HIV resistance molecules and uses thereof

Abstract

This invention discloses an HIV resistance molecule and its applications. The amino acid sequence of this HIV resistance molecule, from the N-terminus to the C-terminus, includes region A and region B; region A is a membrane fusion inhibitory polypeptide 2P23 targeting the HIV gp41 domain-induced membrane fusion process; region B is the GPI signal peptide of BST2, and its amino acid sequence is shown in SEQ ID NO:4. The HIV resistance molecule disclosed in this invention comprises the viral membrane fusion inhibitory polypeptide 2P23 and the GPI signal peptide of BST2, exerting a broad-spectrum inhibitory effect on HIV entry into cells and intracellular replication through these two functional regions. The multifunctional HIV-resistant cells described in this invention are designed to target the mechanisms of HIV extracellular invasion and intracellular replication, possessing potent and broad-spectrum antiviral activity, thereby improving therapeutic efficacy and reducing viral escape, providing a new approach to HIV treatment.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to an HIV resistance molecule and its application. Background Technology

[0002] Human immunodeficiency virus (HIV) is the pathogen of acquired immunodeficiency syndrome (AIDS), also known as AIDS. It is divided into two types: HIV-1 and HIV-2, with HIV-1 being the predominant predator. Due to the high variability of HIV and its ability to evade recognition by the host's immune system, there is still no safe and effective vaccine. Since 1996, highly active antiretroviral therapy (HAART), which combines multiple drugs, remains an effective treatment for suppressing HIV replication, significantly reducing AIDS-related morbidity and mortality, and the risk of HIV transmission. The drawback of this regimen is that it cannot clear the viral reservoir. Once the medication is stopped, the virus in the patient's body rebounds quickly. However, long-term medication can easily cause toxic side effects and drug resistance [1]. Therefore, the cure of AIDS still faces huge challenges and difficulties.

[0003] The process of HIV entering target cells is mediated by viral envelope proteins (Env). Among them, the surface subunit gp120 binds to the cell receptor CD4 and co-receptors CCR5 or CXCR4, thereby causing conformational changes in the viral envelope complex [2]. The transmembrane subunit gp41 participates in the viral membrane-cell membrane fusion by inserting the fusion peptide into the cell membrane and then folding it into a 6-helix bundle structure (6-HB), thereby realizing the process of HIV invading cells [3]. Because it can block viral infection in the early stage, and drugs designed and developed for this stage can be used not only for post-infection treatment but also for pre-exposure prophylaxis, HIV entry inhibitors based on the invasion process have become a hot research topic. At present, a variety of entry inhibitors have been widely reported, including proteins, peptides and small molecule compounds. Based on their different targets, these drugs are mainly divided into two categories: those that target different epitopes on the viral Env, such as broad-spectrum neutralizing antibodies VRC01, 10-1074, 3BNC117, m36.4, N6, 10E8, and membrane fusion inhibitory peptides enfuvirtide (T-20), abacteriophage (ABT), and 2P23; and monoclonal antibodies or small molecules that target host cell surface receptors or co-receptors, such as ipalizumab and maraviroc. However, subsequent experiments have shown that drug resistance caused by long-term use and potential viral tropism changes are important factors limiting the use of these drugs.

[0004] The "Berlin Patient," "London Patient," "New York Patient," and "Düsseldorf Patient" [4-6] demonstrate the importance of CCR5Δ32 in controlling HIV and that reinfusing HIV-resistant cells may be an effective strategy for achieving a cure for HIV. However, due to the difficulty in finding donors carrying the CCR5Δ32 mutation, potential surgical risks, and the fact that CCR5Δ32 hematopoietic stem cell transplantation does not completely cure HIV, this treatment method is only applicable to a very limited population and cannot be widely used. The applicant previously focused on the research of HIV entry inhibitors and has achieved a series of important results. Using glycosylphosphatidylinositol (GPI) anchoring modification, HIV entry inhibitors targeting specific sites of viral envelope proteins (Env) are modified on the surface of target cells. Among them, GPI-2P23[7], GPI-m36.4[8] and GPI-10E8[9] can exert strong and broad-spectrum anti-HIV-1, HIV-2 and simian immunodeficiency virus (SIV) effects by targeting gp120 or gp41. Therefore, the development of HIV-resistant cells with broad-spectrum antiviral activity has potential application value. However, although the target cells modified with single-target viral Env inhibitors have antiviral ability, they are also prone to drug resistance. How to effectively reduce the risk of viral escape is a problem that needs to be further solved. GPI anchoring proteins are a class of post-translational modification products that are covalently linked to the C-terminus of proteins by GPI. They are located on the outer leaf of the cell membrane and participate in biological processes such as cell recognition, adhesion, signal transduction and pathogen infection. Currently, more than 250 eukaryotic membrane proteins are known to be linked to the plasma membrane via GPI anchoring. Existing techniques all use GPI-anchored decay accelerating factors, namely the signal peptide of CD55 (named G1) or the signal peptide of CD52, to modify HIV neutralizing antibodies or viral membrane fusion inhibitory peptides onto the cell surface [7,10]. Interferon-induced antiviral host restriction factor BST2 (bone marrow stromal cell antibody 2) is a transmembrane protein that can effectively inhibit the release of HIV viral particles

[11] , and it is also a GPI-anchored protein. To date, there are no reports on using BST2 signal peptide for anchoring modification to prepare HIV-resistant cells.

[0005] 1.Collier DA, Monit C, Gupta RK. The impact of HIV-1 drug escape on the global treatment landscape. Cell Host Microbe 2019, 26:48-60.

[0006] 2.Checkley MA, Luttge BG, Freed EO. HIV-1 envelope glycoproteinbiosynthesis, trafficking, and incorporation. J Mol Biol 2011, 410:582-608.

[0007] 3.Eckert DM, Kim PS. Mechanisms of viral membrane fusion and itsinhibition. Annu Rev Biochem 2001, 70:777-810.

[0008] 4.Peterson CW, Kiem HP. Lessons from London and Berlin: Designing ascalable gene therapy approach for HIV cure. Cell Stem Cell 2019, 24(5): 685-687.

[0009] 5.Gupta RK, Abdul-Jawad S, McCoy LE, et al. HIV-1 remission followingCCR5Δ32 / Δ32 haematopoietic stem-cell transplantation. Nature 2019, 568(7751):244-248.

[0010] 6.Jensen BO, Knops E, Cords L, et al. In-depth virological andimmunological characterization of HIV-1 cure after CCR5Δ32 / Δ32 allogeneichematopoietic stem cell transplantation. Nat Med 2023, 29(3):583-587.

[0011] 7. Tang X, Jin H, Chen Y, et al. A Membrane-Anchored Short-PeptideFusion Inhibitor Fully Protects Target Cells from Infections of HumanImmunodeficiency Virus Type 1 (HIV-1), HIV-2, and Simian ImmunodeficiencyVirus. J Virol 2019, 93(22):e01177-19.

[0012] 8. Jin H, Tang X, Li L, et al. Generation of HIV-resistant cells witha single-domain antibody: implications for HIV-1 gene therapy. Cell MolImmunol 2021, 18(3):660-674.

[0013] 9. Chen Y, Jin H, Tang X, et al. Cell membrane-anchored anti-HIVsingle-chain antibodies and bifunctional inhibitors targeting the gp41 fusionprotein: new strategies for HIV gene therapy. Emerg Microbes Infect 2022, 11(1): 30-49.

[0014] 10. Maslennikova A, Kruglova N, Kalinichenko S, et al. Engineering T-Cell Resistance to HIV-1 Infection via Knock-In of Peptides from the HeptadRepeat 2 Domain of gp41. mBio 2022, 13(1):e0358921.

[0015] 11. Neil SJ, Zang T, Bieniasz PD. Tetherin inhibits retrovirusrelease and is antagonized by HIV-1 Vpu. Nature 2008, 451(7177):425-30. Summary of the Invention

[0016] To address the aforementioned technical problems, this invention has discovered that the GPI signal peptide of BST2 (named G2) can very effectively anchor HIV entry inhibitors (such as the 2P23 peptide) to the cell surface, while the G2 signal peptide itself also possesses antiviral activity. Based on this, this invention provides an HIV resistance molecule and its applications.

[0017] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0018] On one hand, the present invention provides an HIV resistance molecule, wherein the amino acid sequence of the HIV resistance molecule includes segment A and segment B from the N-terminus to the C-terminus; segment A is a membrane fusion inhibitory polypeptide 2P23 polypeptide targeting the membrane fusion process induced by the HIV gp41 domain; and segment B is a GPI signal peptide of BST2, the amino acid sequence of which is shown in SEQ ID NO:4.

[0019] In the technical solution of the present invention, the 2P23 polypeptide can inhibit HIV from entering cells.

[0020] In a preferred embodiment, in the HIV resistance molecule, segment A and segment B are linked by a linker peptide.

[0021] In the technical solution of the present invention, the amino acid sequence of the 2P23 polypeptide is shown in SEQ ID NO:1.

[0022] And / or, the linker peptide is a human IgG3 hinge region linker peptide, the amino acid sequence of which is shown in SEQ ID NO:5.

[0023] In some specific embodiments, the HIV resistance molecule comprises, from the N-terminus to the C-terminus, segment A, a linker peptide, and segment B.

[0024] In some specific embodiments, the HIV resistance molecule further includes a tag sequence; the tag sequence is located between the linker peptide and segment B; the HIV resistance polypeptide includes, from the N-terminus to the C-terminus: segment A, the linker peptide, the tag sequence, and segment B.

[0025] For example, the tag may be a His tag, such as the His6 tag.

[0026] In some specific embodiments, the amino acid sequence of the HIV resistance molecule is shown in SEQ ID NO:6. In SEQ ID NO:6, the amino acid residue TR between segment A and the linker peptide, the amino acid residue SG between the linker peptide and the tag sequence, and the amino acid residue YV between the tag sequence and segment B are restriction enzyme sites inserted during the construction of an expression vector capable of obtaining the polypeptide sequence, and do not affect the function of the resistance polypeptide; those skilled in the art can adjust them according to actual conditions; the amino acid residue SSQDS before segment B is a sequence inserted during the construction of the expression vector to enable segment B to be recognized, and also does not affect the function of the HIV resistance polypeptide.

[0027] In another aspect, the present invention provides a polymer formed from the above-mentioned HIV resistance molecules.

[0028] In the technical solution of the present invention, the polymer is a dimer, trimer or tetramer.

[0029] In another aspect, the present invention provides an HIV resistance gene, comprising a gene coding sequence of segment A and a gene coding sequence of segment B; the gene coding sequence is a DNA coding sequence or an RNA coding sequence; the gene coding sequence of segment A is a gene coding sequence of a 2P23 polypeptide; the DNA coding sequence of segment B is shown in SEQ ID NO:11.

[0030] In a preferred embodiment, the gene coding sequence of segment A and the gene coding sequence of segment B are linked by the gene coding sequence of a linking peptide.

[0031] In the technical solution of the present invention, the DNA coding sequence of the 2P23 polypeptide is shown in SEQ ID NO:8.

[0032] And / or, the linker peptide is a human IgG3 hinge region linker peptide; the DNA coding sequence of the linker peptide is shown in SEQ ID NO:12.

[0033] In some specific embodiments, the HIV resistance gene comprises, in sequence: the gene coding sequence of segment A, the gene coding sequence of the linker peptide, and the gene coding sequence of segment B.

[0034] In some specific embodiments, the HIV resistance gene further includes a tag sequence; the tag is located between the linker peptide and segment B. For example, the tag may be a His tag, such as a His6 tag.

[0035] In some specific embodiments, the DNA coding sequence of the HIV resistance gene is shown in SEQ ID NO:13.

[0036] In another aspect, the present invention provides a recombinant vector containing the above-mentioned HIV resistance gene.

[0037] For example, the recombinant vector is a recombinant vector obtained by inserting the above-mentioned HIV resistance gene into a lentiviral expression vector.

[0038] The lentiviral expression vector may specifically be the pLVX-EF1α-WPER plasmid.

[0039] For example, the insertion site is a multiple cloning site.

[0040] For example, the insertion sites are BamHI and SalI.

[0041] In another aspect, the present invention provides an HIV-resistant cell, wherein the HIV-resistant cell is a lentiviral expression cell comprising the above-mentioned recombinant vector.

[0042] For example, the lentivirus expression cells are mammalian cells.

[0043] For example, the mammalian cell is a HEK293T cell.

[0044] In another aspect, the present invention provides the use of the above-mentioned HIV-resistant cells in the preparation of treatments and / or preventions of diseases caused by HIV.

[0045] In the technical solution of the present invention, the application is as follows:

[0046] (1) Inhibit HIV from entering cells;

[0047] (2) Inhibit HIV replication.

[0048] In the technical solution of the present invention, the HIV virus is HIV-1, HIV-2 and / or SIV strain.

[0049] The HIV strains mentioned include, but are not limited to, the following: 398-F1_F6_20, TRO.11, X2278_C2_B6, CE1176_A3, CE703010217_B6, X1632-S2-B10, 246_F3_C10_2, CNE8, CNE55, CH119.10, BJOX002000.03, HIV_25710-2.43, NL4-3, JRFL, R3A, CNE107, CH70, RHPA.c / 2635, THRO.c / 2626, LAI.2, SG3.1, 89.6, HIV-2 ROD HIV-2 ST SIV smmPBJ SIV mac239 .

[0050] In practical applications, the HIV-resistant cells of this invention can be directly administered to patients to achieve the purpose of treating and / or preventing HIV infection.

[0051] The dosage of the resistant cells of the present invention depends on many factors, such as the nature and severity of the disease to be prevented or treated, the sex, age, weight and individual response of the patient or animal, the specific active ingredient used, the route of administration and the frequency of administration, etc.

[0052] The resistant cells of this invention can be used alone for the treatment and prevention of HIV infection, or in combination with one or more other antiviral drugs to improve the overall therapeutic effect. These antiviral drugs include, but are not limited to, nucleoside reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs), protease inhibitors (PIs), integrase inhibitors (INSTIs), fusion inhibitors (FIs), and CCR5 receptor antagonists.

[0053] For any given patient, the specific effective therapeutic dose level must be determined based on a number of factors, including the disorder being treated and its severity; the activity of the specific active ingredient used; the specific composition used; the patient's age, weight, general health condition, sex, and diet; the timing of administration and excretion rate of the specific active ingredient used; the duration of treatment; medications used in combination with or concurrently with the specific active ingredient used; and similar factors known in the medical field. For example, it is practiced in the art to start the dose of the active ingredient below the level required to achieve the desired therapeutic effect and gradually increase the dose until the desired effect is achieved.

[0054] The above technical solution has the following advantages or beneficial effects:

[0055] The resistance molecule provided by this invention comprises the membrane fusion inhibitor peptide 2P23 and the GPI signal peptide (G2) of the host restriction factor BST2. This molecule can exert a broad-spectrum inhibition of HIV entry into cells and intracellular replication through the aforementioned two functional regions. G2 itself not only possesses antiviral activity but also effectively anchors the HIV entry inhibitor 2P23 peptide to the cell surface. Based on this, the resistant cells obtained by the BST2 GPI signal peptide anchoring strategy of this invention not only allow the HIV entry inhibitor modification on the cell surface to exert a potent antiviral effect, but also enable the intracellular free G2 after post-translational modification to exert an antiviral effect, achieving multi-pathway inhibition of HIV and improving the problem of viral escape from single targets.

[0056] The HIV-resistant cells provided by this invention are designed to target the mechanisms of HIV extracellular invasion and intracellular replication. They not only have extremely strong activity and broad spectrum, but also a high drug resistance barrier, thereby achieving the purpose of improving efficacy and reducing viral escape. Attached Figure Description

[0057] Figure 1 This is a schematic diagram of the construction pattern of the lentiviral vector for the resistance gene in Example 1.1.

[0058] Figure 2 This shows the expression of the membrane fusion inhibitory peptide on the surface of stable cells in Example 1.3.

[0059] Figure 3 This is for the identification of the membrane fusion inhibition peptide in Example 1.3 located in the lipid raft region of the cell.

[0060] Figure 4 This refers to the antiviral activity of resistant cells against HIV-1 in Example 2.2.

[0061] Figure 5 This refers to the inhibitory activity of resistant cells against HIV-1 envelope-mediated cell fusion in Example 2.3.

[0062] Figure 6 This is an evaluation of the inhibitory activity of resistant cells against HIV-2 and SIV in Example 2.4.

[0063] Figure 7 This is an evaluation of the antiviral activity of G2 against HIV-1 in Example 3.2.

[0064] Figure 8 This illustrates the inhibitory effect of G2 on the progeny virus P24 released extracellularly in Example 3.3.

[0065] Figure 9 This illustrates the inhibitory effect of G2 on intracellular viral proteins in Example 3.4. Detailed Implementation

[0066] The following embodiments are merely some, not all, of the embodiments of the present invention. Therefore, the detailed descriptions of the embodiments provided below are not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0067] In this invention, unless otherwise specified, all equipment and raw materials are commercially available or commonly used in the industry. The methods described in the following embodiments are conventional methods in the art, unless otherwise specified.

[0068] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available. Cell culture lysis buffer: Promega, catalog number E1531. Luciferase detection substrate reagent: Promega, catalog number E1501. pLVX-EF1α-WPER plasmid (lentiviral expression vector): BioVector NTCC Type Culture Collection. APC-conjugated anti-6X His tag antibody: Abcam, catalog number Ab72579. Mouse anti-His tag antibody: Thermo Fisher Scientific, catalog number MA1-21315. Alexa Fluor 488-conjugated goat anti-mouse IgG antibody: Thermo Fisher Scientific, catalog number A28175. Alexa Fluor 555-conjugated CtxB: Thermo Fisher Scientific, catalog number C34776. P24 antigen quantitative reagent kit: Keyue Zhongkai Company, catalog number K12P2401.

[0069] Unless otherwise specified, all quantitative experiments in the following examples were performed in triplicate, with three replicates per experiment. Results were taken as mean ± standard deviation. Unless otherwise specified, the PBS buffer used in the examples was pH 7.2, 0.1M PBS buffer. Unless otherwise specified, the DMEM complete medium used in the examples was DMEM medium containing 10% FBS and 1% penicillin-streptomycin.

[0070] Example 1 Construction of HIV-resistant cells

[0071] 1.1 Construction of recombinant expression vectors

[0072] In this embodiment, the DNA coding sequences of the resistance gene 2P23 and the GPI anchoring signal peptide (named G2) of BST2 were linked via the IgG3 hinge region linker (SEQ ID NO:12), and the resulting fusion gene was labeled 2P23-G2 (BST2) and linked between the BamHI and SalI sites of the lentiviral transfer vector pLVX-EF1α-WPER. Simultaneously, a 2P23 mutant 2P23S-G2 was constructed as a control group. Next, an IgG3 leader secretory peptide sequence was added to the N-terminus of 2P23 or 2P23S to promote protein synthesis and extracellular secretion, and a 6×His tag sequence was added to the C-terminus of the IgG3 linker to facilitate subsequent flow cytometry sorting and detection of cells successfully expressing the membrane fusion inhibitory peptide. Under the action of glycosylphosphatidylinositol transaminases (GPI-T), the fusion protein is cleaved into two parts. The membrane fusion inhibitory peptide is secreted into the extracellular space and anchored to the cell membrane under the action of GPI-T and the secretion signal peptide, while the signal peptide is free in the cytoplasm.

[0073] In this embodiment, the G2 signal peptide was replaced with the DAF GPI signal peptide (named G1) using the above method to construct the recombinant expression vector 2P23-G1.

[0074] A schematic diagram of the recombinant expression vector constructed in this embodiment is shown below. Figure 1 As shown, the recombinant expression vector consists of the following components from N-terminus to C-terminus: IgG3 leader secretory peptide, membrane fusion inhibitory peptide 2P23 or mutant 2P23S, IgG3 hingelinker, 6×His, and GPI signal peptide (BST2 or DAF).

[0075] In this embodiment, the amino acid sequence encoded by the resistance gene 2P23 is shown in SEQ ID NO:1, and its DNA sequence is shown in SEQ ID NO:8.

[0076] The amino acid sequence encoded by 2P23S is shown in SEQ ID NO:2, and its DNA coding sequence is shown in SEQ ID NO:9.

[0077] The amino acid sequence of the G2 signal peptide is shown in SEQ ID NO:4, and its DNA coding sequence is shown in SEQ ID NO:11.

[0078] The amino acid sequence of the G1 signal peptide is shown in SEQ ID NO:3, and its DNA coding sequence is shown in SEQ ID NO:10.

[0079] The gene sequences of the recombinant expression vectors constructed in this embodiment are shown in SEQ ID NO:13 (2P23-G2) and SEQ ID NO:14 (2P23S-G2).

[0080] The amino acid sequence encoded by SEQ ID NO:12 is shown in SEQ ID NO:5.

[0081] The amino acid sequence encoded by SEQ ID NO:13 is shown in SEQ ID NO:6.

[0082] The amino acid sequence encoded by SEQ ID NO:14 is shown in SEQ ID NO:7.

[0083] 1.2 Construction of HIV-resistant cells

[0084] Construction of stable antiviral gene expression cell lines (TZM-bl, 293FT-CCR5 / CXCR4 / DSP8-11): Add 5×10⁻⁶ cells to a 24-well plate. 4 One cell, then add 1×10 6 Lentiviral cells expressing the antiviral gene from TU were added to a working concentration of 8 μg / mL polybrene solution, mixed well, and cultured in a cell culture incubator for 24 hours. The culture medium was then replaced with fresh DMEM, and culturing continued. Once the cells showed good growth, the proportion of cells expressing the antiviral gene was analyzed by flow cytometry using anti-His antibody staining. Two cell lines stably expressing the exogenous gene were then sorted and purified. After a single lentivirus transduction, the proportion of cells expressing the antiviral gene was consistently above 95%.

[0085] 1.3 Analysis of the expression and localization of resistance genes on the surface of stable resistance cell lines

[0086] The expression of exogenous genes in stable HIV target cell lines was analyzed using flow cytometry. The specific procedure involved taking approximately 5 × 10⁶ cells... 5 Target cells stably expressing the resistance gene (TZM-bl, 293FT-CCR5 / CXCR4 / DSP8-11) were placed in clean EP tubes and washed twice with FACS buffer (phosphate buffer containing 0.5% bovine serum albumin and 2mM EDTA). 100 μL of FACS buffer (containing APC-conjugated anti-6X His tag antibody) was added to the EP tubes, and the cells were incubated on ice or at 4°C on a shaker for 1 hour. The cells were washed twice with FACS buffer, the supernatant was discarded, 200 μL of fresh FACS buffer was added, the cells were resuspended, and the cells were transferred through a filter to a flow cytometer to detect the expression level of the resistance gene on the surface of the target cells.

[0087] The results are as follows Figure 2 As shown, the exogenous His-tagged genes were stably and effectively expressed on the cell membrane surface of all TZM-bl or 293FT groups, with a positive cell rate of over 90%. These results indicate that the resistance gene can be stably expressed in stable cell lines.

[0088] The confocal assay was used to demonstrate that the antiviral gene (GPI-anchored anti-HIV inhibitor) is co-localized with the lipid raft region of the target cell TZM-bl. The specific steps are as follows: 8000 stably transfected TZM-bl cells were seeded into a glass-bottomed cell culture dish and cultured in a cell culture incubator for about 36-48 hours. Then, the cells were processed for confocal analysis. Processing steps: (1) Discard the culture medium in the culture dish, wash twice with 300 μL of PBS solution, and fix with 4% paraformaldehyde solution at room temperature for 15 minutes; (2) Wash three times with PBS, and block with 5% BSA solution at room temperature for 1 hour; (3) Wash three times with PBS, and incubate with primary antibody mouse anti-histag antibody at room temperature for 1-2 hours (2.5% BSA solution diluted 1:500); (4) Wash three times with PBS, and incubate with secondary antibody Alexa488-conjugated goat anti-mouse IgG antibody at room temperature for 1 hour (2.5% BSA solution diluted 1:500); (5) Wash three times with PBS, and incubate with Alexa 555-conjugated cholera toxin subunit B (CtxB) at room temperature for 1 hour (2.5% BSA solution diluted 1:500, binding to ganglioside GM1 on cell membrane lipid rafts); (6) Wash three times with PBS, and incubate with DAPI at room temperature. 7 minutes; (7) Wash 3 times with PBS and take pictures using a confocal microscope.

[0089] The results are as follows Figure 3 As shown, CtxB can effectively bind to the lipid raft marker GM1, and 2P23-G2 and 2P23S-G2 co-localize with GM1 on the cell surface, indicating that the G2-anchored antiviral peptide is located in the lipid raft region of the cell membrane.

[0090] Example 2. Evaluation of the antiviral effect of resistant cells

[0091] 2.1 Preparation of different HIV-1 subtypes

[0092] To evaluate the antiviral activity of the bifunctional molecules, this invention prepared 17 HIV-1 pseudoviruses, including internationally representative HIV-1 strains, and 6 HIV-1 replicating viruses. Among them, 6 HIV-1 replicating viral strains include RHPA.c / 2635, THRO.c / 2626, LAI.2, SG3.1, 89.6, and NL4-3; 12 internationally representative HIV-1 strains include 398-F1_F6_20, TRO.11, X2278_C2_B6, CE1176_A3, CE703010217_B6, X1632-S2-B10, 246_F3_C10_2, CNE8, CNE55, CH119.10, BJOX002000.03, and HIV_25710-2.43; and 5 CXCR4-tropic HIV-1 pseudovirus strains include NL4-3, JRFL, R3A, CNE107, and CH70. All of these were provided by the US NIH AIDS Reagents and References Project. These strains encompass three tropisms: CCR5, CXCR4, and R5 / X4. HEK293T cells were co-transfected with recombinant expression plasmids expressing the envelope protein (Env) of the aforementioned 17 HIV-1 strains and the HIV-1 backbone plasmid pSG3Δenv (provided by the US NIH AIDS Reagents and References Project, catalog number 11051) or recombinant expression plasmids of 6 HIV-1 replicating viruses using cell transfection reagents. The cells were incubated at 37°C in a 5% CO2 cell culture incubator for 6 hours, then the medium was changed, followed by another 48 hours of incubation. The cell culture supernatant containing viral particles was pipetted, filtered through a 0.45 μm filter, and the supernatant was collected. Fetal bovine serum (FBS) was added (to bring the final FBS volume ratio to 20%), and the supernatant was transferred to polypropylene tubes. The tubes were stored at -80°C for later use or directly for viral titration. For viral titration, the virus was tripled in 96-well plates with 3 replicates and 9 gradients, with a final volume of 100 μL. TZM-bl cells were digested with trypsin and counted, then diluted to 1×10⁶ cells with DMEM complete medium. 5 100 μL of cells (containing 15 μg / mL DEAE-dextran) were added to each well and incubated at 37°C and 5% CO2 for 48 hours. Then, the 96-well plate was removed from the cell culture incubator, the supernatant was aspirated from the sample wells, 30 μL of luciferase cell culture lysis reagent was added, and after incubation for 10 minutes, 50 μL of luciferase detection substrate reagent was added. 50 μL of the liquid was pipetted from each well and added to the corresponding 96-well plate. The fluorescence value was read using a microplate spectrophotometer, and the relative fluorescence units (RLU) of each well were read. The viral titer was calculated using the Reed-Muench method.

[0093] 2.2 Detection of antiviral activity of resistant cells against HIV-1

[0094] The resistant cells corresponding to the resistance gene were the 2P23-G2 cells obtained in Example 1, and the 2P23 mutant 2P23S-G2 cells obtained in Example 1 were used as a control. The operation steps are as follows:

[0095] Cells that stably express the resistance gene (1×10) 4 (cells) and 200 TCID 50 A mixture of pseudotyped or replicating viruses was prepared, with DEAE-dextran added to achieve a final concentration of 15 μg / mL. This mixture was then added to 96-well plates, with three replicates for each cell type. The resulting reaction system was incubated at 37°C in a 5% CO2 cell culture incubator for 48 hours. Afterward, the 96-well plates were removed, the supernatant was discarded, and 30 μL of cell lysis buffer was added. After incubation at room temperature for 15 minutes, 50 μL of luciferase assay reagent was added. 50 μL of the liquid was pipetted from each well and added to the corresponding 96-well plate. The relative fluorescence units (RLU) of each well were read using a microplate spectrophotometer. The viral infection rate was calculated based on the measured RLU values ​​using the formula: Infection rate = [(RLU produced by cells stably expressing the resistance gene infected with the virus - RLU produced by cells stably expressing the resistance gene uninfected with the virus) / (RLU produced by non-stable cells infected with the virus - RLU produced by non-stable cells uninfected with the virus)] × 100%.

[0096] The results are as follows Figure 4 As shown, resistant cells modified with 2P23-G2 were almost unable to infect the above-mentioned different subtypes and tropisms of pseudotyped or replicating HIV-1 strains, indicating that 2P23-G2 exhibited extremely high antiviral activity against different subtypes of pseudotyped or replicating HIV-1, with an inhibition rate of up to 100%, which was significantly higher than that of the control group 2P23S-G2.

[0097] 2.3 Inhibitory activity of resistant cells against HIV-1 envelope-mediated cell fusion

[0098] The inventors used a DSP-based cell-cell fusion inhibition experiment to detect the inhibitory activity of 2P23-G2 and 2P23S-G2 on HIV-1 envelope protein-mediated cell fusion. The specific procedure was as follows: HEK293T cells (effective cells) were digested and counted, and 100 μL (1.5 x 10⁻⁶ cells) was taken. 5(2.5-3 × 10⁶ / mL) were seeded into 96-well plates and incubated overnight at 37°C in a 5% CO₂ cell culture incubator. Using PEI transfection reagent, plasmids expressing HIV-1 envelope proteins and plasmids expressing DSP1-7 (in this experiment, the plasmids used to express HIV-1 envelope proteins are the same as those used in the antiviral experiment) were co-transfected (in experimental wells and positive control wells), and cultured at 37°C for 24 hours. 293FT resistant cells stably expressing the resistance gene and CXCR4 / CCR5 / DSP8-11 were resuspended and mixed with EnduRen live cell substrate, then incubated at 37°C for 15-30 minutes; resistant cells (2.5-3 × 10⁶ / mL) were incubated. 4 HIV-1 Env was added to each well of transfected HEK293T cells, centrifuged at 300g for 1 minute at room temperature, and then incubated at 37°C for 6 hours. The relative fluorescence units (RLU) of each well were read using a microplate photometer. Based on the measured RLU values, the efficiency of HIV-1 Env in inducing cell fusion in effector cells was calculated using the following formula: Fusion rate = [(RLU produced by a mixture of HEK293T cells expressing HIV-1 envelope protein and 293FT cells stably expressing the resistance gene - RLU produced by a mixture of blank HEK293T cells and 293FT cells stably expressing the resistance gene) / (RLU produced by a mixture of HEK293T cells expressing HIV-1 envelope protein and blank 293FT cells - RLU produced by a mixture of blank HEK293T cells and blank 293FT cells)] × 100%.

[0099] The results are as follows Figure 5 As shown, 2P23-G2 completely inhibited the fusion of envelope proteins of different HIV-1 tropism strains with target cells. However, the control group 2P23S-G2 showed incomplete inhibition of the above strains, indicating that 2P23-G2 can effectively inhibit HIV-1 envelope-mediated cell fusion.

[0100] 2.4 Evaluation of the inhibitory activity of resistant cells against HIV-2 and SIV

[0101] To evaluate the antiviral advantages of the resistant cells of this invention, this experiment further examined their inhibitory activity against HIV-2 and SIV. The HIV-2 molecular cloning plasmids HIV-2 ROD and HIV-2 ST were obtained from the US NIH AIDS Reagents and References Project; the SIV expression strain SIV... smmPBJ and SIV mac239The plasmids containing the envelope proteins (pSIVpbj-Env and pSIV239-Env, respectively) were kindly provided by Professor Xu Jianqing of Fudan University. Preparation of infectious HIV-2 strains: HEK293T cells were transfected with HIV-2 ROD or HIV-2 ST plasmids using PEI transfection reagent and cultured at 37°C and 5% CO2 for 6 hours. After changing the medium, the cells were cultured for another 48 hours. The supernatant was collected, centrifuged at 1000g for 10 minutes, and filtered through a 0.45μm filter. Fetal bovine serum was added to bring the final volume ratio to 20%. The cells were used directly or aliquoted and stored at -80°C for later use or for viral titer determination. The preparation of SIVpbj and SIV239 pseudoviruses was the same as in Example 2.

[0102] The results are as follows Figure 6 As shown, resistant cells modified with 2P23-G2 were almost unable to infect HIV-2 and SIV, which was significantly different from resistant cells modified with 2P23S-G2, indicating that 2P23-G2 has a strong and broad-spectrum inhibitory effect on both HIV-2 and SIV.

[0103] Example 3 Evaluation of the intracellular antiviral effect of BST2 GPI signal peptide (G2)

[0104] 3.1 Construction of recombinant expression vectors

[0105] The BST2 GPI signal peptide (G2) was constructed into the expression vector pcDNA3.1 or the lentiviral vector pLVX-EF1α-WPER, and HA or His tag was attached to the C-terminus for fusion expression. The commonly used DAF GPI signal peptide (G1 signal peptide) expression vector was constructed as a control in the same way.

[0106] In this embodiment, the amino acid sequence of the G1 signal peptide is shown in SEQ ID NO:3, and its DNA coding sequence is shown in SEQ ID NO:10.

[0107] 3.2 Evaluation of the antiviral activity of G2 against HIV-1

[0108] G2 cells were co-transfected with SG3.1, RHPA, or 89.6 at a 1:1 ratio using PEI. Six hours after transfection, the medium was changed, and the cells were cultured for another 24 hours. The supernatant was collected, and 100 μL of supernatant was added to each well of a 96-well plate. TZM-bl cells were digested with trypsin and counted. The cells were then diluted to 1 × 10⁶ cells / well with DMEM complete medium. 5Cells were added at a density of 100 μL / ml (containing 15 μg / mL DEAE-dextran) to each well, and incubated at 37°C and 5% CO2 for 48 hours. The 96-well plate was removed from the cell culture incubator, the supernatant was discarded, and 30 μL of luciferase cell culture lysis reagent was added. After incubation for 10 minutes, 50 μL of luciferase detection substrate reagent was added. 50 μL of the liquid was pipetted from each well and added to the corresponding 96-well plate. The fluorescence value was read using a microplate photometer, and the relative fluorescence units (RLU) of each well were measured.

[0109] The results are as follows Figure 7 As shown ( Figure 7 In the second figure, the statistical results for RHPA+G2-HA are low (and cannot be shown in the figure), compared to the empty vector control group, the viral infectivity of cells transfected with G2 was significantly reduced, while the viral infectivity of cells transfected with G1 was not significantly reduced. These results indicate that the BST2 GPI signal peptide (G2) alone possesses antiviral activity.

[0110] 3.3 Inhibition of P24 content in HIV-1 progeny virus release by G2

[0111] Following the transfection method described in section 3.2, after culturing for 24 hours, the supernatant was collected and centrifuged at 4000 rpm for 5 min. 30 μL of supernatant was transferred to a new 96-well plate, and 120 μL / well of 1% Triton-X100 solution was added to inactivate the virus. Positive control wells and the initial concentration were used for P24 antigen quantification to determine the dilution factor. P24 quantification was performed according to the kit instructions using a 96-well microplate reader. Then, each culture supernatant was diluted to an appropriate concentration for P24 quantification. The concentration was calculated using a linear regression formula.

[0112] The results are as follows Figure 8 As shown ( Figure 8 The third figure shows that the statistical result for 89.6+G2-HA is low (and cannot be shown in the figure). Compared with the empty vector control group, the amount of P24 released into the extracellular space by cells transfected with G2 was significantly reduced, while the amount of P24 released into the extracellular space by cells transfected with G1 was not significantly reduced. The results indicate that G2 can reduce the amount of P24 released by HIV-1 progeny viruses.

[0113] 3.4 Inhibition of intracellular viral proteins by G2

[0114] Following the transfection procedure in 3.2, after culturing for 24 hours, cells were collected, lysed using RIPA strong lysis buffer, and Western blotting was performed to detect intracellular virus-related proteins.

[0115] The results are as follows Figure 9As shown, the expression of viral Gag protein was significantly reduced in cells transfected with G2, indicating that G2 inhibits the expression of HIV-1 viral proteins.

[0116] The sequences in this invention are as follows:

[0117] SEQ ID NO:1:

[0118] EMTWEEWEKKVEELEKKIEELLK.

[0119] SEQ ID NO:2:

[0120] ETMEWWEKEVKEEELKKEILEKL.

[0121] SEQ ID NO:3:

[0122] GSGTTSGTTRLLSGHTCFTLTGLLGTLVTMGLLT.

[0123] SEQ ID NO:4:

[0124] SSAAAPQLLIVLLGLSALLQ.

[0125] SEQ ID NO:5:

[0126] TPLGDTTHT.

[0127] SEQ ID NO:6:

[0128] EMTWEEWEKKVEELEKKIEELLKTRTPLGDTTHTSGHHHHHHYVSSQDSSSAAAPQLLIVLLGLLSALLQ.

[0129] SEQ ID NO:7:

[0130] ETMEWWEKEVKEEELKKEILEKLTRTPLGDTTHTSGHHHHHHYVSSQDSSSAAAPQLLIVLLGLSALLQ.

[0131] SEQ ID NO:8:

[0132] gagatgacctgggaagagtgggagaagaaagtggaagagctggaaaagaagatcgaggagctgctgaag.

[0133] SEQ ID NO:9:

[0134] gagaccatggaatggtgggagaaggaggtgaaggaggaagagctgaagaaagagatcctggaaaagctg。

[0135] SEQ ID NO:10:

[0136] ggaagtggaaccacttcaggtactacccgtcttctatctgggcacacgtgtttcacgttgacaggtttgcttgggacgctagtaaccatgggcttgctgact。

[0137] SEQ ID NO:11:

[0138] agctctgccgctgcccctcagctgctgatcgtgctgctgggcctgagcgccctgctgcag。

[0139] SEQ ID NO:12:

[0140] accccgctgggtgacaccacccacacc。

[0141] SEQ ID NO:13:

[0142] gagatgacctgggaagagtgggagaagaaagtggaagagctggaaaagaagatcgaggagctgctgaagacgcgtaccccgctgggtgacaccacccacacctccggacaccatcaccatcaccattacgtatccagccaggacagcagctctgccgctgcccctcagctgctgatcgtgctgctgggcctgagcgccctgctgcag。

[0143] SEQ ID NO:14:

[0144] gagaccatggaatggtgggagaaggaggtgaaggaggaagagctgaagaaagagatcctggaaaagctgacgcgtaccccgctgggtgacaccacccacacctc cggacaccatcaccatcaccattacgtatccagccaggacagcagctctgccgctgcccctcagctgctgatcgtgctgctgggcctgagcgccctgctgcag.

[0145] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. An HIV resistance molecule, characterized in that, The amino acid sequence of the HIV resistance molecule is shown in SEQ ID NO:

6.

2. An HIV resistance gene, characterized in that, The sequence of the HIV resistance gene is shown in SEQ ID NO:

13.

3. A recombinant vector containing the HIV resistance gene as described in claim 2.

4. An HIV-resistant cell, characterized in that, The HIV-resistant cells are lentiviral expression cells comprising the recombinant vector of claim 3.

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