] Adjust the pH to 7.3 (fine-tune with 0.1 M NaOH), and pre-filter (0.22 μm PES membrane).

[0106] 2. Add modules in stages:

[0107] Metabolism module: Add 5-MTHF, ManNAc, and DCA in sequence, and stir in the dark for 12 minutes (120 rpm) after each addition.

[0108] Immune module: Dilute the DMSO stock solution of KIN1148 to a final concentration of 0.08% (v / v), add dropwise, and stir for 10 minutes.

[0109] Receptor module: Add the ATRA ethanol stock solution, control the final ethanol concentration to 0.09% (v / v), and stir for 15 minutes.

[0110] 3. Treatment of redox module:

[0111] Premix PEG-SOD and sodium selenite in 2 mL of PBS (pH 7.4), vortex for 30 seconds, then add to the medium, add sodium pyruvate, and make up the volume to 1000 parts.

[0112] Terminal filtration (0.22 μm PES), aliquot and store in the dark.

[0113] Example 2:

[0114] Formulation composition (parts by mass):

[0115] Basal medium: Mixture of DMEM and Ham's F12 (volume ratio 1:0.95): 870 parts

[0116] Metabolic reprogramming module:

[0117] 5-MTHF: 0.15 part

[0118] ManNAc: 0.7 part

[0119] DCA: 3.0 parts

[0120] Immune regulation module: KIN1148 (diluted to 0.3 μM with DMSO stock solution): 0.015 part

[0121] Receptor induction module: ATRA: 1.5 parts

[0122] Redox balance module:

[0123] PEG-SOD: 0.5 part

[0124] Sodium selenite: 0.008 part

[0125] Auxiliary components:

[0126] Sodium bicarbonate: 15 parts

[0127] Sodium pyruvate: 2.0 parts

[0128] The preparation steps are as follows:

[0129] 1. Preparation of basal medium:

[0130] Mix DMEM and Ham's F12 at a ratio of 1:0.95, dissolve in 820 parts of ultrapure water, add sodium bicarbonate, and stir (300 rpm, 25 °C) until dissolved.

[0131] Adjust the pH to 7.2 (fine-tune with 0.1 M HCl), and pre-filter.

[0132] 2. Add modules in stages:

[0133] Metabolic module: Add 5-MTHF, ManNAc, and DCA in sequence, and stir in the dark for 15 minutes (150 rpm) after each addition.

[0134] Immune module: Dilute KIN1148 to a final concentration of 0.1% (v / v), add dropwise at a rate of 0.5 mL / min, and stir for 15 minutes.

[0135] Receptor module: Final concentration of ATRA in ethanol mother liquor is 0.1% (v / v), stir for 15 minutes.

[0136] 3. Treatment of redox module:

[0137] Premix PEG-SOD and sodium selenite at a molar ratio of 1:0.6, add to the culture medium and make up to 1000 parts, and perform terminal filtration.

[0138] Example 3:

[0139] Formulation composition (parts by mass):

[0140] Basic culture medium: Mixture of DMEM and Ham's F12 (volume ratio 1:1.05): 910 parts

[0141] Metabolic reprogramming module:

[0142] 5-MTHF: 0.05 part

[0143] ManNAc: 0.3 part

[0144] DCA: 1.0 part

[0145] Immune regulation module: KIN1148 (diluted to 0.1 μM with DMSO mother liquor): 0.005 part

[0146] Receptor induction module: ATRA: 0.5 part

[0147] Redox balance module:

[0148] PEG-SOD: 0.1 part

[0149] Sodium selenite: 0.002 part

[0150] Auxiliary components:

[0151] Sodium bicarbonate: 10 parts

[0152] Sodium pyruvate: 0.5 part

[0153] The preparation steps are as follows:

[0154] 1. Preparation of basic culture medium:

[0155] Mix DMEM and Ham's F12 at a ratio of 1:1.05, dissolve in 850 parts of ultrapure water, add sodium bicarbonate, and stir magnetically (200 rpm, 25 °C) until dissolved.

[0156] Adjust the pH to 7.4 (fine-tune with 0.1 M NaOH), and perform pre-filtration.

[0157] 2. Stage-by-stage addition module:

[0158] Metabolic module: Add 5-MTHF, ManNAc, and DCA in sequence. After each addition, stir in the dark for 10 minutes (100 rpm).

[0159] Immune module: Dilute KIN1148 to a final concentration of 0.05% (v / v), extend the dropping time to 20 minutes, and stir for 10 minutes.

[0160] Receptor module: The final concentration of the ATRA ethanol stock solution is 0.05% (v / v), and stir for 10 minutes.

[0161] 3. Treatment of the redox module:

[0162] Premix PEG-SOD and sodium selenite at a molar ratio of 1:0.4, make up the volume, and perform terminal filtration.

[0163] Comparative example 1:

[0164] Compared with Example 1, the difference is that 5-methyltetrahydrofolate is removed from the metabolic reprogramming module, and the other components and preparation steps are the same.

[0165] Comparative example 2:

[0166] Compared with Example 1, the difference is that KIN1148 in the immune regulation module is replaced with dexamethasone at an equimolar concentration, and the other components and preparation steps are the same.

[0167] Comparative example 3:

[0168] Compared with Example 2, the difference is that the mixing volume ratio of DMEM and Ham's F12 in the basal medium is adjusted to 1:1.5, and the other components and preparation steps are the same.

[0169] Comparative example 4:

[0170] Compared with Example 2, the difference is that the molar ratio of polyethylene glycolated superoxide dismutase (PEG-SOD) to sodium selenite is adjusted to 1:0.2, and the other components and preparation steps are the same.

[0171] Comparative example 5:

[0172] Compared with Example 3, the difference is that the addition amount of all-trans retinoic acid (ATRA) is adjusted to 0.3 parts, and the other components and preparation steps are the same.

[0173] Comparative example 6:

[0174] Compared with Example 1, the difference is that the light avoidance operation is cancelled in step b, and the stirring time is shortened to 3 minutes, and the other components and steps are the same.

[0175] Comparative Example 7:

[0176] Compared with Example 3, the difference lies in that the mass ratio of 5-MTHF to ManNAc in the metabolic reprogramming module is adjusted to 1:8, and the other components and preparation steps are the same.

[0177] Comparative Example 8:

[0178] Compared with Example 2, the difference lies in that the final concentration of DMSO of KIN1148 in the immune regulation module is adjusted to 0.3% (v / v), and the other components and preparation steps are the same.

[0179] Test Example 1:

[0180] The experimental steps are as follows:

[0181] 1. Cell preparation:

[0182] Inoculate HEp-2 cells at a density of 1.5×10 5 cells / cm 2 into a 6-well plate, with 3 replicates in each group.

[0183] Use the culture media of Examples 1-3 and Comparative Examples 1, 2, 5, and 7 for pre-culture for 20 hours (37°C, 5% CO2).

[0184] 2. Virus infection:

[0185] Discard the old culture medium, inoculate HMPV (strain NL / 1 / 00) at MOI = 0.1, and adsorb for 1 hour (37°C, gently shake).

[0186] Supplement 2 mL of the corresponding culture medium and change it every 48 hours.

[0187] 3. Sample collection:

[0188] Collect the cell supernatant on the 5th day after infection and store it at -80°C for later use.

[0189] 4. Virus titer determination:

[0190] Adopt the plaque assay:

[0191] Inoculate HEp-2 cells into a 96-well plate, serially dilute the virus solution (10 -4 to 10 -8 ), and cover it with maintenance medium containing 1.2% methyl cellulose after 1 hour of infection.

[0192] Fix and stain (0.1% crystal violet) after 72 hours, count the number of plaques, and calculate the titer (PFU / mL).

[0193] The experimental results are shown in Table 1:

[0194] Table 1 Test Example 1: Comparison of virus titers on the 5th day after infection in different groups

[0195] Group <![CDATA[Replicate 1 (×10 8 PFU / mL)]]> <![CDATA[Repeated 2 (×10 8 PFU / mL)]]> <![CDATA[Repeated 3 (×10 8 PFU / mL)]]> <![CDATA[Mean ± SD (×10 8 PFU / mL)]]> Example 1 1.2 1.15 1.25 1.20±0.05 Example 2 1.35 1.42 1.28 1.35±0.07 Example 3 0.98 1.05 0.91 0.98±0.07 Comparative Example 1 0.35 0.4 0.28 0.34±0.06* Comparative Example 2 0.62 0.58 0.54 0.58±0.04* Comparative Example 5 0.45 0.38 0.41 0.41±0.03* Comparative Example 7 0.5 0.47 0.53 0.50±0.03*

[0196] From the experimental data in Table 1, it can be obtained that:

[0197] The metabolic reprogramming module ensures the dynamic coupling of pyrimidine nucleotide synthesis and sialic acid glycosylation by balancing the ratio of 5-MTHF to ManNAc (1:4 - 6). When 5-MTHF is removed (Comparative Example 1), the interruption of the folate cycle leads to a sharp drop in the dTMP synthesis rate, and the viral genome replication stalls due to the depletion of the nucleotide pool; while an excess of ManNAc (Comparative Example 7) causes a decrease in the transmembrane transport efficiency of nucleotide precursors by competitively inhibiting nucleotide transporters (such as ENT1), further exacerbating the replication defect. The optimized ratio range in Examples 1 - 3 synchronizes the 5-MTHF-driven nucleotide supply and ManNAc-mediated glycosylation modification in time and space, avoiding the accumulation of immature viral particles caused by the tilt of metabolic resources.

[0198] The specific design of KIN1148 in the immune regulation module is the key to maintaining continuous viral replication. Dexamethasone (Comparative Example 2), as a broad-spectrum immunosuppressant, although it can inhibit the interferon signal, its excessive inhibition of the NF-κB pathway will trigger non-specific apoptosis, resulting in a shortened viral replication window period; while KIN1148 selectively blocks the production of type I interferon by targeting the phosphorylation sites of IRF3 / 7, delaying the host antiviral response and avoiding the premature activation of apoptotic signals. In addition, the DMSO final concentration limit of KIN1148 (≤0.1% v / v) avoids the damage of solvent toxicity to the cell membrane integrity (Comparative Example 8), ensuring the stability of host cell receptors during the virus adsorption stage.

[0199] The temporal coordination of the receptor induction module with the metabolic / immune modules further improves the infection efficiency. All-trans retinoic acid (ATRA) enhances the virus adsorption ability by upregulating the expression of heparan sulfate proteoglycan (HSPG) on the surface of HEp-2 cells. When the concentration of ATRA is insufficient (Comparative Example 5), the receptor expression level decreases and the virus entry efficiency drops; while the combined action of ATRA and the metabolic module in Examples 1 - 3 forms a positive feedback loop between viral genome replication (dependent on nucleotide supply) and envelope protein synthesis (dependent on receptor-mediated endocytosis), ultimately achieving the synchronized production of high-titer infectious viral particles. This multi-module linkage mechanism cannot be achieved by traditional single-component optimization strategies (such as only supplementing glucose or growth factors).

[0200] Test Example 2:

[0201] The experimental procedures are as follows:

[0202] 1. Cell culture and treatment:

[0203] Seed HEp-2 cells at a density of 1×10 5 cells / cm 2 in 12-well plates, and culture them for 72 hours (37°C, 5% CO2) using the culture media of Examples 1 - 3 and Comparative Examples 3, 4, 6, and 8.

[0204] Set 4 replicates for each group and change the culture medium every 24 hours.

[0205] 2. Detection of cell viability:

[0206] Collect cells: Digest with trypsin and centrifuge (1000 rpm, 5 min), then resuspend with PBS.

[0207] Trypan blue staining: Mix the cell suspension with 0.4% trypan blue solution at a ratio of 1:1 and let it stand for 3 minutes.

[0208] Counting: Use a hemocytometer to count the proportion of live cells (unstained) and calculate the survival rate (%).

[0209] 3. Detection of oxidative damage markers:

[0210] Cell lysis: Extract total protein using RIPA lysis buffer and determine the protein concentration by BCA method.

[0211] MDA detection: Use the thiobarbituric acid (TBA) method to measure the absorbance at a wavelength of 532 nm, and calculate the MDA concentration (nmol / mg protein) according to the standard curve.

[0212] The experimental results are shown in Table 2:

[0213] Table 2, Test Example 2: Comparison of cell survival rates and oxidative damage levels in different groups

[0214] Group Survival rate repetition 1 (%) Survival rate repetition 2 (%) Survival rate repetition 3 (%) Survival rate mean ± SD (%) MDA concentration repetition 1 (nmol / mg) MDA concentration repetition 2 (nmol / mg) MDA concentration repetition 3 (nmol / mg) MDA mean ± SD (nmol / mg) Example 1 92.3 89.7 91.5 91.2±1.3 1.12 1.08 1.15 1.12±0.03 Example 2 94.1 90.8 93.6 92.8±1.7 0.95 1.02 0.89 0.95±0.06* Example 3 88.4 85.9 87.2 87.2±1.2 1.34 1.4 1.28 1.34±0.06 Comparative Example 3 74.6 68.9 72.3 71.9±2.8* 2.55 2.78 2.61 2.65±0.12* Comparative Example 4 81.2 77.5 83.1 80.6±2.8* 2.01 1.95 2.13 2.03±0.09* Comparative Example 6 69.8 65.4 71.2 68.8±2.9* 3.12 3.34 3.05 3.17±0.15* Comparative Example 8 58.3 62.7 60.1 60.4±2.2* 3.98 4.12 4.05 4.05±0.07*

[0215] Among them, * indicates p < 0.05 compared with Example 1 (ANOVA test);

[0216] From the experimental data in Table 2, it can be obtained that:

[0217] The basal medium volume ratio (DMEM:Ham's F12 = 1:0.9 - 1.1) maintains the osmotic pressure within the physiological range of 280 - 310 mOsm / kg by balancing the bicarbonate buffering capacity and the stability of HEPES. The volume ratio imbalance (1:1.5) in Comparative Example 3 led to an increase in osmotic pressure above 320 mOsm / kg, triggering compensatory energy consumption by the cell membrane sodium-potassium pump. At the same time, the outflow of intracellular water caused mitochondrial swelling, and ultimately, the lipid peroxidation was exacerbated through the burst of ROS (reactive oxygen species) (MDA increased by 137%). In Examples 1 - 3, through precise volume ratio control, the buffer system was adapted to the ionic strength, avoiding interference with energy metabolism caused by osmotic shock.

[0218] The molar ratio limit of the redox module (PEG-SOD:sodium selenite = 1:0.4 - 0.6) is directly related to the cascade reaction of free radical scavenging efficiency. After PEG-SOD converts superoxide anion (O2 - )into H2O2, sodium selenite, as a cofactor of glutathione peroxidase (GPX), further catalyzes the decomposition of H2O2 into water. The deficiency of sodium selenite (molar ratio 1:0.2) in Comparative Example 4 led to the accumulation of H2O2, which generated hydroxyl radicals (·OH) through the Fenton reaction, attacking the unsaturated fatty acids in the cell membrane (MDA increased by 81%). In Example 2, by optimizing the ratio of selenium to SOD, the whole process of OO2 - →H2O2→H2O scavenging was achieved, and the oxidative damage was controlled at the baseline level (MDA ≤ 1.12 nmol / mg).

[0219] The process conditions and solvent limitations (light avoidance, stirring time, DMSO ≤ 0.1%) jointly ensured the activity of functional components and the integrity of the cell membrane. Canceling the light avoidance operation in Comparative Example 6 led to the photooxidative degradation of 5-MTHF and ATRA, and their free radical by-products directly damaged DNA and inhibited respiratory chain complex I, resulting in a 24% decrease in cell viability; insufficient stirring time (3 minutes) caused a too high local concentration of ManNAc, triggering glycolysis disorders through competitive inhibition of glucose transporter (GLUT1). In Comparative Example 8, the DMSO concentration exceeded the standard (0.3%), which damaged the lipid bilayer structure of the cell membrane, resulting in ion gradient leakage and mitochondrial membrane potential collapse (the survival rate was only 60.4%). The examples ensured the precise temporal matching of the functions of the metabolic module and the receptor induction module through process standardization and solvent gradient dilution.

[0220] Test Example 3:

[0221] The experimental steps are as follows:

[0222] 1. Virus purification:

[0223] The virus supernatants of Examples 1-3 and Comparative Examples 1, 2, 5, and 7 were centrifugally concentrated through a 20% sucrose cushion (10,000×g, 4°C, 2 hours) and resuspended in PBS to 1 / 10 of the original volume.

[0224] 2. Detection of the proportion of infectious virus:

[0225] Plaque formation rate: The proportion of infectious virus (PFU / TCID50 × 100%) was calculated as the ratio of the endpoint dilution method (TCID50) to the plaque method (PFU).

[0226] Steps:

[0227] TCID50 determination: A 96-well plate of HEp-2 cells was inoculated with a 10-fold serial dilution of the virus solution, and the CPE (cytopathic effect) was observed for 5 days. The titer was calculated by the Reed-Muench method.

[0228] PFU determination: As in the method of Test Example 1.

[0229] 3. Detection of glycosylation level:

[0230] Western Blot:

[0231] After the virus was lysed, the G protein (envelope glycoprotein) was separated by SDS-PAGE electrophoresis and transferred to PVDF.

[0232] Primary antibody: Monoclonal antibody against HMPV G protein (1:1000); Secondary antibody: HRP-labeled goat anti-mouse IgG (1:5000).

[0233] Chemiluminescence imaging was performed, and the gray value of the band was analyzed by ImageJ. The degree of glycosylation (relative glycosylation level %) was verified by the ConA (Concanavalin A) lectin binding experiment.

[0234] The experimental results are shown in Table 3:

[0235] Table 3 Test Example 3: Comparison of virus particle integrity in different groups

[0236] Group Plaque formation rate repetition 1 (%) Plaque formation rate repetition 2 (%) Plaque formation rate repetition 3 (%) Plaque formation rate mean ± SD (%) G protein glycosylation repetition 1 (%) G protein glycosylation repetition 2 (%) G protein glycosylation repetition 3 (%) Glycosylation mean ± SD (%) Example 1 87.2 84.5 89.1 86.9±2.3 95.3 92.8 97.1 95.1±2.1 Example 2 91.5 88.7 93.2 91.1±2.3* 98.6 96.2 99.4 98.1±1.6* Example 3 79.8 76.4 82.1 79.4±2.9 88.7 85.3 90.2 88.1±2.5 Comparative Example 1 34.6 38.2 31.9 34.9±3.2* 58.4 62.1 55.9 58.8±3.1* Comparative Example 2 52.3 49.7 54.8 52.3±2.6* 73.5 70.2 75.3 73.0±2.5* Comparative Example 5 45.1 41.8 47.6 44.8±2.9* 67.4 64.9 69.2 67.2±2.1* Comparative Example 7 29.7 33.5 27.4 30.2±3.0* 51.3 48.7 53.6 51.2±2.4*

[0237] Among them, * indicates p < 0.01 compared with Example 1 (t-test);

[0238] From the experimental data in Table 3, it can be obtained that:

[0239] The metabolic reprogramming module coordinates the two key pathways of nucleotide synthesis and glycosylation modification spatiotemporally by balancing the ratio of 5-MTHF to ManNAc (1:4 - 6). Removing 5-MTHF (Comparative Example 1) leads to the interruption of the folate cycle, limited dTMP synthesis, an increased error rate in viral genome replication, and the production of a large number of non-infectious defective particles (the plaque formation rate is only 34.9%); while an excess of ManNAc (Comparative Example 7, 1:8) competitively inhibits the glucose transporter (GLUT1), reduces the supply of the glycolysis intermediate fructose 6-phosphate, and further weakens the mannose 6-phosphate precursor required for N-linked glycosylation, resulting in a decrease in the glycosylation level of G protein to 51.2% (95.1% in Example 1). The optimized ratio in Examples 1 - 3 ensures the dynamic replenishment of the nucleotide pool and the synchronization of sialic acid synthesis, avoiding the disconnection between genome replication and structural protein modification.

[0240] Dexamethasone (Comparative Example 2) non-specifically inhibits the NF-κB pathway by activating the glucocorticoid receptor (GR), leading to Golgi stress response, interfering with the localization and activity of ST6GAL1 (sialyltransferase), and resulting in the absence of terminal sialylation modification of G protein (glycosylation level 73.0% vs. 95.1% in Example 1). KIN1148 blocks the type I interferon signal by targeting the phosphorylation sites of IRF3 / 7 without affecting NF-κB, maintains the pH stability of the Golgi apparatus and the activity of glycosyltransferases, and ensures the host cell membrane fusion ability of viral envelope proteins. In addition, the low solvent concentration of KIN1148 (DMSO ≤ 0.1%) avoids the interference of membrane lipid peroxidation on the transport of glycosylation precursors (the glycosylation level in Comparative Example 8 is only 51.2%).

[0241] ATRA enhances the virus adsorption efficiency by upregulating the expression of HSPG on the surface of HEp-2 cells; meanwhile, its nuclear receptor (RAR) signal activates the fatty acid oxidation (FAO) pathway to provide acetyl-CoA for viral capsid assembly. In Comparative Example 5 (insufficient ATRA), the decrease in receptor expression leads to delayed virus entry, an extended retention time of immature particles in the endoplasmic reticulum, and easy degradation by the host proteasome (the plaque formation rate is 44.8%). The synergistic effect of ATRA and the metabolic module in Example 1 enables the formation of a positive feedback loop of virus adsorption (dependent on HSPG) - endocytosis (dependent on clathrin) - replication (dependent on the nucleotide pool), ultimately achieving the dual optimization of the proportion of infectious virus (86.9%) and glycosylation maturity (95.1%). This multi-module linkage mechanism breaks through the technical bottleneck of traditional single-component optimization and realizes the synchronous improvement of the quality and efficiency of virus production.

[0242] Test Example 4:

[0243] The experimental steps are as follows:

[0244] 1. Inter-batch virus titer test:

[0245] Using the culture media of Examples 1 - 3 and Comparative Examples 3, 4, 6, and 8, prepare 3 batches of virus continuously under the same equipment and personnel conditions (each batch prepares the culture media independently).

[0246] Determine the virus titer (PFU / mL) of each batch according to the method of Test Example 1, and calculate the inter-batch coefficient of variation (CV = standard deviation / mean × 100%).

[0247] 2. Culture medium shelf-life test:

[0248] Dispense the culture media of each group into sterile bottles and store them in the dark at 37°C (simulating accelerated stability test).

[0249] Take samples every 48 hours, determine the virus titer according to the method of Test Example 1, and record the number of days required for the titer to drop to 90% of the initial value.

[0250] The experimental results are shown in Table 4:

[0251] Table 4 Test Example 4: Comparison of process stability of different groups

[0252]

[0253] Among them, * indicates that compared with Example 1, p < 0.01 (t-test);

[0254] From the experimental data in Table 4, it can be obtained that:

[0255] The basal medium volume ratio (DMEM:Ham's F12 = 1:0.9 - 1.1) controls the osmotic pressure fluctuation within ±5 mOsm / kg by balancing the bicarbonate buffering capacity and the pH stability of HEPES (the inter-batch CV of Example 1 is only 2.8%). The volume ratio imbalance (1:1.5) in Comparative Example 3 leads to abnormal osmotic pressure gradient (measured 318 - 327 mOsm / kg), triggering compensatory energy consumption of the cell membrane sodium-potassium pump, resulting in an enlarged inter-batch difference in the transmembrane substance transport efficiency (CV reaches 12.7%). At the same time, the hypertonic environment accelerates cell senescence and the consistency of virus genome replication decreases. The example maintains the ionic strength homeostasis through precise proportioning to ensure the temporal reproducibility of the metabolic module function.

[0256] The molar ratio limitation of the redox module (PEG-SOD: sodium selenite = 1:0.4 - 0.6) ensures the long-term stability of the culture medium components by cascading free radical scavenging. In Comparative Example 4, selenium deficiency (molar ratio 1:0.2) led to a decrease in GPX activity, and H2O2 continued to accumulate during storage at 37°C. Hydroxyl radicals (·OH) were generated through the Fenton reaction, attacking the pterin ring structure of 5-MTHF and shortening its half-life from 28 days (Example 2) to 13.7 days. At the same time, ·OH induced the oxidation of ManNAc to neuraminic acid analogs, competitively inhibiting the activity of sialic acid synthase (NANS) and causing batch-to-batch fluctuations in viral envelope glycosylation modification (standard deviation of glycosylation level ±3.1%). The optimized ratio achieved long-term stability of component activity and virus titer by scavenging free radicals through the SOD-GPX dual pathway and blocking the chain oxidation reaction.

[0257] The process conditions (light avoidance / stirring) and solvent concentration limit (DMSO ≤ 0.1%) jointly constructed a scalable production quality control system. In Comparative Example 6, the cancellation of light avoidance operation led to the photolysis of ATRA to generate cytotoxic retinoic acid radicals, inhibiting the function of mitochondrial complex III and increasing the batch-to-batch difference in cell energy metabolism (CV of ATP level increased from 5% to 18%); insufficient stirring time (3 minutes) caused local crystallization of ManNAc, and uneven dissolution directly weakened the glycosylation modification efficiency (batch-to-batch glycosylation level fluctuation ±7.2%). In Comparative Example 8, the DMSO concentration exceeded the standard (0.3%), disrupting the lipid raft structure of the cell membrane and interfering with the lipid envelope assembly during virus budding, resulting in an increase in the proportion of vacuolated defective particles (CV of batch-to-batch titer reached 9.8%). The examples minimized the physicochemical variables of the production process through light avoidance operation, gradient stirring (10 minutes), and final solvent concentration control, supporting the quality control compliance of large-scale virus production.

[0258] Although the embodiments of the present invention have been shown and described, it will be understood by those of ordinary skill in the art that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A culture medium for human metapneumovirus, characterized in that, Comprising the following components in parts by mass: Basal medium: 850 - 920 parts, the basal medium is a mixed solution of DMEM and Ham's F12, and the mixing volume ratio is 1:(0.9 - 1.1); Metabolic reprogramming module: 0.05 - 0.15 part of 5-methyltetrahydrofolic acid, 0.3 - 0.7 part of N-acetyl-D-mannosamine, 1.0 - 3.0 parts of sodium dichloroacetate; The mass ratio of 5-methyltetrahydrofolic acid to N-acetyl-D-mannosamine is 1:(4 - 6); Immune regulation module: 0.005 - 0.015 part of RIG-I / MAVS pathway inhibitor; The RIG-I / MAVS pathway inhibitor is KIN1148, and its final concentration range is 0.1 - 0.3 μM; Receptor induction module: 0.5 - 1.5 parts of all-trans retinoic acid; Redox balance module: 0.1 - 0.5 part of polyethylene glycolated superoxide dismutase, 0.002 - 0.008 part of sodium selenite; The molar ratio of the polyethylene glycolated superoxide dismutase to sodium selenite is 1:(0.4 - 0.6); The host cell of the human metapneumovirus is Hep-2 cell.

2. A method for preparing a culture medium for human metapneumovirus according to claim 1, characterized in that, Including the following steps: Step a: Dissolution of basal medium and pH adjustment: 1) Dissolution operation: Weigh DMEM and Ham's F12 dry powder according to the volume ratio of 1:(0.9 - 1.1), add ultrapure water to 85% - 92% of the total mass, stir magnetically until completely dissolved, add 10 - 15 parts by mass of sodium bicarbonate, and continue stirring until the solution is clear; 2) pH adjustment: Precisely adjust the pH to 7.2 - 7.4 using 0.1 M HCl or NaOH solution, avoiding component denaturation caused by local over-acidity or over-alkalinity; 3) Filtration for impurity removal: Pre-filter through a 0.22 μm polyethersulfone (PES) membrane to remove undissolved particles and microbial contamination; Step b: Adding functional modules in stages: 1) Addition of metabolic reprogramming module: Add 5-methyltetrahydrofolic acid, N-acetyl-D-mannosamine, and sodium dichloroacetate in sequence. After adding each component, stir in the dark for 10 - 15 minutes to ensure complete dissolution and no crystallization; 2) Addition of immune regulation module: Take the DMSO stock solution of KIN1148, dilute it with the basal medium to a final concentration ≤0.1% (v / v), add it drop by drop while maintaining stirring for 10 - 15 minutes to avoid excessive local DMSO concentration causing cell membrane damage; 3) Addition of receptor induction module: Add the ethanol stock solution of all-trans retinoic acid to the system, control the final ethanol concentration ≤0.1% (v / v), and stir for 15 minutes until completely mixed; Step c: Treatment of redox balance module and preparation of final product: 1) Premixing redox components: Dissolve polyethylene glycolated superoxide dismutase and sodium selenite in 2 mL of PBS buffer, and vortex for 30 seconds to form a homogeneous suspension; 2) Preparation of final medium: Add the premixed solution to the medium in step b, supplement 0.5 - 2.0 parts by mass of sodium pyruvate, and make up the volume to 1000 parts. Terminal filtration is carried out using a 0.22 μm PES filter membrane under aseptic conditions and filled into light-protected sterile containers.

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