Key inhibitor of effector cd8+ t cell dedifferentiation into cd8+ tcm and use in drugs for severe viral infections

Abstract

The present application discloses that using chemokine receptor inhibitor in influenza virus FM1 infected mouse model can promote effector CD8 + T cell dedifferentiation into CD8 + TCM, improve CD8 + TCM ratio, and reduce the viral load in mice. The present application constructs an influenza virus FM1 infected mouse model, and uses a broad-spectrum chemokine receptor inhibitor vMIP-II for treatment. Animal experiments find that the broad-spectrum chemokine receptor inhibitor vMIP-II can reduce the viral load of mice, improve the CD8 + TCM ratio, and reconstruct cellular immunity. vMIP-II can reconstruct specific cellular immunity by regulating the TCM subgroup level of CD8 + T cells, which can be used for the treatment of severe viral infection and the application in related therapeutic drugs.

Description

Technical Field

[0001] This invention pertains to the basic research field of the broad-spectrum chemokine receptor inhibitor vMIP-II in the prevention and treatment of influenza virus FM1 infection in mouse models. More specifically, this invention relates to the regulation of CD8+ in mice by the broad-spectrum chemokine receptor inhibitor vMIP-II. + The TCM subgroup of T cells provides level-based defense against influenza viruses. Background Technology

[0002] Viral macrophage inflammatory protein-II (vMIP-II) is a broad-spectrum chemokine receptor inhibitor encoded by the K4 gene of Kaposi's sarcoma herpesvirus (KSHV). It shares high amino acid sequence homology with the human CC-class chemokine macrophage inflammatory protein I. vMIP can bind to its receptor using a structural backbone similar to other chemokines. vMIP-II is widely recognized as a broad-spectrum chemokine receptor inhibitor, capable of binding to multiple human chemokine receptor subfamilies. Studies have demonstrated that vMIP-II can antagonize multiple chemokine receptors, including CCR1, CCR2, CCR3, CCR5, CCR8, CXCR4, and CX3CR1, but it has no antagonistic effect on CXCR1 and CXCR2. Its effect on CCR7 is currently unknown.

[0003] After viral infection, CD8 + T cells undergo three phases: expansion, contraction, and memory formation. CD8 cells are typically characterized using surface molecules KLRG1 and CD127 (Interleukin-7 receptor subunit alpha, IL-7Rα). + The activation state of T cells, and the activated CD8 +T cells are divided into two main categories: short-lived effector cells (SLECs, characterized by KLRG1hiCD127low) and memory precursor effector cells (MPECs, characterized by KLRG1lowCD127hi). SLECs produce large amounts of cytotoxic molecules and cytokines, and most of them undergo apoptosis during the contraction phase, while MPECs further differentiate into memory cells after clearing pathogens. Memory cells further differentiate into different memory cell subsets, typically characterized by surface molecules CD45RA, CD45RO, chemokine receptor CCR7, and angiogenic L-selectin (CD62L). Specifically, they are classified into central memory T cells (TCM, phenotype CD45RA-CD45RO+CCR7+CD62L+), effector memory T cells (TEM, phenotype CD45RA-CD45RO+CCR7-CD62L-), and tissue-specific memory T cells (TRM, phenotype CD103+CD69+CD62L-CD27-). TCMs are generally distributed in peripheral tissues, immune organs, and lymph nodes, and can rapidly proliferate and differentiate upon re-stimulation by antigens. TEM cells are mainly found in non-lymphatic tissues and organs, participate in systemic circulation, and can migrate to peripheral inflammatory tissues to exert immediate effector functions.

[0004] CD8 under continuous antigen stimulation + T cells exhibit a state of exhaustion (TEX), characterized by low levels of IL-2, TNF-α, and IFN-γ, while their cell surface expresses high levels of inhibitory molecules such as PD-1, TAG3, CD244, and CD160. Studies have shown that they retain the memory of CD8+. + T cells are a subset of effector T cells; suppressing the expression of naivety-related genes can reverse the effector CD8 expression. + T cells differentiate into longevity memory CD8 cells. + T cells.

[0005] Previous research in our laboratory utilized vMIP-II to competitively inhibit the binding of HIV to co-receptors CCR5, CXCR4, and CCR3 on target cells, thereby preventing viral entry into target cells and demonstrating its anti-HIV infection effect. In vivo experiments showed that the expression levels of certain TCRVβ subfamilies changed in cynomolgus monkeys during the early stages of SIV infection, with specific proliferation of some Vβ subfamilies and altered clonal characteristics, suggesting that this proliferation may be virus-specific. vMIP-II promoted the proliferation of these Vβ subfamilies, indicating that it enhances the immune system response and promotes the proliferation of specific immune cells. In fact, our studies on recombinant vMIP-II in monkey SIV models and in human AIDS treatment have shown that it significantly increases the memory CD8 of the infected organism. + T-cell activity plays a crucial role in viral septicemia during the morbid phase.

[0006] Previous research in our laboratory found that vMIP-II can promote the effect of CD8 in the treatment of COVID-19. + T cells dedifferentiate into CD8+ + TCM method to improve CD8 + The therapeutic effect is achieved by adjusting the TCM ratio. Gene chip analysis suggests that vMIP-II exerts its therapeutic effect by influencing chemokine receptors, phosphorylation pathways, the mTOR pathway, and the methyltransferase Dnmt3a. Chemokine receptors and G protein α are key targets for this action. To further explore the effects of these targets in animal models, we used an influenza virus FM1-infected mouse model to validate the chemokine receptor target. Identifying the drug target is crucial for developing antiviral drugs targeting severe viral infections. Summary of the Invention

[0007] This invention utilizes vMIP-II intervention to treat a mouse model infected with influenza virus FM1, and modulates viral load and CD8 levels within cells. + TCM ratio and mitochondrial function were used to assess the CD8+ receptor response of chemokine receptors in a mouse model of influenza virus. + The role of T cell subset differentiation. Attached Figure Description

[0008] Figure 1 This is the standard protein curve for the phosphorylated protein in Example 1.

[0009] Figure 2 The CD8 effect in different groups of mice in Example 1 + Concentration of phosphorylated proteins in T cells.

[0010] Figure 3To illustrate the memory of CD8 in different groups of mice in Example 1. + T cells and CD8 + The percentage of TCM, *p<0.05, n=3.

[0011] Figure 4 The mitochondrial membrane potential in mouse cells from different groups in Example 1 is shown, **p<0.01, n=3. Detailed Implementation

[0012] The present invention will be further described in detail below with reference to the embodiments.

[0013] Example 1: Response of a mouse model infected with influenza virus FM1 to key drug targets

[0014] Materials and Methods

[0015] Virus and Animals: Laboratory mice, 6-8 weeks old and female, were purchased from the Guangdong Provincial Medical Laboratory Animal Center. Before the experiments, the mice were housed in an SPF-grade animal laboratory at a temperature of 20±1℃, humidity of 50%±5%, and light intensity of 3000Lx and 0Lx for 12 hours each. IAV [FM1 / 1 / 47 strain (mouse lung-adapted strain)] was used in all experiments. FM1 was provided by the Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, and stored in our laboratory at -80℃.

[0016] Experimental reagents: vMIP-II injection stock solution and lyophilized powder for injection; vMIP-II antigen standard (physicochemical control) was developed by the Institute of Genomic Drugs, Jinan University and passed the national drug and biological product testing. vMIP-II monoclonal antibodies, CD45RO-PE-Cy5, CD62L-APC-H7, PD-1-PE-Cy5, Tim-3-APC-H7, HLA-DR-APC-H7, CD38-APC, GNLY-FITC, PRF-FITC, etc., were all purchased from Biolegend; BD FACS flow cytometer (Bio-Rad Laboratories, USA), etc.

[0017] Establishment of a mouse model of influenza virus infection

[0018] After viral resuscitation, the virus was passaged twice via the allantoic cavity of 9-day-old chicken embryos. The hemagglutination titer of the obtained virus was measured to be 1:640. The mortality rate of mice was determined by serial dilution. 50 μl of the virus concentration representing 20% ​​of mouse mortality (hemagglutination titer 1:40) was administered intranasally to mice. The experimental mice were labeled and weighed, and anesthetized by intraperitoneal injection of 1% sodium pentobarbital solution. After anesthesia, mice were infected intranasally with 50 μl of FM1 virus. The control group received an equal volume of distilled water. After infection, the mice were returned to individually air-conditioned isolation cages (IVCs) for normal rearing. The general survival status, weight, and coat condition of the mice were observed and recorded daily after the virus administration.

[0019] RT-qPCR detection of viral FM1

[0020] Total RNA extraction from animal tissues

[0021] During total RNA extraction, avoid enzyme-free operations and store samples on ice to prevent contamination and degradation. Sample tissue processing: Remove dissected mouse lung tissue from a -80℃ freezer; clean the steel balls with 95% alcohol, add 3-4 steel balls and 1 ml of RNAiso plus to each lung tissue, homogenize, incubate at room temperature for 5 min, and centrifuge for 5 min (4℃, 12000 rpm / min); carefully obtain the supernatant using a pipette and transfer it to a new 1.5 ml EP tube, only aspirating the supernatant. Add 200 μL of chloroform to each supernatant and mix manually until the solution turns milky white; do not pipette. Incubate the EP tubes at room temperature for 5 minutes, then centrifuge for 15 minutes (4℃, 12000 rpm / min). Carefully collect the supernatant using a pipette and transfer it to a new 1.5 ml EP tube with a conical bottom, paying attention to the white middle layer and ensuring enzyme-free operation. Add 1 ml of isopropanol to the supernatant using a pipette, mix well, and incubate at room temperature for 10 minutes. Centrifuge for 10 minutes (4℃, 12000 rpm / min), and the RNA precipitate will form at the bottom of the tube. To wash the RNA precipitate, add 1 ml of 75% ethanol to the EP tube, wash thoroughly, and centrifuge for 5 minutes (4℃, 7500 rpm / min). Discard the supernatant, being careful not to touch the RNA precipitate. Allow to dry at room temperature, then add 100 μL of RNase-free water to dissolve the precipitate completely. After mixing, determine the RNA concentration and obtain high-purity RNA according to the kit instructions.

[0022] cDNA synthesis (Total 20ul)

[0023] Prepare the reaction mixture on ice according to the following ingredients. To ensure the accuracy of the reaction mixture preparation, prepare the Master Mix by the reaction number + 2 before each reaction, then aliquot it into each reaction tube, and finally add the RNA sample. Reaction conditions: 42℃ for 2 min (or room temperature for 5 min-30 min), store at 4℃.

[0024]

[0025] Reverse transcription reaction: Prepare reaction solutions on ice. To ensure accuracy, prepare Master Mix twice the amount needed for each reaction, then aliquot 10 μl into each reaction tube. Gently mix and immediately proceed with the reverse transcription reaction. Reaction conditions: 37°C for 15 min; 85°C for 5 s; store at 4°C (for long-term storage, store at -20°C or lower).

[0026]

[0027]

[0028] Real-time PCR amplification of cDNA

[0029] The reaction solution should be prepared on ice. Procedure for reacting with Premix Ex Taq II. Reaction conditions: 95℃, 30s; 95℃, 5s; 60℃, 30s; Repeat → Step 2, 39 times (40 cycles); 95℃, 10s; 55℃ to 95℃, 5s; 4℃, Forever, End.

[0030]

[0031] The primer sequences used in the experimental detection are shown in the table below:

[0032]

[0033] During the experiment, each sample was placed in three sub-wells, and the experiment was conducted three times. Finally, the relative expression level of the virus was calculated using the 2-ΔΔCt method; GADPH was used as an internal reference.

[0034] Mouse grouping and administration

[0035] Mice successfully infected with FM1 virus were randomly divided into two groups: a vMIP-II treatment group and a positive control group. Mice in the treatment group were intraperitoneally injected with vMIP-II 25 μg / kg daily for 3 days. Two hours after the last administration, 0.5 mL of venous blood was collected by tail clipping.

[0036] Isolation and culture of mouse PBMCs

[0037] Two groups of mice were subjected to tail-cropping blood collection. 0.5 mL of venous blood was collected from each mouse and diluted with an equal volume of Hanks' solution. Then, an equal volume of mouse lymphocyte separation medium was added, and the mixture was centrifuged at 2000 rpm for 20 min. After removing the centrifuge tube, the PBMCs at the liquid-liquid interface were carefully aspirated, and 3 mL of Hanks' solution was added and mixed. The mixture was centrifuged at 1500 rpm for 5 min. The supernatant was discarded, and the PBMCs concentration was adjusted to 1 × 10⁹ / L using RPMI 1640 medium containing 10% fetal bovine serum and a final concentration of 5 μg / mL concanavalin A.

[0038] Detection of protein phosphorylation levels in mouse effector CD8+ T cells

[0039] Two groups of mouse cells were divided at 1×10 7 Adjust the concentration of phosphopeptides / mL to a desired level and resuspend in 1 mL of Phospho-Lysis Buffer. Incubate at room temperature for 10 min, then centrifuge at 14000 rpm for 10 min at 4 °C and discard the precipitate. Mix the protein solution with 0.2 volumes of 10× Phospho-Wash Buffer and incubate at room temperature for 10 min. After 10 min, centrifuge at 14000 rpm for 10 min at 4 °C and discard the precipitate. Titrate the phosphopeptide solution with 1% acetic acid to pH < 5. Dilute an appropriate amount of 10× Phospho-Wash Buffer to 1× with deionized water, and dilute an appropriate amount of 5× Phospho-Elution Buffer to 1× with deionized water. Equilibrate the Phospho-Column with 10 mL of 1× Phospho-Wash Buffer, allowing the buffer to pass through under gravity. Load the prepared phosphopeptide solution onto the column and allow it to pass through under gravity. Collect the protein and wash the column with 10 mL of 1×Phospho-Wash Buffer and 5 mL of deionized water. Elute the bound phosphoproteins with 10 mL of phosphate elution buffer. Detect the concentration of phosphorylated proteins using the BCA method.

[0040] Flow cytometry detection of mouse CD8 + T and CD8 + TCM ratio

[0041] Cultured mouse PBMC cells (1.5 mL) were placed in a test tube, and H7 cells from CD3-PE-sorted T cells were added with 10 μL of each of four monoclonal fluorescent antibodies, including mouse anti-CD3-PE, CD4-FITC, CD8-APC, CD45RO-PE-Cy5, and CD62L-APC. After mixing thoroughly, the mixture was incubated at room temperature in the dark for 15-30 minutes. 2 mL of hemolysin was added to lyse the red blood cells. The culture was mixed on a shaker and incubated at room temperature in the dark for 10 minutes, followed by centrifugation at 1000 rpm for 10 minutes. The supernatant was discarded. The cells were washed with 1 mL of PBS buffer containing 0.1% NaN3, centrifuged at 1000 rpm for 10 minutes, and the supernatant was discarded. The cells were resuspended in fixative (300 μL) and then analyzed using a BD-FACsCalibur flow cytometer.

[0042] Detection of mouse effect CD8 + Mitochondrial membrane potential of T cells

[0043] Mitochondrial membrane potential was measured using the JC-1 kit (Beyotime Biotechnology, Shanghai, China) according to the manufacturer's instructions. After removing the culture medium from the cells, they were washed once with PBS. Then, 1 mL of cell culture medium and 0.5 mL of JC-1 staining working solution were added to the cells. After thorough mixing, the cells were incubated at 37°C for 20 minutes. The supernatant was then removed, and the cells were washed twice with diluted JC-1 staining buffer (1x). 2 mL of cell culture medium was added to the washed cells, and the cells were observed under a fluorescence microscope.

[0044] Statistical analysis

[0045] All experiments were performed in triplicate. Statistical significance was determined using Prism software. P-values ​​were determined using a two-tailed t-test. *P<0.05; **P<0.01.

[0046] Results Analysis

[0047] Effect of vMIP-II on viral load in lung tissue of mice infected with influenza virus FM1 strain

[0048] After mice were infected with the FM1 strain of influenza virus, the viral load in the lung tissue was significantly high. However, after treatment with vMIP-II, the viral load was significantly reduced, indicating the therapeutic effect of vMIP-II on influenza virus mice.

[0049] Table 1. Effects of vMIP-II on viral load in lung tissue of mice infected with influenza virus strain FM1

[0050] **P < 0.01, n = 3.

[0051] Mouse effect CD8 + Changes in T cell protein phosphorylation levels

[0052] Phosphorylated proteins were extracted using a phosphorylated protein extraction kit, and protein concentrations were determined using the Bradford method. Figure 1 The results showed that, compared to the positive control group, the concentration of phosphorylated proteins in the cells of mice in the treatment group was reduced. This indicates that competitive inhibition of chemokine receptor expression by vMIP-II can reduce the effector CD8 in mice. + Protein phosphorylation levels within T cells.

[0053] CD8 in mice after vMIP-II treatment + T cell subset detection

[0054] We used flow cytometry to sort mouse PBMCs from the treatment group and the positive control group, and detected CD8. + T cell subset proportions, results as follows Figure 3 As shown. Compared to the positive control group, the treatment group's memory CD8... + The total number of T cells did not change significantly, but the CD8 count in the treatment group... + The proportion of TCM was significantly higher than that of the positive control group. This indicates that inhibiting chemokine receptors via vMIP-II can promote effector CD8. + T cells convert CD8 + TCM differentiation.

[0055] Mouse effect CD8 + T-cell mitochondrial function detection

[0056] The changes in mitochondrial membrane potential in cells of the treatment group and the positive control group were detected using the JC-1 probe. The results are as follows: Figure 4 The positive control group showed red fluorescence, while the treatment group showed green fluorescence, indicating that the mitochondrial membrane potential in the treatment group was higher than that in the positive control group (**P<0.01). This demonstrates that vMIP-II treatment reduces mitochondrial membrane potential and impairs mitochondrial function.

Claims

1. The application of vMIP-II, a broad-spectrum chemokine inhibitor that can induce the dedifferentiation of effector CD8+ T cells into CD8+ TCMs in mice infected with influenza virus FM1, in the preparation of a therapeutic drug for mice infected with FM1 influenza virus.

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