Culture medium for human metapneumovirus and application

Abstract

The invention relates to the technical field of biological medicine, and discloses a culture medium for human metapneumovirus and application, the culture medium comprises the following components by mass: 850-920 parts of a basic culture medium; the metabolism reprogramming module is prepared from the following components in parts by weight: 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; the immune regulation and control module is prepared from 0.005 to 0.015 part of an RIG-I / MAVS pathway inhibitor; the receptor induction module is prepared from 0.5 to 1.5 parts of all-trans retinoic acid; the redox balance module is prepared from the following components in parts by weight: 0.1 to 0.5 part of pegylated superoxide dismutase and 0.002 to 0.008 part of sodium selenite. According to the method, the proportion of infectious virus particles can be remarkably increased, the generation of defective particles is reduced, meanwhile, the production cycle of host cells is prolonged, large-scale continuous culture is supported, and a high-quality and high-efficiency solution is provided for industrial production of gene therapy vectors and virus vaccines.

Description

Technical Field

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

[0002] 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 between host cell metabolic pressure, antiviral immune response and the functional integrity of viral particles.

[0003] Traditional processes usually use single component optimization or broad-spectrum inhibitors to force amplify the viral replication window, which can easily lead to accelerated oxidative damage to host cells, disruption of key metabolic pathways, and problems such as an excessively high proportion of non-infectious defective particles and significant titer fluctuations between batches.

[0004] In addition, the insufficient stability of culture medium components leads to a rapid decay of virus yield during long-term storage or continuous production, further increasing the cost and risk of large-scale applications.

[0005] Existing technologies have not yet effectively coordinated the metabolic reprogramming, immunosuppression precision and host cell homeostasis required for viral replication, and a systematic solution is urgently needed to break through the quality and efficiency bottleneck. Summary of the Invention

[0006] In response to the deficiencies of the existing technology, the present invention provides a culture medium and application for human metapneumovirus, which solves the technical problems of low infectious particle ratio, severe host cell damage and poor batch stability in traditional virus production processes.

[0007] To achieve the above objectives, the present invention is implemented through the following technical solutions: The first aspect of the present invention provides a culture medium for human metapneumovirus, comprising the following components in parts by mass: Basic culture medium: 850-920 copies; Metabolic reprogramming module: including 0.05-0.15 parts of 5-methyltetrahydrofolate, 0.3-0.7 parts of N-acetyl-D-mannosamine, and 1.0-3.0 parts of sodium dichloroacetate; Immune regulation module: including 0.005-0.015 parts of RIG-I / MAVS pathway inhibitor; Receptor induction module: including 0.5 to 1.5 parts of all-trans retinoic acid; Redox balance module: comprising 0.1 to 0.5 parts of pegylated superoxide dismutase and 0.002 to 0.008 parts of sodium selenite.

[0008] Among them, the metabolic reprogramming module includes 5-methyltetrahydrofolate (5-MTHF), N-acetyl-D-mannosamine (ManNAc), and sodium dichloroacetate (DCA); its core mechanism is to reconstruct the metabolic network of the host cell and directionally enhance the biosynthesis capacity of nucleotides, glycosylation intermediates and lipid precursors required for viral replication.

[0009] 5-MTHF, as a one-carbon unit donor, 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. ManNAc, as a key precursor for sialic acid synthesis, promotes the maturation of viral envelope proteins and the sugar chain binding efficiency of host cell receptors by increasing the level of glycosylation modification on the host cell surface; DCA inhibits pyruvate dehydrogenase kinase (PDK), relieves the oxidative phosphorylation inhibition of pyruvate metabolism, promotes the massive production of mitochondrial acetyl-CoA, and provides sufficient raw materials for the synthesis of viral envelope lipids.

[0010] Traditional culture media cannot effectively suppress the host cell's innate immune response, leading to premature blockage of viral replication. This invention introduces RIG-I / MAVS pathway inhibitors (such as KIN1148). Its innovative mechanism lies in specifically blocking MAVS protein aggregation downstream of RIG-I signaling, inhibiting the release of interferon-β (IFN-β), thereby extending the "golden window" for viral replication. Compared to existing approaches that use high-dose interferon-α (which actually inhibits the virus) or broad-spectrum immunosuppressants (such as dexamethasone), KIN1148 achieves precise immunosuppression at a concentration of 0.1-0.3 μM without interfering with viral adsorption and packaging.

[0011] One of the core reasons for the low infection efficiency of HMPV is insufficient expression of host cell surface receptors (such as heparan sulfate proteoglycans, or HSPGs). This study uses all-trans retinoic acid (ATRA) to activate the retinoic acid receptor α (RARα) signaling pathway, inducing upregulation of HSPG glycosaminoglycan chain synthases (such as EXT1 / 2), thereby increasing the density of viral attachment sites on the host cell surface by 2-3 times. This strategy breaks away from the passive model of traditional technologies that rely solely on the expression of natural cell receptors and achieves active regulation of receptor expression for the first time.

[0012] Among them, oxidative stress in host cells during the late stage of viral infection is the main reason for the decrease in virus production. This module uses a composite system of PEGylated superoxide dismutase (PEG-SOD) and sodium selenite: PEG-SOD is modified with polyethylene glycol to prolong its half-life and to target the scavenging of mitochondrial superoxide anion free radicals (O2 -), to avoid oxidative damage caused by viral replication; Sodium selenite acts as an essential cofactor for glutathione peroxidase (GPX), enhancing the cell's ability to detoxify lipid peroxides.

[0013] Through the above scheme, the present invention starts from the biological nature of virus hijacking host metabolism, specifically strengthens the nucleotide, glycosylation and lipid synthesis pathways, breaking through the blindness of traditional nutritional supplements; and uses small molecule targeted inhibitors (KIN1148) to replace broad-spectrum immunomodulators to extend the viral replication window while maintaining cell activity; at the same time, through chemical inducers (ATRA), actively upregulates host receptor expression to solve the core pain point of low virus adsorption efficiency; based on the enzyme-coenzyme synergistic antioxidant system, it achieves cell homeostasis maintenance under high viral load.

[0014] Preferably, in the metabolic reprogramming module, the mass ratio of 5-methyltetrahydrofolate to N-acetyl-D-mannosamine is 1:(4-6).

[0015] 5-MTHF, as a one-carbon unit carrier, directly participates in the synthesis of thymidylic acid (dTMP), and its concentration must match ManNAc-driven sialic acid metabolism. When the 5-MTHF:ManNAc mass ratio is lower than 1:4, the pyrimidine synthesis rate exceeds the glycosylation capacity, resulting in the accumulation of unglycosylated viral envelope proteins and affecting the assembly of viral particles. When the ratio is higher than 1:6, excess ManNAc may competitively inhibit nucleotide transporters (such as ENT1), thereby limiting the intracellular transport efficiency of pyrimidine precursors.

[0016] Within the mass ratio range of 1:4 to 6, the methyl group provided by 5-MTHF (through the folic acid cycle) and the N-acetylmannosamine provided by ManNAc (through the UDP-GlcNAc pathway) can complement each other and jointly support the simultaneous replication of the viral genome (requiring pyrimidine) and the maturation of the envelope glycoprotein (requiring sialic acid modification).

[0017] Preferably, the molar ratio of the PEGylated superoxide dismutase to sodium selenite is 1:(0.4-0.6).

[0018] PEG-SOD catalyzes the superoxide anion (O2 - ) is converted into hydrogen peroxide (H2O2), playing the role of the first line of antioxidant defense; Sodium selenite, as a selenium source, is integrated into the active center of glutathione peroxidase (GPX), driving the further reduction of H2O2 to water (H2O), thereby preventing lipid peroxidation caused by H2O2 accumulation.

[0019] When the molar ratio is lower than 1:0.4, insufficient selenium supply leads to limited GPX activity, delayed H2O2 clearance rate, and secondary oxidative damage; when the molar ratio is higher than 1:0.6, excess selenium may competitively inhibit SOD active sites, weakening O2 - Primary removal efficiency.

[0020] PEG modification can prolong the half-life of SOD (about 72 hours), but its sustained catalytic activity depends on the timely removal of H2O2 by GPX to avoid its own inactivation due to excessive H2O2 concentration; The molar ratio of 1:0.4 to 0.6 can ensure that the H2O2 produced by each unit of SOD catalysis is dynamically matched with the decomposition capacity of GPX, maintaining redox homeostasis.

[0021] Preferably, the basal culture medium is a mixture of DMEM and Ham's F12, with a mixing volume ratio of 1:(0.9-1.1).

[0022] DMEM provides high concentrations of glucose (4.5 g / L) and essential amino acids (such as glutamine), which support the rapid energy metabolism of host cells and the supply of carbon skeletons required for viral replication; Ham's F12 supplements the trace elements (such as sodium selenite and putrescine) and fat-soluble vitamins (such as biotin) that are lacking in DMEM, enhancing the cell's antioxidant capacity and membrane structure stability.

[0023] In the range of volume ratio 1:0.9 to 1.1, the concentration gradient of the two components tends to be gentle, avoiding cell stress caused by extreme concentrations in a single culture medium (such as the high osmotic pressure of DMEM or the low buffering capacity of Ham's F12).

[0024] DMEM relies on a bicarbonate / CO2 buffer system, while Ham's F12 provides additional buffering capacity through HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid).

[0025] 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 fluid changes on virus adsorption.

[0026] The second aspect of the present invention provides a method for preparing a culture medium for human metapneumovirus according to the first aspect of the present invention, comprising the following steps: Step a: Dissolution of basal medium and pH adjustment 1. Dissolution operation: Weigh DMEM and Ham's F12 dry powder in 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.

[0027] Sodium bicarbonate (10-15 parts by mass) was added and stirred continuously until the solution became clear.

[0028] 2. pH adjustment: Use 0.1 M HCl or NaOH solution to accurately adjust the pH to 7.2-7.4 (pH meter calibration accuracy ±0.02) to avoid local over-acidity or over-alkalinity that may cause denaturation of components.

[0029] 3. Filter and remove impurities: The sample was pre-filtered through a 0.22 μm polyethersulfone (PES) membrane to remove undissolved particles and microbial contamination.

[0030] Step b: Add functional modules in stages 1. Metabolic reprogramming module added: Add 5-methyltetrahydrofolate (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) in sequence. After adding each ingredient, stir in the dark for 10-15 minutes (at 100-150 rpm) to ensure that it is fully dissolved and no crystals are precipitated.

[0031] 2. Addition of immune regulation module: Take the DMSO stock solution of KIN1148 (10 mM) and dilute it with basal culture medium to a final concentration of ≤0.1% (v / v). Add it dropwise (drop speed ≤1 mL / min) while maintaining stirring (120-180 rpm) to avoid local high DMSO concentration that may cause cell membrane damage.

[0032] 3. Add receptor induction module: Add 0.5-1.5 parts by weight of a stock solution of all-trans retinoic acid in ethanol (1 mM) to the system, controlling the final ethanol concentration to ≤0.1% (v / v), and stir for 15 minutes until completely mixed.

[0033] Step c: Redox balance module processing and final product preparation 1. Premixed redox components: Dissolve PEGylated 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.

[0034] 2. Final culture medium preparation: Add the premixed solution to the culture medium in step b, and add sodium pyruvate (0.5-2.0 parts by mass) to make the volume up to 1000 parts by mass.

[0035] The product was filtered through a 0.22 μm PES membrane under sterile conditions and dispensed into light-proof sterile containers.

[0036] 3. Storage conditions: Store at 4°C away from light. The shelf life is 30 days. Before use, the solution must be equilibrated to 37°C and aerated with 5% CO2 to stabilize the pH.

[0037] The third aspect of the present invention provides use of the culture medium for human metapneumovirus according to the first aspect of the present invention in preparing a human metapneumovirus vaccine or a diagnostic reagent.

[0038] The culture medium of the present invention significantly improves the infection efficiency and yield of human metapneumovirus (HMPV) by optimizing the viral replication microenvironment. It is suitable for vaccine production (such as live attenuated vaccines and viral vector vaccines) and diagnostic reagent development (such as antigen detection reagents and neutralizing antibody titer determination). Its application method is as follows: 1. Cell pre-culture stage Cell inoculation: HEp-2 cells were seeded at a density of (1-2) × 10 5 cells / cm 2 Inoculate into a culture vessel (such as a roller bottle or a bioreactor) containing the culture medium of the present invention, and pre-culture at 37°C and 5% CO2 for 18 to 24 hours.

[0039] Purpose of pre-culture: To allow cells to enter the logarithmic growth phase, the expression of surface receptors (HSPG) reaches its peak, and the metabolic reprogramming module fully activates the nucleotide synthesis pathway, providing metabolic reserves for efficient viral adsorption and replication.

[0040] 2. Viral infection and amplification Inoculation parameters: HMPV virus solution was inoculated at an MOI (multiplicity of infection) of 0.01 to 1.0 and adsorbed for 1 hour (37°C with intermittent shaking to enhance virus-cell contact).

[0041] MOI range is based on: low MOI (0.01-0.1) is suitable for high-purity virus seed batch preparation to reduce interference from defective viruses; high MOI (0.5-1.0) is used for rapid amplification to shorten the production cycle.

[0042] Maintenance culture: Add fresh culture medium to the working volume and replace the medium every 48-72 hours for a total culture time of 5-7 days.

[0043] Medium change frequency design: Regularly remove cell debris and metabolic byproducts (such as lactic acid), while replenishing redox balance module components to maintain cell viability until the late stage of infection.

[0044] 3. Virus Harvest and Quality Control Harvest timing: Day 5 after infection is the peak window for viral titer (≥1×10 8 PFU / mL), remove cell debris by centrifugation or filtration, and collect the supernatant as the virus stock solution.

[0045] Titer standard basis: ≥1×10 8 PFU / mL is the minimum threshold for vaccine antigen potency to meet the standard (refer to the WHO viral vaccine production guidelines), which can ensure the feasibility of the subsequent purification process.

[0046] Inactivation / Purification: Vaccine production: β-propiolactone (final concentration 0.01% to 0.03%) is used to inactivate the virus, preserving the antigenicity of the envelope protein; Diagnostic reagents: Purify viral particles by ultracentrifugation or chromatography for ELISA coating or immunoblotting of antigens.

[0047] The present invention provides a culture medium for human metapneumovirus and its application, which has the following beneficial effects: 1. The present invention significantly optimizes the viral replication microenvironment of host cells through the synergistic effects of metabolic reprogramming, immune regulation and receptor induction modules, ensures a dynamic balance between viral genome synthesis, structural protein modification and host resource supply, greatly improves the output efficiency and functional integrity of infectious viral particles, and overcomes the technical bottleneck of the high proportion of non-infectious defective particles in traditional processes.

[0048] 2. The present invention is based on a multi-module linkage design. While suppressing the host's antiviral immune response, it maintains cellular energy metabolism and redox homeostasis, avoids cytotoxicity or metabolic disorders caused by broad-spectrum immunosuppressants, and achieves the dual goals of continuous viral replication and host cell survival, providing a sustainable cellular basis for large-scale production of high-titer viruses.

[0049] 3. The present invention activates the expression of specific membrane proteins through the receptor-inducing module, enhances the binding efficiency of virus particles and host cells, and optimizes the conformational stability of viral envelope proteins in combination with glycosylation modification, thereby improving the specificity and success rate of viral infection and reducing the waste of resources caused by ineffective adsorption.

[0050] 4. The present invention adopts an osmotic pressure-adapted culture medium formula and a free radical scavenging system to effectively alleviate cell membrane stress and oxidative damage during viral replication, extend the host cell production cycle, support continuous multi-batch high-quality virus output, and reduce the contamination risk and cost loss caused by frequent passage.

[0051] 5. This invention significantly reduces batch-to-batch variability through standardized process parameters (such as light-avoiding operation and gradient stirring) and key component stability design, ensuring the consistency of virus titer and function across different production batches. It also extends the shelf life of the culture medium and meets the stringent stability and compliance requirements of industrial production. DETAILED DESCRIPTION

[0052] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0053] Example 1: Formula composition (parts by mass): Basal culture medium: DMEM and Ham's F12 mixture (volume ratio 1:1): 890 parts Metabolic Reprogramming Module: 5-Methyltetrahydrofolate (5-MTHF): 0.10 parts N-acetyl-D-mannosamine (ManNAc): 0.55 parts Sodium dichloroacetate (DCA): 2.0 parts Immune regulation module: KIN1148 (DMSO stock solution diluted to 0.2 μM): 0.010 parts Receptor induction module: all-trans retinoic acid (ATRA, ethanol stock solution): 1.0 part Redox balance module: PEGylated superoxide dismutase (PEG-SOD): 0.3 parts Sodium selenite (Na2SeO3): 0.005 parts Auxiliary ingredients: Sodium bicarbonate: 12 parts Sodium pyruvate: 1.2 parts The preparation steps are as follows: 1. Preparation of basal culture medium: DMEM and Ham's F12 dry powder were mixed in a volume ratio of 1:1, dissolved in 800 parts of ultrapure water, and sodium bicarbonate was added. The mixture was stirred magnetically (250 rpm, 25°C) until dissolved.

[0054] The pH was adjusted to 7.3 (fine-tuning with 0.1 M NaOH) and pre-filtered (0.22 μm PES membrane).

[0055] 2. Add modules in stages: Metabolic module: 5-MTHF, ManNAc, and DCA were added sequentially, and stirred in the dark for 12 minutes (120 rpm) after each addition.

[0056] Immunoassay module: Dilute the DMSO stock solution of KIN1148 to a final concentration of 0.08% (v / v), add dropwise, and stir for 10 minutes.

[0057] Receptor module: Add ATRA ethanol stock solution to control the final ethanol concentration to 0.09% (v / v) and stir for 15 minutes.

[0058] 3. Redox module treatment: PEG-SOD and sodium selenite were premixed in 2 mL PBS (pH 7.4), vortexed for 30 seconds, added to the culture medium, and sodium pyruvate was added to make up to 1000 copies.

[0059] Final filter (0.22 μm PES), aliquot and store in the dark.

[0060] Example 2: Formula composition (parts by mass): Basal culture medium: DMEM and Ham's F12 mixture (volume ratio 1:0.95): 870 parts Metabolic Reprogramming Module: 5-MTHF: 0.15 parts ManNAc: 0.7 parts DCA: 3.0 parts Immune regulation module: KIN1148 (DMSO stock solution diluted to 0.3 μM): 0.015 parts Receptor induction module: ATRA: 1.5 parts Redox balance module: PEG-SOD: 0.5 parts Sodium selenite: 0.008 parts Auxiliary ingredients: Sodium bicarbonate: 15 parts Sodium pyruvate: 2.0 parts The preparation steps are as follows: 1. Preparation of basal culture medium: DMEM and Ham's F12 were mixed at a ratio of 1:0.95, dissolved in 820 parts of ultrapure water, and sodium bicarbonate was added. The mixture was stirred (300 rpm, 25°C) until dissolved.

[0061] Adjust pH to 7.2 (fine-tune with 0.1 M HCl) and pre-filter.

[0062] 2. Add modules in stages: Metabolic module: 5-MTHF, ManNAc, and DCA were added sequentially, and stirred in the dark for 15 minutes (150 rpm) after each addition.

[0063] Immunoassay module: KIN1148 was diluted to a final concentration of 0.1% (v / v) at a dropwise rate of 0.5 mL / min and stirred for 15 minutes.

[0064] Receptor module: ATRA ethanol stock solution with a final concentration of 0.1% (v / v) was stirred for 15 minutes.

[0065] 3. Redox module treatment: PEG-SOD and sodium selenite were premixed at a molar ratio of 1:0.6, added to the culture medium and fixed to 1000 parts, and then terminally filtered.

[0066] Example 3: Formula composition (parts by mass): Basal culture medium: DMEM and Ham's F12 mixture (volume ratio 1:1.05): 910 parts Metabolic Reprogramming Module: 5-MTHF: 0.05 parts ManNAc: 0.3 parts DCA: 1.0 part Immune regulation module: KIN1148 (DMSO stock solution diluted to 0.1 μM): 0.005 parts Receptor induction module: ATRA: 0.5 parts Redox balance module: PEG-SOD: 0.1 parts Sodium selenite: 0.002 parts Auxiliary ingredients: Sodium bicarbonate: 10 parts Sodium pyruvate: 0.5 parts The preparation steps are as follows: 1. Preparation of basal culture medium: DMEM and Ham's F12 were mixed at a ratio of 1:1.05 and dissolved in 850 parts of ultrapure water. Sodium bicarbonate was added and magnetic stirring was performed (200 rpm, 25°C) until dissolved.

[0067] Adjust pH to 7.4 (fine-tune with 0.1 M NaOH) and pre-filter.

[0068] 2. Add modules in stages: Metabolic module: 5-MTHF, ManNAc, and DCA were added sequentially, and stirred in the dark for 10 minutes (100 rpm) after each addition.

[0069] Immunoassay module: KIN1148 was diluted to a final concentration of 0.05% (v / v), the addition time was extended to 20 minutes, and stirring was performed for 10 minutes.

[0070] Receptor module: ATRA ethanol stock solution final concentration 0.05% (v / v), stirred for 10 minutes.

[0071] 3. Redox module treatment: PEG-SOD and sodium selenite were premixed at a molar ratio of 1:0.4, fixed to volume, and then terminally filtered.

[0072] Comparative Example 1: 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.

[0073] Comparative Example 2: Compared with Example 1, the difference is that KIN1148 in the immune regulation module is replaced by dexamethasone of equimolar concentration, and the other components and preparation steps are the same.

[0074] Comparative Example 3: Compared with Example 2, the difference is that the mixing volume ratio of DMEM and Ham's F12 in the basal culture medium is adjusted to 1:1.5, and the other components and preparation steps are the same.

[0075] Comparative Example 4: Compared with Example 2, the difference is that the molar ratio of PEG-superoxide dismutase (PEG-SOD) to sodium selenite is adjusted to 1:0.2, and the other components and preparation steps are the same.

[0076] Comparative Example 5: Compared with Example 3, the difference is that the added amount of all-trans retinoic acid (ATRA) is adjusted to 0.3 parts, and the other components and preparation steps are the same.

[0077] Comparative Example 6: Compared with Example 1, the difference is that the light-shielding operation is cancelled in step b, and the stirring time is shortened to 3 minutes. The other components and steps are the same.

[0078] Comparative Example 7: Compared with Example 3, the difference is 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.

[0079] Comparative Example 8: Compared with Example 2, the difference is that the final DMSO concentration of KIN1148 in the immune regulation module is adjusted to 0.3% (v / v), and the other components and preparation steps are the same.

[0080] Test Example 1: The experimental steps are as follows: 1. Cell Preparation: HEp-2 cells were cultured at 1.5×10 5 cells / cm 2 The cells were seeded in 6-well plates with 3 replicate wells in each group.

[0081] The culture medium of Examples 1-3 and Comparative Examples 1, 2, 5, and 7 was used for pre-culture for 20 hours (37° C., 5% CO 2 ).

[0082] 2. Viral infection: The old culture medium was discarded and HMPV (strain NL / 1 / 00) was inoculated at an MOI of 0.1 and adsorbed for 1 hour (37°C, gently shaking).

[0083] Supplement with 2 mL of the corresponding culture medium and replace it every 48 hours.

[0084] 3. Sample collection: The cell supernatant was collected on the 5th day after infection and stored at -80°C for future use.

[0085] 4. Virus titer determination: Plaque Assay: HEp-2 cells were seeded in 96-well plates and the virus solution was serially diluted (10 -4 to 10 -8 ), and covered with maintenance medium containing 1.2% methylcellulose 1 hour after infection.

[0086] After 72 hours, the cells were fixed and stained (0.1% crystal violet), the number of plaques was counted, and the titer (PFU / mL) was calculated.

[0087] The experimental results are shown in Table 1: Table 1 Test Example 1: Comparison of virus titers on the 5th day after infection in different groups Group <![CDATA[Replicate 1 (×10 8 PFU / mL)]]> <![CDATA[Replicate 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* From the experimental data in Table 1, we can get: 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 folate cycle is interrupted, causing a sharp drop in the rate of dTMP synthesis, and viral genome replication is stalled due to nucleotide pool depletion. Meanwhile, an excess of ManNAc (Comparative Example 7) competitively inhibits nucleotide transporters (such as ENT1), resulting in a decrease in the efficiency of nucleotide precursor transmembrane transport, further exacerbating replication defects. The optimized ratio ranges in Examples 1-3 synchronize the temporal and spatial supply of 5-MTHF-driven nucleotides with ManNAc-mediated glycosylation modifications, avoiding the accumulation of immature viral particles due to a skewed metabolic resource base.

[0088] The specific design of KIN1148 within the immune regulation module is key to maintaining sustained viral replication. Dexamethasone (Comparative Example 2), a broad-spectrum immunosuppressant, can inhibit interferon signaling, but its excessive inhibition of the NF-κB pathway can trigger nonspecific apoptosis, shortening the viral replication window. KIN1148, by targeting IRF3 / 7 phosphorylation sites, selectively blocks type I interferon production, delaying the host antiviral response while preventing premature activation of apoptotic signaling. Furthermore, the limited final DMSO concentration of KIN1148 (≤0.1% v / v) prevents solvent toxicity from disrupting cell membrane integrity (Comparative Example 8), ensuring host cell receptor stability during viral adsorption.

[0089] The temporal synergy between the receptor-inducing module and the metabolic / immune module further enhances infection efficiency. All-trans retinoic acid (ATRA) enhances viral adsorption by upregulating heparan sulfate proteoglycan (HSPG) expression on the surface of HEp-2 cells. Insufficient ATRA concentration (Comparative Example 5) reduces receptor expression and viral entry efficiency. However, the combined action of ATRA and the metabolic module in Examples 1-3 creates 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 is unachievable with traditional single-component optimization strategies (such as supplementing glucose or growth factors alone).

[0090] Test Example 2: The experimental steps are as follows: 1. Cell culture and treatment: HEp-2 cells were cultured at 1×10 5 cells / cm 2 The cells were inoculated into 12-well plates and cultured using the culture medium of Examples 1-3 and Comparative Examples 3, 4, 6, and 8 for 72 hours (37° C., 5% CO 2 ).

[0091] Each group had 4 replicate wells, and the culture medium was replaced every 24 hours.

[0092] 2. Cell viability detection: Cell collection: trypsinize and centrifuge (1000 rpm, 5 min), then resuspend in PBS.

[0093] Trypan blue staining: Mix the cell suspension with 0.4% trypan blue solution in a ratio of 1:1 and let it stand for 3 minutes.

[0094] Counting: The proportion of living cells (unstained) was counted using a hemocytometer and the survival rate (%) was calculated.

[0095] 3. Detection of oxidative damage markers: Cell lysis: Total protein was extracted using RIPA lysis buffer, and protein concentration was determined by BCA method.

[0096] MDA detection: thiobarbituric acid (TBA) method, absorbance was measured at 532 nm, and MDA concentration (nmol / mg protein) was calculated according to the standard curve.

[0097] The experimental results are shown in Table 2: Table 2 Test Example 2: Comparison of cell survival rate and oxidative damage levels in different groups Group Survival rate repeat 1 (%) Survival rate replicate 2 (%) Survival rate replicate 3 (%) Mean ± SD survival rate (%) MDA concentration replicate 1 (nmol / mg) MDA concentration replicate 2 (nmol / mg) MDA concentration repeated 3 times (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* Wherein, * indicates p < 0.05 compared with Example 1 (ANOVA test); From the experimental data in Table 2, we can get: The basal medium volume ratio (DMEM:Ham's F12 = 1:0.9-1.1) maintains osmotic pressure within the physiological range of 280-310 mOsm / kg by balancing bicarbonate buffering capacity with HEPES stability. The unbalanced volume ratio in Comparative Example 3 (1:1.5) caused osmotic pressure to rise above 320 mOsm / kg, triggering compensatory energy consumption by the cell membrane sodium-potassium pump. This, combined with an outflow of intracellular water and mitochondrial swelling, ultimately exacerbated lipid peroxidation through a ROS (reactive oxygen species) burst (MDA increased by 137%). Examples 1-3 precisely controlled the volume ratio to adapt the buffer system to the ionic strength, preventing osmotic shock from interfering with energy metabolism.

[0098] The molar ratio 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. PEG-SOD converts superoxide anions (O2 -) is converted into H2O2, and sodium selenite acts as a cofactor for glutathione peroxidase (GPX), further catalyzing the decomposition of H2O2 into water. In Comparative Example 4, insufficient sodium selenite (molar ratio 1:0.2) leads to the accumulation of H2O2, which generates hydroxyl radicals (·OH) through the Fenton reaction and attacks the unsaturated fatty acids in the cell membrane (MDA increased by 81%). Example 2 achieves OO2 by optimizing the ratio of selenium to SOD. - →H2O2→H2O is completely eliminated, controlling oxidative damage to the baseline level (MDA≤1.12 nmol / mg).

[0099] Process conditions and solvent restrictions (light protection, stirring time, DMSO ≤ 0.1%) jointly ensure the activity of functional ingredients and cell membrane integrity. In Comparative Example 6, the omission of light protection resulted in photooxidative degradation of 5-MTHF and ATRA. Their free radical byproducts directly damaged DNA and inhibited respiratory chain complex I, resulting in a 24% decrease in cell viability. Insufficient stirring time (3 minutes) resulted in excessive local concentrations of ManNAc, which competitively inhibited the glucose transporter (GLUT1) and caused glycolysis impairment. In Comparative Example 8, excessive DMSO concentration (0.3%) disrupted the lipid bilayer structure of the cell membrane, leading to ion gradient leakage and mitochondrial membrane potential collapse (survival rate was only 60.4%). Through process standardization and solvent gradient dilution, this example ensures precise functional timing matching between the metabolic module and the receptor-inducing module.

[0100] Test Example 3: The experimental steps are as follows: 1. Virus purification: The viral supernatants of Examples 1-3 and Comparative Examples 1, 2, 5, and 7 were concentrated by centrifugation through a 20% sucrose cushion (10,000×g, 4° C., 2 hours) and resuspended in PBS to 1 / 10 of the original volume.

[0101] 2. Detection of infectious virus proportion: Plaque formation rate: The ratio of the endpoint dilution method (TCID50) to the plaque formation method (PFU) was used to calculate the proportion of infectious virus (PFU / TCID50 × 100%).

[0102] step: TCID50 assay: HEp-2 cells were inoculated into 96-well plates with 10-fold serial dilutions of the virus solution, and CPE (cytopathic effect) was observed for 5 days. The titer was calculated by the Reed-Muench method.

[0103] PFU determination: as in Test Example 1.

[0104] 3. Glycosylation level detection: Western Blot: After viral lysis, G protein (envelope glycoprotein) was separated by SDS-PAGE electrophoresis and transferred to PVDF membrane.

[0105] Primary antibody: anti-HMPV G protein monoclonal antibody (1:1000); secondary antibody: HRP-conjugated goat anti-mouse IgG (1:5000).

[0106] Chemiluminescence was developed, and the grayscale value of the bands was analyzed by ImageJ. The degree of glycosylation (relative glycosylation level %) was verified by ConA (concanavalin A) lectin binding assay.

[0107] The experimental results are shown in Table 3: Table 3 Test Example 3: Comparison of Virus Particle Integrity in Different Groups Group Plaque formation rate repeat 1 (%) Plaque formation rate repeated 2(%) Plaque formation rate repeated 3 (%) Mean ± SD of plaque formation rate (%) G protein glycosylation repeat 1 (%) G protein glycosylation repeat 2 (%) G protein glycosylation repeat 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* Wherein, * indicates p < 0.01 compared with Example 1 (t test); From the experimental data in Table 3, we can get: The metabolic reprogramming module coordinates the two key pathways of nucleotide synthesis and glycosylation modification in both time and space by balancing the ratio of 5-MTHF to ManNAc (1:4-6). Removing 5-MTHF (Comparative Example 1) disrupts the folate cycle, restricts dTMP synthesis, increases the error rate of viral genome replication, and produces a large number of non-infectious defective particles (with a plaque formation rate of only 34.9%). Meanwhile, an excess of ManNAc (Comparative Example 7, 1:8) competitively inhibits the glucose transporter (GLUT1), reducing the supply of the glycolytic intermediate fructose-6-phosphate, thereby impairing the mannose-6-phosphate precursor required for N-linked glycosylation, resulting in a decrease in G protein glycosylation to 51.2% (compared to 95.1% in Example 1). The optimized ratios in Examples 1-3 ensure the synchronization of dynamic nucleotide pool replenishment and sialic acid synthesis, avoiding a disconnect between genome replication and structural protein modification.

[0108] Dexamethasone (Comparative Example 2) nonspecifically inhibits the NF-κB pathway by activating the glucocorticoid receptor (GR), leading to a Golgi stress response and interfering with the localization and activity of ST6GAL1 (sialyltransferase), resulting in a loss of terminal sialylation of the G protein (glycosylation level 73.0% vs. 95.1% in Example 1). KIN1148, on the other hand, targets phosphorylation sites of IRF3 / 7, blocking type I interferon signaling without affecting NF-κB. This maintains Golgi pH stability and glycosyltransferase activity, thereby ensuring host cell membrane fusion of viral envelope proteins. Furthermore, KIN1148's low solvent concentration (DMSO ≤ 0.1%) prevents membrane lipid peroxidation from interfering with the transport of glycosylated precursors (glycosylation level in Comparative Example 8 was only 51.2%).

[0109] ATRA enhances viral adsorption efficiency by upregulating HSPG expression on the HEp-2 cell surface. Simultaneously, its nuclear receptor (RAR) signaling activates the fatty acid oxidation (FAO) pathway, providing acetyl-CoA for viral capsid assembly. In Comparative Example 5 (ATRA deficiency), decreased receptor expression delayed viral entry and prolonged retention of immature particles in the endoplasmic reticulum, making them susceptible to host proteasome degradation (plaque formation rate 44.8%). In Example 1, the synergistic effect of ATRA and the metabolic module creates a positive feedback loop: viral adsorption (HSPG-dependent), endocytosis (clathrin-dependent), and replication (nucleotide pool-dependent), ultimately achieving dual optimization of both infectious virus percentage (86.9%) and glycosylation maturity (95.1%). This multi-module linkage overcomes the technical bottlenecks of traditional single-component optimization, achieving simultaneous improvements in viral production quality and efficiency.

[0110] Test Example 4: The experimental steps are as follows: 1. Inter-batch virus titer testing: Using the culture medium of Examples 1-3 and Comparative Examples 3, 4, 6, and 8, three batches of virus were prepared continuously under the same equipment and personnel conditions (culture medium was prepared independently for each batch).

[0111] The virus titer (PFU / mL) of each batch was determined according to the method of Test Example 1, and the inter-batch coefficient of variation (CV = standard deviation / mean × 100%) was calculated.

[0112] 2. Culture medium shelf life test: The culture medium of each group was divided into sterile bottles and stored at 37°C in the dark (simulating accelerated stability test).

[0113] Samples were taken every 48 hours, and the virus titer was determined according to the method of Test Example 1. The number of days required for the titer to drop to 90% of the initial value was recorded.

[0114] The experimental results are shown in Table 4: Table 4 Test Example 4: Comparison of process stability in different groups

[0115] Wherein, * indicates p < 0.01 compared with Example 1 (t test); From the experimental data in Table 4, we can get: The basal culture medium volume ratio (DMEM: Ham's F12 = 1:0.9-1.1) controls osmotic pressure fluctuations within ±5 mOsm / kg by balancing the bicarbonate buffering capacity with the pH stability of HEPES (the inter-batch CV in Example 1 was only 2.8%). In Comparative Example 3, the unbalanced volume ratio (1:1.5) resulted in an abnormal osmotic pressure gradient (measured at 318-327 mOsm / kg), triggering compensatory energy consumption by the cell membrane sodium-potassium pump, which increased the inter-batch variation in transmembrane material transport efficiency (CV reached 12.7%). At the same time, the hypertonic environment accelerated cell aging and reduced the consistency of viral genome replication. The examples maintain ionic strength homeostasis through precise ratios, ensuring the temporal repeatability of metabolic module functions.

[0116] The defined molar ratio of the redox module (PEG-SOD: sodium selenite = 1:0.4-0.6) ensures the long-term stability of the culture medium components through cascade free radical scavenging. In Comparative Example 4, selenium deficiency (molar ratio 1:0.2) leads to reduced GPX activity. H₂O₂ accumulates continuously during storage at 37°C, generating hydroxyl radicals (·OH) via the Fenton reaction. This hydroxyl radical attacks the pterin ring of 5-MTHF, shortening its half-life from 28 days (Example 2) to 13.7 days. Simultaneously, ·OH induces the oxidation of ManNAc to neuraminic acid analogs, competitively inhibiting sialic acid synthase (NANS) activity and contributing to batch-to-batch variability in viral envelope glycosylation (standard deviation of glycosylation levels ±3.1%). The optimized ratio, through dual SOD-GPX free radical scavenging, blocks the chain oxidation reaction, achieving long-term stability in component activity and viral titer.

[0117] The process conditions (light protection / stirring) and solvent concentration limits (DMSO ≤ 0.1%) together establish a scalable production quality control system. In Comparative Example 6, the omission of light protection resulted in the photolysis of ATRA to generate cytotoxic retinoic acid free radicals, which inhibited mitochondrial complex III function and increased inter-batch variability in cellular energy metabolism (ATP level CV increased from 5% to 18%). Insufficient stirring time (3 minutes) triggered localized crystallization of ManNAc, and uneven dissolution directly impaired glycosylation efficiency (inter-batch glycosylation level fluctuation of ±7.2%). In Comparative Example 8, excessive DMSO concentration (0.3%) disrupted cell membrane lipid rafts, interfering with lipid envelope assembly during viral budding and resulting in an increased proportion of defective vacuolated particles (inter-batch titer CV reached 9.8%). By utilizing light protection, gradient stirring (10 minutes), and controlled final solvent concentration, this example minimizes physicochemical variables in the production process, supporting quality control compliance for large-scale viral production.

[0118] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A culture medium for human metapneumovirus, characterized in that: The composition includes the following components in parts by weight: Basic culture medium: 850-920 copies; Metabolic reprogramming module: including 0.05-0.15 parts of 5-methyltetrahydrofolate, 0.3-0.7 parts of N-acetyl-D-mannosamine, and 1.0-3.0 parts of sodium dichloroacetate; Immunomodulatory module: including 0.005-0.015 parts of RIG-I / MAVS pathway inhibitor; Receptor induction module: including 0.5 to 1.5 parts of all-trans retinoic acid; Redox balance module: comprising 0.1 to 0.5 parts of pegylated superoxide dismutase and 0.002 to 0.008 parts of sodium selenite.

2. The culture medium for human metapneumovirus according to claim 1, characterized in that In the metabolic reprogramming module, the mass ratio of 5-methyltetrahydrofolate to N-acetyl-D-mannosamine is 1:(4-6).

3. The culture medium for human metapneumovirus according to claim 1, characterized in that The RIG-I / MAVS pathway inhibitor is KIN1148, and its final concentration range is 0.1-0.3 μM.

4. The culture medium for human metapneumovirus according to claim 1, characterized in that The molar ratio of the PEGylated superoxide dismutase to sodium selenite is 1:(0.4-0.6).

5. The culture medium for human metapneumovirus according to claim 1, characterized in that The basic culture medium is a mixture of DMEM and Ham's F12, and the mixing volume ratio is 1:(0.9-1.1).

6. A method for preparing a culture medium for human metapneumovirus according to any one of claims 1 to 5, characterized in that: The following steps are involved: a) Dissolve the basal medium and adjust the pH to 7.2-7.4; b) adding the components of the metabolic reprogramming module, immune regulation module, and receptor induction module in stages, stirring for 10 to 15 minutes after each addition; c) Premix the components of the redox balance module and add them to the solution, make up to volume and filter to sterilize.

7. The preparation method according to claim 6, characterized in that: The immune regulation module in step b) is added dropwise after diluting the DMSO stock solution of KIN1148 to a final concentration of ≤0.1% (v / v).

8. Use of the culture medium for human metapneumovirus according to any one of claims 1 to 5 in the preparation of human metapneumovirus vaccines or diagnostic reagents.

9. The use according to claim 8, characterized in that: The following steps are involved: Inoculating HEp-2 cells in a culture system containing the culture medium and pre-culturing for 18 to 24 hours; Human metapneumovirus was inoculated at an MOI of 0.01 to 1.0, and the culture medium was replaced every 48 to 72 hours. The total culture time was 5 to 7 days.

10. The use according to claim 9, characterized in that: The virus titer of the culture medium on the 5th day after infection was ≥ 1×10 8 PFU / mL.

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