Application of TNFR2 in preparation of medicine for treating HIV infection

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of TNFR2 in preparation of a medicine for treating HIV infection. It is found for the first time that TNFR2 protein can inhibit infection of HIV pseudoviruses on CD4 cells by blocking combination of HIV envelope protein gp120 and CD4. Compared with the prior art, the invention has the following advantages: 1, experimental evidence is provided for the combination of TNFR2 and gp120 for the first time; 2, the binding site of the TNFR2 on the gp120 is close to that of the CD4, and the binding of the gp120 and the CD4 cell can be blocked; and 3, it is proved for the first time that the TNFR2 protein can inhibit infection of HIV pseudovirus on cells. The invention has important theoretical and practical meanings for researching the inhibition of the TNFR2 protein on HIV (Human Immunodeficiency Virus) infection.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and particularly relates to the application of TNFR2 in preparing medicines for treating HIV infection. Background Art

[0002] Human immunodeficiency virus type 1 (HIV-1), the causative agent of AIDS, infects cells by first binding to the CD4 receptor and then to the coreceptors CCR5 or CXCR4. A gradual decrease in CD4+ cells is a hallmark of AIDS, but the mechanisms underlying this decrease remain largely unresolved. Among all CD4+ cells, certain cells with high coreceptor expression, such as macrophages and regulatory T cells (Tregs), have been found to be resistant to HIV-1 infection. Previous studies have found that CD4+ macrophage subsets are less sensitive to the cytopathic effects of HIV-1 and are able to survive longer. Furthermore, multiple studies have demonstrated that tumor necrosis factor (TNF) downregulates CD4 expression on the macrophage surface during HIV-1 infection, potentially contributing to this mechanism. However, despite the presence of TNF, effector CD4+ T cells do not exhibit resistance to HIV, whereas CD4+ FoxP3+ regulatory T cells (Tregs) have been found to be resistant to HIV-1 infection. Furthermore, Tregs isolated from the blood and lymphoid tissues of HIV-infected individuals maintained their suppressive capacity, indicating that these cells are not intrinsically impaired by HIV infection. These findings suggest that the mechanism of CD4 cell resistance to HIV is independent of CD4 expression. Currently, there is no clear explanation for the tolerance of macrophages and Tregs to HIV-1.

[0003] Previous studies have shown that TNF and TNFRs can affect HIV-1 infection. For example, pretreatment of macrophages with a TNF mutant protein that selectively binds to TNFR2 delayed the detection of HIV-1 DNA long terminal repeats in the cells compared to controls. In addition, Foxp3, a key transcription factor for the development and function of Treg cells, has been shown to significantly inhibit HIV-1 replication. However, despite these findings, it remains unclear whether the TNFR2 protein can inhibit HIV-1 infection. Elucidating the relationship between TNFR2 and HIV-1 is crucial for accurately understanding HIV pathogenesis and developing an effective HIV-1 vaccine. Summary of the Invention

[0004] The first aspect of the present invention aims to provide use of a TNFR2 activator in the preparation of a medicament for treating HIV infection.

[0005] The second aspect of the present invention aims to provide a method.

[0006] In order to achieve the above-mentioned purpose of the present invention, the technical solution adopted by the present invention is:

[0007] The first aspect of the present invention provides use of a TNFR2 activator in the preparation of a medicament for treating HIV infection.

[0008] Preferably, the TNFR2 activator comprises at least one of increasing the expression level, activity, and / or binding ability of TNFR2 protein with interacting proteins.

[0009] Preferably, the amino acid sequence of the TNFR2 protein includes at least one of 1) to 3);

[0010] 1) the amino acid sequence shown in SEQ ID NO: 1;

[0011] 2) a nuclear sequence in which one or more amino acids are substituted and / or deleted and / or added to SEQ ID NO: 1 and which has the same function as the protein shown in SEQ ID NO: 1; or

[0012] 3) An amino acid sequence that has 95%, 96%, 97%, 98% or more homology to SEQ ID NO: 1 and has the same function as the protein shown in SEQ ID NO: 60.

[0013] Preferably, common substitutions in proteins with the same function in 2) are considered conservative substitutions, such as substitutions between aliphatic amino acids Ala, Val, Leu and Ile, interchange of hydroxyl residues Ser and Thr, exchange of acidic residues Asp and Glu, substitution between amide residues Asn and Gln, exchange of basic residues Lys and Arg, and substitution between aromatic residues Phe and Tyr.

[0014] Preferably, the above 3) means that the given amino acid sequence shares at least 95% identity with the reference sequence, for example, 95%, 96%, 97%, 98%, or 99%. Alternatively, it means that the given amino acid sequence differs from the reference sequence by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. For polypeptides, such differences are preferably amino acid substitutions or deletions. The difference in identity here can come from conservative amino acid substitutions, or from the introduction of a tag protein, or the introduction of a nuclear import sequence, or the introduction or modification of a signal peptide sequence. For example, adding a tag such as His, GFP, or HA to the C-terminus of SEQ ID NO: 1 results in a change in identity, or adding a signal peptide to the N-terminus of SEQ ID NO: 1, or adding a conventional nuclear import sequence to the N-terminus, thereby resulting in a change in identity.

[0015] Preferably, the substance that increases the expression level of TNFR2 protein includes TNFR2 protein itself, such as exogenously added TNFR2 protein.

[0016] Preferably, the TNFR2 protein includes a pharmaceutically acceptable salt or a pharmaceutically acceptable chemical modification thereof.

[0017] Preferably, the pharmaceutically acceptable salt includes at least one of a metal salt, an ammonium salt, a salt formed with an inorganic acid, a salt formed with an organic base, a salt formed with an organic acid, a salt formed with a basic amino acid, and a salt formed with an acidic amino acid.

[0018] Preferably, the pharmaceutically acceptable salt includes at least one of a metal salt, an ammonium salt, a salt formed with an inorganic acid, a salt formed with an organic base, a salt formed with an organic acid, a salt formed with a basic amino acid, and a salt formed with an acidic amino acid.

[0019] Preferably, the metal salt includes an alkali metal salt or an alkaline earth metal salt.

[0020] Preferably, the alkali metal salt includes at least one of a sodium salt and a potassium salt.

[0021] Preferably, the alkaline earth metal salt includes at least one of calcium salt, magnesium salt, barium salt and aluminum salt.

[0022] Preferably, the salt formed with an organic base includes a salt formed with at least one of the following organic bases: trimethylamine, triethylamine, pyridine, picoline, 2,6-lutidine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine, and N,N'-dibenzylethylenediamine.

[0023] Preferably, the salt formed with an inorganic acid includes a salt formed with at least one of the following inorganic acids: hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, and phosphoric acid.

[0024] Preferably, the salt formed with the organic acid includes a salt formed with the following organic acids: at least one of formic acid, acetic acid, trifluoroacetic acid, phthalic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, and p-toluenesulfonic acid.

[0025] Preferably, the salt formed with a basic amino acid includes a salt formed with at least one of the following basic amino acids: arginine, lysine, and ornithine.

[0026] Preferably, the salt formed with the acidic amino acid includes a salt formed with the following acidic amino acids: at least one of aspartic acid and glutamic acid.

[0027] Preferably, the pharmaceutically acceptable modification includes at least one of phosphorylation, sulfonation, acylation, glycosylation, ubiquitination, acetylation, methylation, sulfation, phospholipidation, and halogenation. For example, fluorine atoms (fluoroethyl) can be introduced to enhance lipophilicity and improve blood-brain barrier permeability. The corresponding modification method can be selected according to the actual use of the drug.

[0028] The pharmaceutically acceptable modification includes at least one of phosphorylation, sulfonation, acylation, glycosylation, ubiquitination, acetylation, methylation, sulfation, phospholipidation, and halogenation.

[0029] Preferably, the drug comprises pharmaceutically acceptable excipients, and / or any one or more other active ingredients.

[0030] Preferably, the pharmaceutically acceptable excipients include at least one of solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integrators, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoaming agents, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculating agents, filter aids, release retardants, and carriers.

[0031] In some embodiments of the present invention, the drug further comprises a pharmaceutically acceptable excipient. The excipient is generally recognized for this purpose and as an inactive ingredient of the medicament. A compilation of pharmaceutically acceptable excipients can be found in reference books such as the Handbook of Pharmaceutical Excipients (2nd edition, edited by A. Wade and PJ Weller; published by the American Pharmaceutical Association, Washington and The Pharmaceutical Gess, London, 1994); and the Pharmacopoeia of the People's Republic of China - List of Pharmaceutical Excipients.

[0032] Preferably, the drug is administered by one or more methods selected from the following groups: oral administration, injection, gold-coated gene gun bombardment, replication-defective bacteria carrying plasmid DNA, replication-defective adenovirus carrying target DNA or target gene-encoded protein, electroporation, intravenous, pulmonary, mucosal, nasal, intraperitoneal, intracranial, intratumoral, sublingual, buccal, and transdermal administration.

[0033] Preferably, the dosage form of the product includes a dosage form for gastrointestinal administration or a dosage form for parenteral administration.

[0034] Preferably, the dosage form for administration through the gastrointestinal tract includes at least one of powders, tablets, granules, capsules, sustained-release preparations, solutions, dry suspensions, effervescent tablets, emulsions, suspensions, syrups, drops, and chewable tablets.

[0035] Preferably, the non-gastrointestinal administration dosage form includes at least one of an injection dosage form, a respiratory tract administration dosage form, a skin administration dosage form, a mucosal administration dosage form, and a cavity administration dosage form.

[0036] The beneficial effects of the present invention are:

[0037] This invention, for the first time, discovers that TNFR2 protein can inhibit HIV pseudovirus infection of CD4 cells by blocking the binding of HIV envelope protein gp120 to CD4. Compared with previous technologies, this invention has the following advantages: 1. It provides the first experimental evidence for the binding of TNFR2 to gp120; 2. It reveals that the binding site of TNFR2 on gp120 is close to CD4 and can block the binding of gp120 to CD4 cells; 3. It demonstrates for the first time that TNFR2 protein can inhibit HIV pseudovirus infection of cells. This invention has important theoretical and practical significance for the study of the inhibitory effect of TNFR2 protein on HIV infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] The present invention will be further described below with reference to the accompanying drawings and embodiments, in which:

[0039] Figure 1 The direct interaction between HIV-1 gp120 protein and TNFR2 protein; A is the ELISA assay for the binding of gp120 to TNFR2, with the color development results of TMB substrate shown on the left and the statistical analysis of the final OD450 value on the right; B is the Co-IP analysis to verify the interaction between gp120 and TNFR2, with the IgG group as a control to ensure the specificity of the experiment; C is the biofilm interferometry (BLI) technique to detect the binding of gp120 to TNFR2 Five gp120 concentrations (2 μM, 1 μM, 0.5 μM, 0.25 μM, and 0.125 μM) were used to determine their affinity; D is the Kd value determined from the BLI results, providing a quantitative assessment of the binding affinity between gp120 and TNFR2; one-way analysis of variance (ANOVA) was used to analyze the differences between the groups; significance levels: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns, no significant difference.

[0040] Figure 2Comparison results of the TNFR2 epitope and the CD4 binding epitope on gp120, where: A is the interaction between TNFR2 and the gp120 binding site evaluated using the AlphaFold3 structure, and pLDDT is used as a measure of conformational flexibility; B is the interaction between TNFR2 (blue) and gp120 (orange); C is the hydrogen bond between residues in the TNFR2-gp120 complex; D is the comparison of the binding epitopes of TNFR2 and CD4 on gp120.

[0041] Figure 3 Blockade of gp120 binding to CD4 by TNFR2 (A) ELISA was used to assess whether TNFR2 (50 μg / mL to 30 μg / mL) could inhibit the interaction of gp120 with plate-immobilized CD4, and the final OD450 values ​​are shown. B) Flow cytometry was used to assess His-tagged cell binding, indicating gp120 binding to Jurkat cells, and to examine whether TNFR2 (10 μg / ml) could block gp120 binding to cells. C) Flow cytometric analysis shows the percentage of positive cells, with mean fluorescence intensity (MFI) on the left. D) Further examination of the dose-dependent blocking effect of TNFR2 (20 μg / ml to 1 μg / ml). E) Flow cytometry was used to assess gp120 binding to human peripheral blood mononuclear cells and to assess the effect of TNFR2. One-way analysis of variance (ANOVA) was used to analyze group differences. Significance level: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns, no significant difference.

[0042] Figure 4 TNFR2 protein can inhibit HIV pseudovirus infection CD4 + CCR5 + cells, wherein: A) HIV pseudovirus carrying the green fluorescent protein gene is used to infect CD4 + CCR5 + 293T cells, the proportion of GFP-positive cells was analyzed by flow cytometry to show the efficiency of viral infection, and the proportion of positive cells in each group is shown on the right; B) The blocking effect of TNFR2 was further evaluated to see whether it was dose-dependent; C) 12 hours after infection, the transfected CD4 + CCR5 + 293T cells; significance level: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns, no significant difference. DETAILED DESCRIPTION

[0043] The following will clearly and completely describe the concept and technical effects of the present invention in conjunction with the embodiments to fully understand the purpose, features and effects of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0044] Based on an in-depth analysis of existing HIV infection, this paper proposes an innovative solution to explore the relationship between HIV and TNFR2. Specifically, (1) the present invention systematically explores whether gp120 protein binds to TNFR2 and whether it has high affinity through various methods such as enzyme-linked immunosorbent assay (ELISA), co-immunoprecipitation (Co-IP), and biofilm interferometry. These technologies will help determine the direct interaction between gp120 and TNFR2 and its binding strength; (2) the present invention simulates the binding of gp120 protein to TNFR2 through AlphaFold3 technology, and compares it with the binding of gp120 to CD4. Through this simulation, we further explore the binding patterns and affinity differences of gp120 in different receptors, providing a theoretical basis for understanding the mechanism of action of gp120 on different receptors; (3) The present invention uses Jurkat cell lines and human primary lymphocytes, combined with the His tag analysis on the surface of gp120 protein, to provide an experimental basis for exploring the effect of TNFR2 on the binding of gp120 and CD4; (4) The present invention uses HIV pseudovirus containing gp120 envelope to explore the effect of TNFR2 on virus-infected cells, simulate the real HIV infection process, and analyze the role of TNFR2 in the process of virus entry into host cells. Example 1 HIV-1 envelope protein gp120 directly interacts with TNFR2

[0045] 1. Experimental methods

[0046] (1) Biomembrane interference TNFR2 protein was biotinylated and purified using a desalting column (Thermo, USA) according to the manufacturer's instructions. The concentration of biotinylated protein was measured using a BCA kit (Thermo, USA). 5 μg / ml biotinylated TNFR2 was immobilized on a SA biosensor (SARTOTIUS, Germany) and exposed to 5 μM, 2 μM, 1 μM, 0.5 μM, 0.25 μM, and 0.125 μM gp120 protein for 200 s. PBST was used as a control. Dissociation was performed for 300 s. Data were obtained from Data were collected using R2 (SARTOTIUS, Germany) and analyzed using Data Analysis 9.0 (SARTOTIUS, Germany).

[0047] (2) Immunoprecipitation Cell lysate was used to prepare cell lysate proteins from HEK293T-TNFR2. The cells were incubated with Dynabeads Protein G beads coupled with antibodies (anti-gp120, abcam, USA; or control IgG antibody, CST, Framingham, MA USA) overnight at 4°C, and the antibody-antigen immune complexes enriched by magnetic separation were collected. After washing three times with PBS, the cells were boiled and eluted in 2×SDS sample buffer. The obtained protein complexes were resolved on SDS-PAGE (SDS-polyacrylamide gel electrophoresis) gradient gels (10% bis-tris protein gel; company) and transferred to polyvinylidene difluoride membranes. The membranes were blocked with 3% BSA and probed with primary antibodies (anti-TNFR2 antibodies, CST, Framingham, MA USA) at 4°C overnight. The membranes were then incubated with horseradish peroxidase-labeled secondary antibodies (1:500) at room temperature for 1 hour to detect protein bands.

[0048] (3) Enzyme-linked immunosorbent assay (ELISA) To measure gp120-TNFR2 binding, TNFR2 protein was first coated onto plates overnight. Different concentrations of gp120 (50 μg / mL, 5 μg / mL, 0.5 μg / mL, 0) were added to the TNFR2-coated plates and incubated at room temperature for 2 hours. After washing with buffer (20 mM Tris-HCl, 500 mM NaCl, 0.05% Tween 20 [pH 7.5]), gp120 binding was detected using an anti-His-HRP conjugated antibody and then detected using a 3,3',5,5'-tetramethylbenzidine chromogenic substrate.

[0049] 2. Experimental results

[0050] In this example, the interaction between the extracellular domain of TNFR2 (sTNFR2, protein purchased from Sino Biological Biotechnology Co., Ltd.) and gp120 core (gp120core, protein purchased from Sino Biological Biotechnology Co., Ltd.) was tested by enzyme-linked immunosorbent assay (ELISA).

[0051] The sequence of the TNFR2 protein is shown in SEQ ID NO: 1: APEPG STCRL REYYD QTAQM CCSKC SPGQHAKVFC TKTSD TVCDS CEDST YTQLW NWVPE CLSCG SRCSSDQVET QACTR EQNRI CTCRP GWYCALSKQE GCRLC APLRK CRPGF GVARP GTETSDVVCK PCAPG TFSNT TSSTD ICRPH QICNV VAIPGNASMD AVCTS TSP.

[0052] The results showed that at a concentration of 90 nM (5 μg / ml), gp120core exhibited a significant ability to bind to immobilized sTNFR2, and this binding occurred in a concentration-dependent manner ( Figure 1 gp120core was incubated with lysate proteins from TNFR2-overexpressing cells for Co-IP experiments, and the results further demonstrated the binding of gp120 to TNFR2 ( Figure 1 In this example, biofilm interferometry (BLI) was used to measure biomolecular interactions in real time to assess the binding of gp120 to TNFR2. Specifically, 187 nM (5 μg / ml) of sTNFR2-biotin protein was immobilized on a streptavidin (SA) sensor tip and exposed to varying concentrations of gp120 core (125 nM to 2 μM). It was observed that gp120 bound to TNFR2 with high affinity and exhibited very slow dissociation ( Figure 1 In addition, combined with BLI analysis, the dissociation constant (Kd) of the gp120-TNFR2 interaction was 86.5 nM ( Figure 1 Middle (D), indicating high affinity and strong specificity of the binding interaction between gp120 and TNFR2.

[0053] Example 2 Analysis of Binding between TNFR2 and gp120

[0054] 1. Experimental methods

[0055] AlphaFold3 was used to predict the structure of the protein complex. The amino acid sequences of TNFR2 (PDB ID: 3ALQ) and gp120 (PDB ID: 4RZ8) were collected from the UniProt database and multiple sequence alignments were performed using HHblits. These alignments were then input into AlphaFold3, which was configured for multi-chain complex prediction. The model generated several candidate structures, which were evaluated using the pLDDT scoring system to determine their reliability. The structure with the highest score was selected as the final model. Visualization and further analysis were performed using PyMOL17-19.

[0056] 2. Experimental results

[0057] To visualize the binding of TNFR2 and gp120, this example uses AlphaFold3 to predict the complex structure of TNFR2 and gp120, providing a detailed and reliable characterization of the interaction ( Figure 2 (A) The model shows that TNFR2 binds to a specific epitope on gp120, and this region is critical for the interaction between CD4 and gp120. Detailed analysis of the interaction surface between gp120 and TNFR2 revealed a total binding area of This prominent interaction surface involves electrostatic and hydrophobic interactions, with key residues forming hydrogen bonds, salt bridges, and hydrophobic contacts, thereby stabilizing the gp120-TNFR2 complex ( Figure 2 In addition, comparative analysis of binding epitopes showed that the TNFR2 epitope on gp120 highly overlaps with the CD4 binding epitope ( Figure 2 Middle D).

[0058] Example 3 TNFR2 protein blocks the binding of gp120 to CD4

[0059] 1. Experimental methods

[0060] (1) Detection of the ability of TNFR2 to block gp120 binding to cells by flow cytometry Jurkat cells and human PBMCs were incubated with serially diluted TNFR2 (10 μg / mL, 5 μg / mL, 2.5 μg / mL, 1.25 μg / mL) or TNFR1 (20 μg / mL, 10 μg / mL, 5 μg / mL) for 1 hour. Subsequently, His-tagged gp120 (5 μg / mL) was added to the cells and incubated for another 1 hour. Flow cytometric analysis using specific antibodies against the His tag was performed to assess the binding between gp120 and Jurkat cells or PBMCs. High-throughput flow cytometry was used to determine the mean fluorescence intensity (MFI). In the PBMC experiment, anti-human CD4 antibodies were used to distinguish cell populations. The ethics of the human blood component research has been approved by the Health Bureau of the Macao Special Administrative Region (Protocol ID: 11 / UM / ICMS / OFI / 2022).

[0061] (2) Enzyme-linked immunosorbent assay (ELISA) To determine the ability of TNFR2 to compete with soluble CD4 for gp120 binding, CD4 protein was first coated on a plate. Different concentrations of TNFR2 protein (50 μg / mL, 40 μg / mL, 30 μg / mL) were mixed with gp120 and then added to the plate and left at room temperature for 2 hours. After washing with buffer, gp120 binding was detected using an anti-His HRP conjugate antibody. Detection was performed using a 3,3',5,5'-tetramethylbenzidine chromogenic substrate.

[0062] 2. Experimental results

[0063] In this example, CD4 protein was fixed on the surface of a solid phase plate and incubated with gp120 in the presence or absence of TNFR2 protein. The results showed that the addition of TNFR2 protein significantly inhibited the binding of gp120 to CD4. At high concentrations, the inhibition rate reached 80% ( Figure 3 To verify this competitive interaction at the cellular level, this example used the Jurkat cell line, a cell model widely used to study HIV-1 infection. By detecting the His tag on the gp120 protein, this example quantitatively analyzed the binding of the His-gp120 protein to Jurkat cells. Consistent with the ELISA results, our analysis showed that in the absence of TNFR2, approximately 75% of the cells showed gp120 binding. However, when TNFR2 protein was added, gp120 binding was significantly reduced by 65%, with only approximately 10% of the cells showing gp120 binding ( Figure 3In addition, this inhibitory effect was dose-dependent. When the concentration of TNFR2 protein was decreased from 20 μg / mL to 1 μg / mL, TNFR2 achieved 100% inhibition of gp120 binding at the highest concentration. Even at the lowest concentration, an inhibitory effect of approximately 5% was observed ( Figure 3 To further verify the physiological relevance of these results, this example conducted studies on primary cells. The analysis results showed that gp120 mainly binds to CD4 positive cells, while the addition of TNFR2 protein significantly inhibited this binding ( Figure 3 Middle E).

[0064] Example 4 TNFR2 protein can inhibit HIV pseudovirus infection of CD4+CCR5+ cells

[0065] 1. Experimental methods

[0066] HIV-1 pseudoviruses containing HIV-Env protein and encapsulating green fluorescent protein-associated RNA were obtained from Fubio Biotechnology (Suzhou, Jiangsu, China). Different concentrations of TNFR2 protein (20 μg / mL, 10 μg / mL, 5 μg / mL, and 2.5 μg / mL) were prepared and incubated with the virus for 1 hour. The pretreated virus was added to 293T-CD4-CCR5 cells at an MOI of 5 or 10. After 16 hours, flow cytometry was used to determine the proportion of cells expressing green fluorescent protein.

[0067] 2. Experimental results

[0068] To explore the effect of TNFR2 on HIV infection, this example constructed HIV-1 pseudoviruses carrying the gp120 envelope protein and encapsulating the green fluorescent protein gene. These pseudoviruses mimic the structure and behavior of real HIV-1 viruses, but do not contain the viral genome, making them safer for laboratory use. This example used pseudoviruses to infect CD4 and CCR5 double-positive HEK293T cells, and monitored the infection efficiency by the expression of green fluorescence. The results showed that TNFR2 protein significantly inhibited HIV pseudovirus infection, reducing the infection rate by approximately 20%, and this inhibitory effect was dose-dependent ( Figure 4 (A~C).

Claims

1. Application of TNFR2 activators in the preparation of drugs for treating HIV infection.

2. The use according to claim 1, characterized in that: The TNFR2 activator includes at least one of increasing the expression level, activity, and / or binding ability of TNFR2 protein with interacting proteins.

3. The use according to claim 2, characterized in that: The amino acid sequence of the TNFR2 protein includes at least one of 1) to 3); 1) the amino acid sequence shown in SEQ ID NO: 1; 2) a nuclear sequence in which one or more amino acids are substituted and / or deleted and / or added to SEQ ID NO: 1 and which has the same function as the protein shown in SEQ ID NO: 1; or 3) An amino acid sequence that has 95%, 96%, 97%, 98% or more homology with SEQ ID NO: 1 and has the same function as the protein shown in SEQ ID NO:

60.

4. The use according to claim 2, characterized in that: The substance for increasing the expression level of TNFR2 protein includes TNFR2 protein.

5. The use according to claim 4, characterized in that: The TNFR2 protein includes a pharmaceutically acceptable salt or a pharmaceutically acceptable chemical modification thereof.

6. The use according to claim 5, characterized in that: The pharmaceutically acceptable salt includes at least one of a metal salt, an ammonium salt, a salt formed with an inorganic acid, a salt formed with an organic base, a salt formed with an organic acid, a salt formed with a basic amino acid, and a salt formed with an acidic amino acid; and / or The pharmaceutically acceptable modification includes at least one of phosphorylation, sulfonation, acylation, glycosylation, ubiquitination, acetylation, methylation, sulfation, phospholipidation, and halogenation.

7. The use according to claim 1, characterized in that: The drug includes pharmaceutically acceptable excipients, and / or any one or more other active ingredients.

8. The use according to claim 7, characterized in that: The pharmaceutically acceptable excipients include at least one of solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integrities, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculating agents, filter aids, release retardants, and carriers.

9. The use according to claim 8, characterized in that: The dosage form of the product includes a dosage form for gastrointestinal administration or a dosage form for parenteral administration; Preferably, the dosage form for administration via the gastrointestinal tract includes at least one of powder, tablet, granule, capsule, sustained-release agent, solution, dry suspension, effervescent tablet, emulsion, suspension, syrup, drops, and chewable tablet; Preferably, the non-intestinal administration dosage form includes at least one of an injection dosage form, a respiratory tract administration dosage form, a skin administration dosage form, a mucosal administration dosage form, and a cavity administration dosage form.

10. The method described in 1) to 3), comprising the step of treating cells with TNFR2 protein: 1) A method for binding gp120 protein in vitro; 2) A method for competitively inhibiting the binding of gp120 to CD4 in vitro; 3) A method for inhibiting HIV infection of cells in vitro; The method is used for in vitro non-therapeutic purposes.

Images