WO2019154414A1 - Use of substance for inhibiting or eliminating trabd in hiv reagent and screening method - Google Patents

Abstract

Disclosed is the use of a substance or a vector thereof for inhibiting or eliminating TRABD in the preparation of a reagent and/or a drug for detecting or eliminating HIV-infected cells, a method for detecting or eliminating HIV-infected cells, the use of the substance or a vector thereof in the preparation of a reagent for increasing cell resistance ability to HIV and/or SIV infection or for reducing the release of HIV and/or SIV progeny virus by infected cells, and a screening method for a substance capable of eliminating or detecting cells infected with HIV and/or SIV. The substance is capable of inhibiting or eliminating TRABD activity, triggering the release of HIV and other viruses by HIV reservoir cells infected with HIV and other viruses, thereby enabling the HIV reservoir cells infected with HIV and other viruses to be detected or killed.

Description

Use of a substance for inhibiting or eliminating TRABD in HIV reagents and screening method Technical field

The present invention relates to a technique and related method for detecting or eliminating cells infected with a virus such as HIV, and particularly relates to a reagent and/or a drug for detecting or eliminating cells of an HIV reservoir using a substance capable of inhibiting or eliminating TRABD (TraB domain-containing protein). And methods of screening for such materials.

Background technique

HIV infection has become a major threat to global human health and cannot be cured. Current anti-HIV therapies, such as cocktail therapy, can only effectively control the active replication of the virus, but not radically. Even if the HIV virus in the body is reduced to an undetectable level by joint antiviral therapy, the virus will rebound once the drug is stopped. In the process of rebound, there is a group of cells that are infected with HIV but do not release HIV, because these cells are not isolated or cleared in current anti-HIV therapies. Therefore, there is a need to find effective and safe detection methods and methods for such cells.

In AIDS patients, the viral load in plasma rapidly decreased after the Highly Active Antiretroviral Therapy (HAART), up to the lowest detection limit (<20 copies/mL). However, once antiviral therapy is stopped, the virus in the body will rebound quickly. Therefore, AIDS patients need lifelong antiviral treatment. The rapid rebound of this virus stems from the presence of a class of HIV depot cells. After infection with HIV, the HIV virus enters a latent state and cannot release progeny virus particles (collectively referred to as "HIV reservoir cells"). There is currently no way to identify HIV viruses in such cells, let alone to clear them. Even if anti-HIV drugs continue to be used for many years or even decades, once they are stopped, the HIV virus will rebound quickly and the virus infection will not be eradicated. At the same time, the stock of such cells is very low, further causing such cells to be difficult to detect and difficult to remove. The HIV reservoir cells include peripheral blood cells, such as CD4 ⁺ T cells, and may also be NK cells, brain cells, and the like.

There is currently no clinically available method for detecting and killing such HIV-infected HIV reservoir cells. The existing detection methods basically activate the HIV storage cells under laboratory conditions, for example, by adding substances such as phytohemagglutinin (PHA) to activate the HIV storage cells, and actively sterilizing the cells, and Progeny virus cells are released in large quantities to detect the amount of virus released and estimate the approximate stock of HIV reservoir cells. The disadvantage of this detection method is that (1) it cannot be standardized and quantified. (2) Once the HIV storage cell is activated, it can promote the release of the virus, which has already lost important physiological characteristics of the HIV storage cell. Therefore, it releases the virus and does not have the characteristics of a latent virus.

In terms of treatment methods, the field of shock therapy (Shock & Kill) as the basic treatment principle is widely studied in the international theory. Similar to the existing detection methods, in the Shock&Kill treatment principle, it is first necessary to activate the /Shock HIV reservoir cells, and then the Killer CD8 ⁺ T cells discover and remove the original HIV-infected CD4 ⁺ T that is activated after the virus is released. Repository cells. However, in the treatment of therapeutic methods, the key problem currently encountered is that activation of HIV-infected CD4 ⁺ T reservoir cells activates the entire body's immune system. Therefore, if the current activator is used for Shock & Kill treatment, the patient will be immune to disorder, causing an immune storm or even fatal. Therefore, Shock&Kill's therapy has only succeeded in basic experiments, but it has not been able to enter clinical treatment.

There are three subclasses of TRABD domain-containing protein 2A. Prior to the present invention, only TRABD2A-203 was reported to inhibit the expression of Wnt signaling in cells, and none of TRABD2A-201 and TRABD2A-202. A report on information such as its function.

Summary of the invention

The present invention provides the use of related substances for detecting and resecting HIV storage cells, by treating HIV storage cells to detect and clear the cells, and to provide screening for clearance of cellular material in the HIV reservoir. Methods.

In particular, the invention provides the use of a substance for the preparation of an agent and/or a medicament for detecting or eliminating HIV infected cells, characterized in that the substance is capable of inhibiting human TRABD protein activity or clearing human TRABD protein.

Preferably, the human TRABD protein described above is TRABD2A.

Preferably, the human TRABD2A protein may be selected from the TRABD2A-201 protein, and the amino acid sequence of the TRABD2A-201 protein is shown in SEQ ID NO. It is also preferred to use the TRABD2A-202 protein, and the amino acid sequence of the TRABD2A-202 protein is shown in SEQ ID NO.

Further, such a substance for preparing an agent for detecting or eliminating HIV-infected cells may be selected from a small molecule compound, a metalloproteinase inhibitor, or a human TRABD protein-specific antibody.

Preferably, the small molecule compound described above is capable of competing with Mn ²⁺ for binding to a human TRABD protein.

Preferably, the above small molecule compound is a metal ion compound not containing Mn ²⁺ .

Preferably, the metal ion compound is a metal ion compound containing Ni ²⁺ , Co ²⁺ or Zn ²⁺ .

Preferably, the above metalloproteinase inhibitor is a divalent metal chelating agent, more preferably 1,10-phenanthroline.

Preferably, the above-described substance for preparing an agent for detecting or eliminating HIV-infected cells removes human TRABD protein on the cell surface by an RNAi method. More preferably, the substance is siRNA or shRNA.

Preferably, the siRNA described above transfects the cells.

Preferably, the siRNA described above is transfected into the cells by electroporation or tandem.

Preferably, the shRNA is transfected into the cell.

The invention also provides the use of a vector for the preparation of an agent and/or a medicament for detecting or eliminating HIV infected cells, characterized in that the vector comprises the above-mentioned reagents for preparing cells for detecting or eliminating HIV infection and/or The substance of the drug.

The present invention also provides a method for detecting or eliminating HIV-infected cells, the method comprising: inhibiting human TRABD protein activity in the cells or clearing human TRABD protein in the cells, detecting HIV virus released by the cells for detection And/or eliminate the cells.

Preferably, the method treats said cells using any of the reagents described above for the preparation of cells for detecting or eliminating HIV infection.

Preferably, the cells are HIV depot cells, including but not limited to HIV-infected CD4 ⁺ T cells, further, HIV-infected resting CD4 ⁺ T cells, and may also be HIV-infected peripheral blood cells, NK Cells, brain cells.

The invention also provides the use of a substance for the preparation of an agent for increasing the ability of a cell to resist HIV and/or SIV infection or for reducing the release of HIV and/or SIV progeny virus by an infected cell, characterized in that the substance is an expression plasmid for TRABD protein. .

Preferably, the expression plasmid of the above TRABD protein is an expression plasmid of TRABD2A protein.

Preferably, the expression plasmid of the above TRABD2A protein is an expression plasmid of TRABD2A protein of human, primate or mammalian.

Preferably, the expression plasmid of the above TRABD protein may also be an expression plasmid of the TRABD2B protein.

Preferably, the expression plasmid of the above TRABD2B protein is an expression plasmid of TRABD2B protein of human, primate or mammalian.

Preferably, the above cells are cells that have been infected with HIV and/or SIV.

Preferably, the above cells are CD4 ⁺ T cells that have been infected with HIV and/or SIV.

The invention also provides the use of a vector for the preparation of an agent for increasing the ability of a cell to resist HIV and/or SIV infection or for reducing the release of HIV and/or SIV progeny virus by an infected cell, characterized in that the carrier comprises the above-described preparation test. Or a substance that removes reagents from HIV-infected cells.

The present invention also provides a method of screening for a substance capable of scavenging or detecting cells infected with HIV and/or SIV, the method comprising screening for a substance capable of inhibiting TRABD protein activity or scavenging TRABD protein.

Preferably, the above substances are capable of inhibiting TRABD protein activity in humans, primates or mammals, or scavenging human, primate or mammalian TRABD proteins.

Preferably, the above method comprises performing a cell assay and/or an animal assay to screen for a substance capable of inhibiting TRABD protein activity or clearing TRABD protein.

Preferably, the macaques are used in the above methods for the cell experiments and/or animal experiments.

In particular, the present invention inhibits or eliminates the TRABD2A protein of HIV-1 cells infected with HIV-1 and/or HIV-2 by using related substances, so that these HIV storage cells release a large amount of HIV progeny virus, thereby enabling These cells are tested. At the same time, the present invention can also promote the release of HIV virus from HIV storage cells by inhibiting or eliminating the above-mentioned human TRABD2A protein by using related replication, thereby enabling these cells to be specifically recognized by the human immune system (such as CD8 ⁺ T and other Killer cells). Or related antiviral drugs are identified to achieve the purpose of clearing HIV repository cells for further treatment of HIV-1 and/or HIV-2 infection. Further, the present invention can also inhibit the replication of HIV-1 and/or HIV-2 virus in infected cells by increasing the expression of TRABD2A protein or TRABD2B protein (TraB domain-containing protein 2B) in infected cells. And released.

In addition, the invention also provides methods of screening for clearance of cellular material in an HIV depot. HIV has no animal model, and mice cannot be infected with HIV, so it is equivalent to HIV research work, and there is no animal model. In order to establish animal models, researchers have used SIV to infect macaques (Macaque) and established similar AIDS symptoms, making it the only living laboratory to replace HIV. At present, we have found that TRABD can also fight SIV in macaque cells. Therefore, it is possible to study the effects of these substances on the clearance of human HIV reservoir cells and to adjust the symptoms of HIV infection by studying the effects of different substances on rhesus monkey TRABD infected with SIV and adjusting related symptom manifestations. This provides an effective high-throughput method for screening HIV and SIV drugs. Orangutans can be infected with HIV. With the deepening of research and the development of gorilla clinical symptoms, gorillas have the potential to become experimental models of HIV animals.

We have found that HIV storage cells are surface-specific and contain a large amount of cell membrane metalloproteinase human TRABD2A protein relative to cells that are capable of being produced by viral infection and releasing HIV-1 and/or HIV-2 viruses. The TRABD2A protein prevents the release of the virus, ie the TRABD2A protein is responsible for the minimal release of the virus from the HIV reservoir cells. Further, TRABD needs to bind manganese ions to remove the viral capsid protein, that is, it has two active centers, one center is bound to ions, such as Mn ²⁺ ; the other center is bound to viral Gag protein molecules. The present invention employs TRABD antibodies, metalloproteinase inhibitors, such as 1,10-phenanthroline, or other small molecule compounds that compete with Mn ²⁺ for binding to the TRABD2A protein, such as metal ion Co ^{2 The} inhibition of TRABD2A protein activity by ⁺ , Ni ²⁺ or Zn ²⁺ will cause the HIV reservoir cells to also release a large amount of virus. In fact, small molecules that bind to TRABD are able to achieve viral release for therapeutic purposes. Further, the above small molecule compound has substantially the same binding ability and activity inhibiting ability as TRABD2A-201, TRABD2A-202, and TRABD2A-203, which are different subclasses of TRABD2A. Similarly, the removal of TRABD2A by RNAi will cause the HIV reservoir cells to begin releasing large amounts of virus. And similarly, the RNAi method is also basically the same for the different subclasses of TRABD2A, namely TRABD2A-201, TRABD2A-202 and TRABD2A-203. This allows HIV reservoir cells to be specifically detected and recognized and cleared in the body by the immune system or related antiviral drugs.

Although the HIV reservoir cells themselves do not contain a large amount of TRABD2B protein, the TRABD2B protein also has a similar function as the TRABD2A protein. That is, TRABD2B protein can also prevent the replication and release of lentiviruses such as HIV-1, HIV-2 and SIV. Moreover, similar to the TRABD2A protein, the antiviral activity of the TRABD2B protein is also inhibited by TRABD antibodies, metalloproteinase inhibitors, and metal ions Co ²⁺ , Ni ²⁺ , Zn ²⁺ , and other small molecules capable of binding to the TRABD2B protein. . Therefore, the entire TraB family is likely to have similar effects of inhibiting viral replication and release.

Compared with the existing detection method, a method of detecting the amount of the HIV storage cell by adding a substance such as a plant lectin that activates T cells, thereby causing the HIV storage cell to be activated and starting to release the progeny virus, the method of the present invention The advantage is that it can promote the release of a large number of progeny viruses without activating the HIV storage cells, thereby providing the ability to detect these cells under physiological conditions, and the obtained test data has clinical indicators, which are more sensitive and precise, and provide clinical guidance. Medication is more valuable.

At the same time, the present invention proposes a new detection standard, that is, extracting 5-10 ml of peripheral blood of a virus latent patient, extracting peripheral blood mononuclear cell PBMCs, or CD4 ⁺ T reservoir cells, and treating with TRABD inhibitor for 12-24 hours, can be detected. The medium supernatant has an infectious viral particle count to determine the number of HIV-infected CD4 ⁺ T reservoir cells in different patients. The detection method can shorten the HIV detection window period: after the early infection of the human body, the virus is not detected in the blood, and it takes one to several months to detect the antibody in the blood. With the method provided by the present invention, HIV infection can detect a small amount of infectious virus at the cell level at an early stage, which provides a guarantee for early detection of HIV infection and early treatment of HIV.

In another aspect, the present invention also finds that as long as the antiviral activity of the TRABD2A protein is inhibited in the HIV reservoir cells, the latent virus can be packaged, released from these cells, and presented with a viral antigen on the surface of the HIV storage cell. It promotes the specific recognition of Killer cells such as CD8 ⁺ T, and clears these cells, which can achieve the purpose of clearing the HIV storage cells and completely curing the virus infection. At the same time, this process of activating latent viruses and killing HIV storage cells does not require cell activation, so it fully satisfies shock therapy (Shock & Kill) and can enter clinical treatment. That is, the present invention has an advantage in that it does not cause an activation of the entire human immune system due to the need to activate the HIV reservoir cells, and may cause adverse consequences for the death of the patient, relative to the Shock & kill treatment method which is currently under study. Therefore, the strategy for treating HIV reservoir cells in the present invention is defined as compression therapy (Push & Kill).

In addition, in addition to the method of inhibiting or eliminating the TRABD2A protein of the HIV reservoir cells, such cells release the virus from the human immune cells such as killer T cells, the present invention can also directly inhibit the virus in the cells by enhancing the expression of TRABD. freed. For example, it is possible to inhibit the production of intracellular viruses by expression of eukaryotic cells of the TRABD gene.

DRAWINGS

Figure 1-1: Comparison of the content of HIV-1 in the supernatant after transfection of 293T cells with the TRABD2A protein expression vector in Example 1.

Figure 1-2: Comparison of p24 content after transfection of 293T cells with TRABD2A protein expression vector in Example 1.

Figure 1-3: Comparison of virion-related genomic RNA content of 293T cells transfected with TRABD2A protein expression vector in Example 1.

Figure 1-4: Comparison of HIV Gag RNA content after transfection of 293T cells using the TRABD2A protein expression vector in Example 1.

Figure 1-5: Comparison of HIV-1 Gag, gp120, TRABD2A protein and glyceraldehyde phosphate dehydrogenase (GAPDH) after transfection of 293T cells with TRABD2A protein expression vector in Example 1.

Figure 1-6: Comparison of HBV virus content in supernatant after transfection of 293T cells with TRABD2A protein expression vector in Example 1.

Figure 1-7: Comparison of TRABD2A and glyceraldehyde phosphate dehydrogenase after transfection of 293T cells using the TRABD2A protein expression vector in Example 1.

Figure 2-1: Comparison of the content of HIV-1 in the supernatant after transfection of 293T cells with various TRABD protein expression vectors in Example 2.

Figure 2-2: Comparison of exogenous TRABD protein and glyceraldehyde phosphate dehydrogenase after transfection of 293T cells using various TRABD protein expression vectors in Example 2.

Figure 3-1: Comparison of the contents of VSV-G, HIV-1 _NL4.3 or -89.6 in the supernatant after transfection of 293T cells with TRABD2A and 2B protein expression vectors in Example 3.

Figure 3-2: Comparison of HIV Gag RNA content after transfection of 293T cells with TRABD2A and 2B protein expression vectors in Example 3.

Figure 3-3: Comparison of HIV-1 _BH-10 content in the supernatant after transfection of 293T cells with TRABD2A and 2B protein expression vectors in Example 3.

Figure 3-4: Comparison of HIV-1 _BaL content in supernatants after transfection of 293T cells with TRABD2A and 2B protein expression vectors in Example 3.

Figure 3-5: Comparison of HIV-1 _NL4.3 content in supernatant after transfection of 293T cells with TRABD 2B protein expression vector in Example 3.

Figure 3-6: Comparison of HIV Gag RNA content after transfection of 293T cells using the TRABD 2B protein expression vector in Example 3.

Figure 3-7: TRABD2B, Gag and glyceraldehyde phosphate dehydrogenase content profiles after transfection of 293T cells with the TRABD 2B protein expression vector in Example 3.

Figure 4-1: Comparison of CCR5 HIV-1 _AD8 , HIV-1 _89.6 , and SIV _{mac239 in the} supernatant after transfection of 293T cells with TRABD2A and 2B protein expression vectors in Example 4.

Figure 4-2: Comparison of HIV-2 _ST levels in supernatants after transfection of 293T cells with TRABD2A and 2B protein expression vectors in Example 4.

Figure 5-1: Effect of 1,10-phenanthroline on the inhibition of HIV-1 infection by TRABD protein after transfection of 293T cells with TRABD2A and 2B protein expression vectors in Example 5 and western blotting (western blotting) Blot) map.

Figure 5-2: Effect of Co ²⁺ on the inhibition of HIV-1 infection by TRABD protein after transfection of 293T cells with TRABD2A expression vector in Example 5 and immunoblot assay.

Figure 5-3: Effect of Ni ²⁺ on the inhibition of HIV-1 infection by TRABD protein after transfection of 293T cells with TRABD2A expression vector in Example 5 and immunoblot assay.

Figure 5-4: Effect of Mn ²⁺ on the inhibition of HIV-1 infection by TRABD protein after transfection of 293T cells with TRABD2A expression vector in Example 5 and immunoblot assay.

Figure 5-5: Effect of Zn ²⁺ on the inhibition of HIV-1 infection by TRABD protein after transfection of 293T cells with TRABD2A expression vector in Example 5 and immunoblot assay.

Figure 5-6: Effect of Co ²⁺ on the inhibition of HIV-1 infection by TRABD protein after transfection of 293T cells with TRABD2B expression vector in Example 5 and immunoblot assay.

Figure 5-7: Effect of Ni ²⁺ on the inhibition of HIV-1 infection by TRABD protein after transfection of 293T cells with TRABD2B expression vector in Example 5 and immunoblot assay.

Figure 5-8: Effect of Mn ²⁺ on the inhibition of HIV-1 infection by TRABD protein after transfection of 293T cells with TRABD2B expression vector in Example 5 and immunoblot assay.

Figure 5-9: Effect of Zn ²⁺ on the inhibition of HIV-1 infection by TRABD protein after transfection of 293T cells with TRABD2B expression vector in Example 5 and immunoblot assay.

Figure 6: The supernatant obtained by treating the CD4 ⁺ T reservoir cells of HIV-1 and HIV-2 with a specific monoclonal antibody of Ni ²⁺ , Co ²⁺ , a divalent metal chelating agent, and TRABD2A protein in Example 6. Comparison of HIV-1 and HIV-2 levels in the liquid.

Figure 7: Supernatant obtained from the treatment of CD4 ⁺ T reservoir cells in the blood of HIV-1 patients using a specific monoclonal antibody of Ni ²⁺ , Co ²⁺ , a divalent metal chelating agent, and TRABD2A protein in Example 7. Comparison of HIV-1 levels.

Figure 8-1: Treatment of activated or non-activated CD4 ⁺ T reservoir cells obtained from individual patients using different Ni ²⁺ concentrations in Example 8 and comparison of HIV-1 levels in the supernatant obtained.

Figure 8-2: Treatment of activated or inactivated CD4 ⁺ T reservoir cells obtained from individual patients using different Ni^&lt;2+ concentrations in Example 8 and comparison of TRABD2A RNA content obtained.

Figure 8-3: Comparison of the levels of CD4 ⁺ T surface marker CD69 expression obtained by treatment with activated or inactivated CD4 ⁺ T reservoir cells obtained from individual patients using different Ni ²⁺ concentrations in Example 8.

Figure 8-4: Comparison of HIV-1 levels in the supernatant obtained by treating CD4 ⁺ T reservoir cells obtained from each patient using different Ni ²⁺ concentrations in Example 8.

Figure 9-1: Comparison of the TRABD2A content obtained in Example 9 using CD4 ⁺ T reservoir cells obtained from individual donors with and without activation.

Figure 9-2: Comparison of HIV-1 levels in supernatants obtained after treatment of HIV-infected CD4 ⁺ T reservoir cells injected with luciferase using different concentrations of Ni ²⁺ in Example 9.

Figure 9-3: Comparison of luciferase, TRABD2A RNA and Gag RNA content obtained after treatment of HIV-infected CD4 ⁺ T reservoir cells injected with luciferase using different Ni ²⁺ concentrations in Example 9.

Figure 9-4: Comparison of HIV-1 levels in the supernatant obtained after treatment of HIV-infected CD4 ⁺ T reservoir cells injected with luciferase using different Co ²⁺ concentrations in Example 9, and luciferase, Comparison of TRABD2A RNA and Gag RNA content.

Figure 9-5: Comparison of HIV-1 levels in the supernatant obtained after treatment of HIV-infected CD4 ⁺ T reservoir cells injected with luciferase using different concentrations of 1,10-phenanthroline in Example 9. , as well as a comparison of luciferase, TRABD2A RNA and Gag RNA content.

Figure 9-6: Cell surface obtained after treatment of HIV-infected CD4 ⁺ T reservoir cells injected with luciferase using different Ni ²⁺ , Co ²⁺ , 1,10-phenanthroline concentrations in Example 9. Mark the CD69 expression level comparison chart.

Figure 10-1: Comparison of HIV-2 levels in the supernatant obtained after treatment of HIV-2 infected CD4 ⁺ T reservoir cells with different contents of Ni ²⁺ , Co ²⁺ in Example 10.

Figure 10-2: Comparison of RNA content of TRABD2A obtained after treatment of HIV-2 infected CD4 ⁺ T reservoir cells with different contents of Ni ²⁺ and Co ²⁺ in Example 10.

Figure 11-1: Comparison of HIV-2 levels in supernatants obtained after treatment of HIV-2 infected CD4 ⁺ T reservoir cells using small interfering RNA (siRNA) in Example 11.

Figure 11-2: Comparison of RNA content of TRABD2A obtained after treatment of HIV-2 infected CD4 ⁺ T reservoir cells with small interfering RNA in Example 11.

Figure 12-1: HIV-1 levels in supernatants obtained after treatment of HIV-1 infected CD4 ⁺ T depot cells obtained from donors by electroporation (siRNAs electroporation) using the small interfering RNA in Example 12. Comparison chart.

Figure 12-2: HIV-1 reversed transcription (reverse transcriptase) in supernatant obtained after treatment of HIV-1 infected CD4 ⁺ T reservoir cells obtained from donors by electroporation using small interfering RNA in Example 12. , RT) activity comparison chart.

Figure 12-3: Comparison of HIV-1 Gag RNA content obtained after treatment of HIV-1 infected CD4 ⁺ T reservoir cells obtained from donors by electroporation using small interfering RNA in Example 12.

Figure 12-4: Comparison of RNA content of TRABD2A obtained after treatment of HIV-1 infected CD4 ⁺ T reservoir cells obtained from donors by electroporation using small interfering RNA in Example 12.

Figure 12-5: HIV-1 infected CD4 ⁺ T reservoir cells obtained from donors after treatment with tandem siRNA electroporation in Example 12, and the resulting supernatant was HIV-1 Content comparison chart.

Figure 12-6: Comparison of RNA content of TRABD2A obtained after treatment of HIV-1 infected CD4 ⁺ T reservoir cells obtained from donors using a small interfering RNA in Example 12.

Figure 12-7: Comparison of HIV-1 Gag RNA content obtained after treatment of HIV-1 infected CD4 ⁺ T reservoir cells obtained from donors using a small interfering RNA in Example 12.

Figure 13-1: Comparison of the obtained TRABD2A RNA content in Example 13 in which the activated HIV-1 infected CD4 ⁺ T reservoir cells were slowly returned to rest.

Figure 13-2: Comparison of the CD69 surface marker content of activated HIV-1 infected CD4 ⁺ T reservoir cells treated with shRNAs in Example 13 and allowed to slowly return to rest.

Figure 13-3: Comparison of HIV-1 levels in the supernatant obtained by treatment of activated HIV-1 infected CD4 ⁺ T reservoir cells using shRNAs in Example 13 and allowing them to slowly return to rest.

Figure 13-4: Comparison of the Gag RNA content obtained by treatment of activated HIV-1 infected CD4 ⁺ T reservoir cells with shRNAs in Example 13 and allowing them to slowly return to rest.

Figure 13-5: Comparison of the TRABD2A RNA content obtained by treatment of activated HIV-1 infected CD4 ⁺ T reservoir cells with shRNAs in Example 13 and allowing them to slowly return to rest.

Figure 14-1: In Example 14, the expression plasmids of TRABD2A protein and TRABD2B protein were introduced into HEK293T cells, respectively, and the cells were infected with HIV-1, HIV-2 and SIV _mac239 , respectively, and the obtained supernatant was HIV-1. Comparison of HIV-2 and SIV _mac239 content.

Figure 14-2: In Example 14, the expression plasmids of HIV-1 virus, HIV-2 virus, SIV virus, TRABD2A protein and TRABD2B protein were introduced into HEK293T cells, respectively, and the obtained supernatants were HIV-1 and HIV-2. Comparison with SIV content.

Figure 15: Peripheral blood cells (PBMCs) isolated from HIV-1 infected patients whose viral load (Viral Load < 20 copies/mL) was not detected in plasma, and treated with monoclonal blocking antibody of TRABD2A for 72 hours. , the content of HIV-1 reservoir cells.

Figure 16: In Example 17, peripheral blood cells (PBMCs) isolated from HIV-1 infected patients with no viral load (Viral Load < 20 copies/mL) were removed and CD8 ⁺ T immune cells were removed and used. The content of HIV-1 reservoir cells after 72 hours of TRABD2A monoclonal blocking antibody treatment.

Figure 17: In Example 18, peripheral blood cells (PBMCs) isolated from HIV-1 infected patients in which no viral load (Viral Load < 20 copies/mL) was detected in plasma were removed and CD8 ⁺ T immune cells were not removed and used. The content of HIV-1 depot cells after 72 hours of treatment with TRABD2A and PD-1 blocking antibodies.

Figure 18: In Example 19, resting CD4 ⁺ T and resting CD8 ⁺ T cells isolated from HIV-1 infected patients with no viral load (Viral Load < 20 copies/mL) were cultured in vitro, CD4 ⁺ T cells were treated with the blocking antibody TRABD2A 72 h, CD4 ⁺ T cells with a Gag polypeptide and the activated CD8 ⁺ T cell populations were cultured for 5 days, the content of the case of HIV-1 cell repository.

Figure 19: In Example 20, resting CD4 ⁺ T and resting CD8 ⁺ T cells isolated from HIV-1 infected patients with no viral load (Viral Load < 20 copies/mL) were cultured in vitro, CD4 ⁺ T cells treated with the blocking antibody TRABD2A 72 h, CD4 ⁺ T cells and activated and treated with PD-1 blocking antibody CD8 ⁺ T cells co-cultured for 5 days with a Gag polypeptide group, the content of HIV-1 repository cells Happening.

Figure 20: Example 21, in which HIV-1 infected patients with viral load (Viral Load < 20 copies/mL) were accidentally wounded, and the Microglia cells and the resting CD8 ⁺ T in the blood were obtained from the CNS system. The cells were cultured in vitro and treated with TRABD2A blocking antibody and Gag polypeptide for 96 hours to release the survival rate of HIV-1 Microglia cells.

The contents of the present invention will be described below by way of examples.

Implementation

The present invention will be described in detail below with reference to specific embodiments for the purpose of better understanding of the technical contents of the public, and not limiting the technical content. In fact, improvements are made on the same or similar principles. All are within the scope of the claims as claimed. In particular, in the examples and the figures:

BD means below detection limit (below detection limit);

RLU refers to relative luminescence units;

NS means not significant;

Mock refers to the negative control group;

HIV Release refers to the level of HIV virus release;

Infectivity refers to the level of virus infection

Example 1

The human TRABD2A protein of the HIV reservoir cells has an inhibitory effect on the release of HIV-1 virus and is specifically inhibited against HIV-1. And participate in the specific degradation of Gag precursors.

The Myc-tagged Gag protein was introduced into CD4 ⁺ T reservoir cells infected with HIV-1 virus and cultured for 4 days. Cellular proteins that may interact with Gag proteins at the late stage of HIV-1 replication were identified by immunoprecipitation experiments using anti-Myc antibodies and IgG control antibodies. The results showed that 54 proteins formed a precipitate with the Myc-labeled Gag protein (see Table 1), while 69 proteins formed a precipitate with the IgG antibody (see Table 2). By comparison with the IgG control antibody, it was found that 25 cell proteins specifically bind to the Gag protein. Based on alignment with relevant data materials, it was found that only TRABD2A protein was significantly higher in HIV-infected CD4 ⁺ T reservoir cells than in activated CD4 ⁺ T cells, whereas TRABD2B protein was in HIV-infected CD4 ⁺ T reservoir cells. None of the above or activated CD4 ⁺ T cells were found. The specific test procedure was as follows: isolated CD4 ⁺ T cells obtained from 10 healthy donors, Gag ( ⁴ ×10 ⁶ cells/electroporation) constructed with Myc markers, respectively, after 6 hours with HIV-1 _NL4.3 Centrifuged. Five days after transfection, the cells were lysed in immunoprecipitation (IP) lysis buffer (50 mM Tris-HCl, pH 7.2, 50 mM NaCl, 1% NP-40, 1 mM EDTA, 2% glycerol, 1× protease inhibitor) and collected. All cell lysates. The lysate was incubated on ice for 30 minutes and centrifuged at 12000 rpm at 4 °C for 10 minutes. The supernatant was transferred to a test tube, and the centrifuged residue was mixed with cold immunoprecipitated (IP) lysis buffer and sonicated, and then centrifuged at 12,000 rpm at 4 ° C for 10 minutes. The supernatant obtained after the above two extraction steps was co-reacted with Protein A and protein G Dynabeads (from Invitrogen), and the protein A and protein G Dynabeads were pretreated with the antibody at 4 °C. The immunoprecipitated product was washed 5-10 times with cold immunoprecipitation buffer and PBST (500 μL each). The immunoprecipitated product was first degraded for 5 minutes at 95 ° C in 20 mM dithiothreitol (DTT) (from Sigma) and then alkylated in 50 mM iodoacetamide (IAA) (from Sigma) for 30 minutes at room temperature in a dark room. After alkylation, the sample was transferred to a 10 kD centrifugal spin filter (from Millipore), washed three times with 200 μL of 8 M urea by centrifugation at 14,000 x g, and washed twice with 200 μL of 50 mM ammonium bicarbonate. Next, trypsin (from Promega) was added at a ratio of 1:50 (enzyme/matrix, m/m) in 200 μL of 50 mM ammonium hydrogencarbonate, and trypsinization was carried out at 37 ° C for 16 hours. The filtration system was replaced with a new collection tube and centrifuged at 14,000 x g to collect the polypeptides. In order to increase the yield of the polypeptide, the filtration system can be washed with 100 μL of 50 mM NaHCO ₃ . The obtained polypeptides were desalted using STAGE TIP. MS experiments were performed by nanoscale UHPLC (EASY-nLC1000 from Proxeon Biosystems, Odense, Denmark) coupled to Orbitrap Q-Exactive, equipped with a nanoelectron spray source (from Thermo Fisher Scientific, Bremen, Germany). The polypeptide was dissolved in 0.1% FA with 5% CH ₃ CN and applied to a RP-HPLC analytical column (75 μm x 15 cm) packed with 2 m C18 magnetic beads (from Thermo Fisher Scientific, Bremen, Germany) with a gradient of 2 h. % to 40% of acetonitrile was dissolved in formic acid at a flow rate of 250 nL/min. The spray voltage was set at 2.5 kV and the ion transport capillary was at 275 °C. A complete MS/MS cycle consists of a complete MS scan (resolution, 70,000; AGC value, le6; maximum injection time 50ms) in profile mode, mass range from 300 to 1800m/z, followed by cracking The process uses the top ten strongest ions to normalize the collision energy to 28% of the centroid mode (resolution, 17,500; AGC value le5, maximum injection time 100ms) high energy collision dissociation. The dynamic exclusion window is set at 40 seconds. A micro scan is required for each MS and MS/MS scan. Unspecified ions or other charges of 1+ and >7+ are rejected by the MS/MS cycle. Background correction is used for mass correction (m/z 445.12003). Raw data was processed using Proteome Discoverer (PD, version 2.1) and the obtained MS/MS spectra were compared with the Swiss-Prot human proteome. All searches were done with a mass tolerance of 7 ppm mixture. Fragment quality tolerances are 20millimass unit (mmu), oxidation 20 (Met) (+15.9949 Da) and acetylation (protein N-termini) (+42.0106 Da) as variable coefficients, urea methylation (Cys) (+57.0215 Da As a constant coefficient, the trypsin digestion fragment was not completely set to two. Only polypeptides of at least 6 amino acids in length are allowed. Polypeptide and protein identification was filtered with PD to control the false discovery rate (FDR) <1%. At least one unique protein is required for protein identification.

To further verify the role of TRABD2A, HIV-1 virus vector was treated with 293T cells with different TRABD2A protein expression levels, and these 293T cells containing TRABD2A protein expression were found to be invaded by HIV-1 compared to 293T cells without TRABD2A protein expression. The infectivity can be reduced to a level of up to about 1/4000, and even more so that the infectivity of the virus is completely undetectable. The specific test procedure was as follows: 293T cells were transfected with the Myc-tagged TRABD2A protein expression vector, including pNL4.3, including the negative control group. At 48 hours post-transfection, the supernatant was used to infect TZMbl reporter cells to obtain virus infection levels. The background RLU was subtracted from each measurement (Figure 1-1, Fig. 1b).

In the case of overexpression of the TRABD2A protein, the content of the core antigen p24 of the virions in the culture supernatant and the genomic RNA of the virus particles is also greatly reduced. The specific test procedure is as follows: p24 enzyme-linked immunosorbent assay (ELISA) (Fig. 1-2, Fig. 1c), and qPCR based on specific probes were used to measure virions using a similar pretreatment step as in Figure 1-1. Related genomic RNA (Virion RNA) (Figure 1-3, Fig.d).

However, overexpression of TRABD2A protein did not affect intracellular viral Gag transcription levels. This demonstrates that overexpression of TRABD2A protein significantly reduces the granule packaging of HIV-1, but does not affect the transcription of HIV-1. At the same time, the viral Gag protein positively associated with the TRABD2A protein was also significantly reduced, while the envelope glycoprotein gp120 and the like were not affected, indicating that TARBD2A is involved in the specific degradation of the Gag precursor. The specific test procedure was as follows: All RNA was extracted for qPCR to measure the Gag transcription level of HIV-1 (Fig. 1-4, Fig. 1e). The cells were lysed for immunoblotting and the expression levels of HIV-1 Gag, gp120, TRABD2A protein and glyceraldehyde phosphate dehydrogenase protein were detected using specific antibodies (Fig. 1-5, Fig. 1f).

The effect of overexpression of TRABD2A protein in 293T cells on the CMV promoter-driven HBV (hepatitis B virus) virus was examined to determine whether the TRABD2A protein specifically acts on HIV-1, and a complete HBV replication, TRABD2A, was found. Protein does not affect the replication and release of HBV. The specific test procedure was as follows: The 293T cells of the experimental group were transfected with the Myc-tagged TRABD2A expression vector, and treated with the HBV replicon (CMV promoter) together with the negative control group. After 48 hours, HBV virions in the culture supernatant were examined using HBs enzyme-linked immunosorbent assay (Figures 1-6, Fig. 1g). The cells were lysed for immunoblotting to measure the expression levels of TRABD2A and glyceraldehyde phosphate dehydrogenase (Figures 1-7, Fig. 1h).

Table 1 shows the precipitation of 54 proteins with Myc-tagged Gag protein

Serial number	Protein number	Serial number	Protein number	Serial number		Protein number
1.	P35908	2.	P81605-2	3.	P35030
4.	H6VRG1	5.	P26447	6.	P60174
7.	L0R599	8.	Q86YZ3	9.	B7Z6Z4
10.	Q86V40	11.	A8MU27	12.	B3KMQ6
13.	P35527	14.	P02533	15.	P31947
16.	P13645	17.	P62851	18.	P04083
19.	Q53HU8	20.	Q65ZC9	twenty one.	Q6MZX7
twenty two.	A0N7I9	twenty three.	B4DPP6	twenty four.	A0A024R1N1
25.	D1MGQ2	26.	P28799	27.	P07355-2
28.	P13647	29.	B4DR52	30.	O43290
31.	Q0ZCJ1	32.	B7Z8Q2	33.	Q05639
34.	P19474	35.	P62269	36.	Q5D862
37.	Q6GMV7	38.	E7EX29	39.	Q9BUH8
40.	P62805	41.	Q6N092	42.	P06733
43.	P62979	44.	A8K486	45.	P14625
46.	H7C2N1	47.	Q8TCD0	48.	M1VPF4
49.	A0A0A0MS14	50.	P20700	51.	I0B0K7
52.	A0A075B7D0	53.	Q59G88	54.	V9HWB4

Table 2: 69 proteins that precipitate with IgG antibodies

Serial number	Protein number	Serial number	Protein number	Serial number		Protein number
1.	H6VRG1	2.	J3QRS3	3.	P06703
4.	P35908	5.	Q6MZW0	6.	Q59G88
7.	P13645	8.	P28799	9.	P62269
10.	P35527	11.	Q6N092	12.	J3KSD8
13.	Q53HU8	14.	Q5D862	15.	P06313
16.	P02533	17.	P16402	18.	P84098
19.	P13647	20.	P81605-2	twenty one.	Q59EJ3
twenty two.	P08779	twenty three.	B4DR52	twenty four.	Q8NF17
25.	A0A024R1N1	26.	H7C2N1	27.	P62263
28.	B2R853	29.	D1MGQ2	30.	B2R7F8
31.	D3DTX7	32.	A0A5E4	33.	Q5T749
34.	B4DPP6	35.	Q05639	36.	Q86YZ3
37.	P63261	38.	P08123	39.	P23435
40.	P02452	41.	A0A0A0MT36	42.	Q05D60
43.	A2NJV5	44.	A9UFC0	45.	P0C0L4
46.	B7Z8Q2	47.	A0A0A0MS14	48.	Q03001
49.	P62805	50.	P07355-2	51.	P06748
52.	P19474	53.	P02461	54.	D3DQ70
55.	P35030	56.	A0A075B7D0	57.	P62280
58.	P01613	59.	Q5CZ94	60.	P00338-3
61.	A0A096LP77	62.	A0A087X130	63.	M1VPF4
64.	B7Z6Z4	65.	P07737	66.	A0A087WYA1
67.	Q14094	68.	Q59GY2	69.	A0A096LNH6

Example 2

The TRABD2A-201 and 202 and TRABD2B proteins in the human TRABD2A protein family have an inhibitory effect on HIV-1 virus release and are specifically inhibited against HIV-1. At the same time, chimpanzee's TRABD2A protein also has the effect of inhibiting the release of HIV-1 virus, and has specific inhibition to HIV-1.

There are three specific human TRABD2A proteins, TARBD2A-201 protein, TRABD2A-202 protein and TRABD2A-203 protein. There is only one human TRABD2B protein. The anti-HIV-1 activity of all three human TRABD2A proteins, human TRABD2B protein and chimpanzee TRABD2A protein (201) was examined. The human TRABD2A-201 protein and the human TRABD2B protein reduced the infectivity of HIV-1 to 1/1087 and 1/186 of the negative control group, respectively, relative to the negative control group. The human TRABD2A-203 protein showed no anti-HIV-1 activity. Human TRABD2A-202 protein and chimpanzee TRABD2A protein also reduced HIV-1 invasiveness, except that the magnitude of the decrease was slightly different from that of human TRABD2A-201. This suggests that the primate TraB family cell membrane metalloproteinases may have anti-HIV-1 activity. The specific test procedures are as follows: 293T cells were transfected with GFP-tagged human TRABD2A-201, FLAG-tagged human TRABD2A-202 and TRABD2A-203, MYC-FLAG-labeled human TRABD2B, FLAG-labeled chimpanzee TRABD2A protein, including the negative control group. Both were treated with pNL4.3. At 48 hours post-transfection, the supernatant was used to infect TZMbl reporter cells to obtain virus infection levels. The background RLU was subtracted from each measurement (Figure 2-1, Extended Data Fig. 5b). Detection of exogenous TRABD protein and glyceraldehyde phosphate dehydrogenase by immunoblotting with specific antibodies (Fig. 2-2, Extended Data Fig. 5c)

Example 3

The TRABD2A protein and the TRABD2B protein are inhibitory to HIV-1, which is manifested in the specific degradation of the viral core molecule Gag protein, which affects the transcription of Gag.

Both human TRABD2A and 2B proteins exhibit a very strong ability to block HIV-1 infection and do not affect Gag transcription. The specific test procedure was as follows: 293T cells were transfected with GFP-tagged TRABD2A expression vector and Myc-labeled TRABD2B expression vector, including pNL4.3ΔE-GFP and negative expression of VSV-G, HIV-1 _NL4.3, or the negative control group, respectively. Carrier treatment of -89.6. After 48 hours of transfection, the supernatant was used to infect TZMbl reporter cells to obtain virus infection levels, and background RLU was subtracted from each measurement (Figure 3-1, Fig. 2a). All RNAs extracted were subjected to qPCR to quantify the Gag transcript and normalized to glyceraldehyde phosphate dehydrogenase (Figure 3-2, Fig. 2b).

Expression of human TRABD2A and 2B also demonstrated a limiting capacity for HIV-1 _BH10 and HIV-1 _BaL infection. The specific test procedure was as follows: 293T cells were transfected with GFP-tagged TRABD2A and Myc-labeled TRABD2B, including the viral vector BH10 (Fig. 3-3, Fig. 2c), BaL (Fig. 3-4, Fig. .2d) Processing. After 48 hours of transfection, the supernatant was used to infect TZMbl reporter cells to obtain the level of infection of the virus, and the background RLU was subtracted from each measurement.

The human TRABD2B protein also showed a function of preventing HIV-1 infection and enhancing viral Gag protein degradation without affecting Gag transcription. The specific test procedure was as follows: 293T cells were transfected with Myc-tagged TRABD2B expression vector, including pNL4.3, including the negative control group. At 48 hours post-transfection, the supernatant was used to infect TZMbl reporter cells to obtain virus infection levels, and the background RLU was subtracted from each measurement (Figure 3-5, Extended Data Fig. 2a). All RNA was extracted for qPCR to quantify viral Gag transcripts and normalized using glyceraldehyde phosphate dehydrogenase (Figure 3-6, Extended Data Fig. 2b). The content of TRABD2B, Gag and glyceraldehyde phosphate dehydrogenase protein was determined by immunoblotting assay (Fig. 3-7, Extended Data Fig. 2c).

Example 4

The human TRABD2A protein and the TRABD2B protein have the effect of inhibiting other lentiviruses, in particular, inhibiting the release of HIV-2 virus, and have specific inhibition against HIV-2.

Overexpression of human TRABD2A and 2B has the ability to limit infection for SIV _mac239 , HIV-2 _ST , HIV-1 _89.6 , CCR5 HIV-1 _AD8 , and the like. This illustrates the broad anti-lentiviruses ability of the TRABD cell membrane metalloprotein family. The specific test procedures were as follows: 293T cells were transfected with GFP-tagged TRABD2A and Myc-labeled TRABD2B, including the viral vectors CCR5HIV-1 _AD8 , HIV-1 _89.6 , and SIV _mac239 (Fig. 4-1, Fig.). 2e) or HIV-2 _ST (Fig. 4-2, Fig. 2f) treatment. After 48 hours of transfection, the supernatant was used to infect TZMbl reporter cells to obtain the level of infection of the virus, and the background RLU was subtracted from each measurement.

Example 5

TRABD requires Mn ²⁺ to achieve its inhibition of HIV-1. Divalent metal chelators, Ni ²⁺ , Co ²⁺ and Zn ²⁺ can inhibit the inhibition of HIV-1 by TRABD.

Human TRABD2A and TRABD2B are membrane cell metalloproteinases. Treatment with 1,10-phenanthroline or other divalent metal chelators, although the expression levels of TRABD2A and 2B did not change, but their antiviral ability completely disappeared. The results were treated with Co ²⁺ , Ni ²⁺ , Mn ²⁺ and Zn ²⁺ , respectively. Co ²⁺ and Ni ²⁺ can inhibit the anti-HIV-1 virus ability of human TRABD2A, while Mn ²⁺ enhances the anti-HIV-1 virus ability of human TRABD2A. When Zn ^{2+ is} at a higher concentration, it exhibits a certain inhibitory effect on the anti-HIV-1 virus ability of TRABD2A. The specific test procedure was as follows: 293T cells were infected with GFP-tagged TRABD2A expression vector and Myc-labeled TRABD2B expression vector, including pNL4.3, including the negative control group. Six hours after transfection, the cells were washed with various concentrations of the following solutions: 1,10-phenanthroline, Co ²⁺ , Ni ²⁺ , Mn ^{2+ ,} and Zn ²⁺ . After 42 hours of transfection, the cells were lysed and the expression levels of TRABD2A and glyceraldehyde phosphate dehydrogenase protein were detected by immunoblotting with specific antibodies. The supernatant was used to infect TZMbl reporter cells to obtain the release level of the virus. The effect of each ion on the inhibition of HIV-1 infection by TRABD protein was obtained. The results are as follows: 1,10-phenanthroline (Fig. 5-1, Fig. 3a), Co ²⁺ (Fig. 5-2, Fig. 3b), Ni ²⁺ (Fig. 5-3, Fig. 3c) , Mn ²⁺ (Fig. 5-4, Fig. 3d) and Zn ²⁺ (Fig. 5-5, Fig. 3g).

These metal ions also exhibit a similar effect on TRABD2A for the effect of TRABD2B against HIV-1 virus capacity. The specific test procedure was as follows: 293T cells were transfected with Myc-tagged TRABD2B expression vector, including pNL4.3, including the negative control group. Six hours after transfection, the cells were washed with various concentrations of the following solutions: Co ²⁺ , Ni ²⁺ , Mn ²⁺ , and Zn ²⁺ . After 42 hours of transfection, the cells were lysed and the expression levels of TRABD2B and glyceraldehyde phosphate dehydrogenase protein were detected by immunoblotting with specific antibodies. The supernatant was used to infect TZMbl reporter cells to obtain the release level of the virus. The effect of each ion on the inhibition of HIV-1 infection by TRABD protein was obtained. Detected separately for TRABD2B and control group. The results are as follows: Co ²⁺ (Fig. 5-6, Extended Data Fig. 6a), Ni ²⁺ (Fig. 5-7, Extended Data Fig. 6b), Mn ²⁺ (Fig. 5-8, Extended Data Fig. 6c) And Zn ²⁺ (Fig. 5-9, Extended Data Fig. 6f).

Example 6

Inhibition of human TRABD2A protein activity by metal ions, metal chelators and related monoclonal antibodies can promote the infection of virus-infected reservoir cells to release HIV-1 and HIV-2 viruses in vitro and be detected.

By using Ni ²⁺ , Co ²⁺ , a divalent metal chelating agent, and a monoclonal antibody specific for the TRABD2A protein, HIV-1, HIV-2 type virus can be released from a reservoir cell that promotes HIV infection. The specific test procedure is as follows: HIV-1 and HIV-2 viruses are infected with resting CD4+ T cells by ultracentrifugation. Six hours after the infection was completed, the excess virus was washed away with the medium, and then 100 μM of Ni ²⁺ , Co ²⁺ , 5 μM of 1,10-phenanthroline, or 100 ng of the specific monoclonal antibody of TRABD2A were added. Antibody (Antibody), after 48 hours of culture, the supernatant was used to infect TZMbl reporter cells to obtain the release level of the virus, as shown in FIG.

Example 7

The inhibition of human TRABD2A protein activity by metal ions, metal chelators and related monoclonal antibodies can promote the release of HIV-1 virus in the reservoir cells of HIV-1 patients, thereby accurately detecting the amount of cells in the patient's body.

From patients with no detectable viral RNA load in 8 plasmas, peripheral blood mononuclear cells (PBMC) were isolated and CD4 ⁺ T reservoir cells were purified. The obtained CD4 ⁺ T reservoir cells were treated with 100 μM Ni ²⁺ , Co ²⁺ , 5 μM 1,10-phenanthroline, or 100 ng of a monoclonal antibody (Antibody) of TRABD2A protein, respectively. After 24 hours of culture, the supernatant was used to infect TZMbl reporter cells to obtain the release level of the virus as shown in FIG.

Example 8

The use of Ni ²⁺ ions to treat the reservoir cells obtained from the patient will promote the release of HIV-1 virus without activating the reservoir cells and will be detected.

HIV-1 latent infection reservoir cells were isolated from patients receiving cART treatment for more than two years to further analyze the anti-HIV-1 physiological effects of TRABD2A protein in humans.

In addition to patient No. 1, the viral RNA content in other patients' plasma was much lower than 20 cp/ml. TZMbl reporter cells were used to detect activated and inactivated depot cells in the presence or absence of Ni ²⁺ . The results showed that the release of HIV-1 was associated with the treatment of Ni ²⁺ except for Patient No. 3, and HIV-1 was not released in the absence of Ni ²⁺ . In contrast, activated CD4 ⁺ T cells released a large amount of HIV-1 virus after activation with CD3/CD28 plus IL2. At the same time, the expression of TRABD2A on activated CD4 ⁺ T cells was also greatly reduced. Ni ²⁺ does not activate CD4 ⁺ T reservoir cells, suggesting that HIV-1 release after Ni ²⁺ treatment is not due to activation of CD4 ⁺ T reservoir cells. The specific test procedure was as follows: The isolated CD4 ⁺ T cells were aliquoted and treated with various concentrations of Ni ²⁺ with or without activation of the CD3/CD28 promoter and IL2. After 24 hours of treatment, the medium was used to infect TZMbl reporter cells to obtain the release level of the virus, and the background RLU was subtracted from each measurement data (Fig. 8-1, Extended Data Fig. 21b). All RNA in CD4 ⁺ T cells was extracted and the transcript of TRABD2A was measured by qPCR and normalized with glyceraldehyde phosphate dehydrogenase (Fig. 8-2, Extended Data Fig. 21c). The CD4 ⁺ T surface marker CD69 expression level was measured by flow cytometry (Fig. 8-3, Extended Data Fig. 21d).

HIV-1 latent-infected depot cells were isolated from 16 patients who underwent cART treatment for more than two years and whose viral RNA content in the plasma was well below 20 cp/mL, and the validation test was continued. Observations were started 24 hours after treatment with Ni ²⁺ and without Ni ²⁺ , respectively. It was found that after treatment with Ni ²⁺ , all patients' reservoir cells released HIV-1 virus, and the reservoir cells were not activated; At the same time, the reservoir cells that were not treated with Ni ²⁺ were not detected to release any HIV-1 virus. The specific test procedure is as follows: The latent HIV-1-infected reservoir cells were extracted from 16 cART-treated patients (all patients with plasma viral load <20 copies/mL), and all types of cells were used without activation of cells. The solution of concentration Ni ²⁺ was treated for 24 hours. The medium was used to infect TZMbl reporter cells to obtain the release level of the virus, and the background RLU was subtracted from each measurement data (Fig. 8-4, Extended Data Fig. 21e).

Example 9

The use of metal ions, metal chelators, and related monoclonal antibodies to inhibit human TRABD2A protein activity, enabling HIV-infected reservoir cells, including reservoir cells obtained in patients, without activating HIV-infected reservoir cells , released HIV-1 virus and detected.

CD4 ⁺ T cells and activated CD4 ⁺ TRABD2A expression levels on T cells obtained from a healthy donor Comparative in vivo, for further analysis TRABD2A activity against HIV-1 in CD4 ⁺ T cells. After 72 hours of activation of CD4 ⁺ T cells, the TRABD2A transcription level was reduced to a level less than about one percent. This suggests that the effect of TRABD2A involved in limiting HIV-1 release may occur only in depot cells and not in activated CD4 ⁺ T cells. The specific test procedure is as follows: CD4 ⁺ T cells are isolated from two unrelated healthy donors, and cultured for 12 hours, 24 hours, 48 hours, or 72 hours under the conditions of Activation/Resting. All RNA was extracted for qPCR to detect the transcriptional content of TRABD2A, normalized to glyceraldehyde phosphate dehydrogenase. The data could not be called a repository relative to normal people. Cells were compared (set to 100%) (Figure 9-1, Fig. 5a).

It was further verified that Ni ^{2+ was} used to eliminate the metalloproteinase activity of TRABD2A. HIV infection of CD4 ⁺ T cells to release a large repository of HIV-1 cells in the presence of Ni ²⁺ and Ni ²⁺ are positively correlated with the concentration and treatment time. At the same time, Ni ²⁺ does not affect the level of vector-driven luciferase, TRABD2A protein or viral Gag transcription in HIV-infected CD4 ⁺ T reservoir cells. However, HIV-infected CD4 ⁺ T reservoir cells do not release any HIV-1 virus that is recognized in the absence of Ni ²⁺ , which is consistent with the viral restriction of TRABD2A. The specific test procedure was as follows: HIV-infected CD4 ⁺ T reservoir cells were injected by electroporation with a luciferase expression vector and transfected with HIV-1 _NL4.3 . After 6 hours of transfection, the virus was washed twice to remove the virus, and then treated with no concentration of Ni ²⁺ after 24 hours of transfection. One or two days after treatment, the supernatant was used to infect TZMbl reporter cells to obtain the release level of the virus (background RLU was subtracted from each measurement) (Fig. 9-2, Fig. 5b). HIV-infected CD4 ⁺ T reservoir cells were lysed to quantify luciferase activity, and all RNA was extracted for qPCR to measure TRABD2A RNA and Gag RNA levels, normalized to glyceraldehyde phosphate dehydrogenase (Figure 9-3, Fig. 5c-e).

Similar to Ni ²⁺ , Co ²⁺ can also limit TRABD2A activity and enhance the ability of HIV-infected CD4 ⁺ T reservoir cells to release virus. At the same time, in addition to the highest concentration of 200 μM, Co ²⁺ did not affect the transcription levels of TRABD2A and viral Gag. The specific test procedure was as follows: HIV-infected CD4 ⁺ T reservoir cells were injected by electroporation with a luciferase expression vector and transfected with HIV-1 _NL4.3 . After 6 hours of transfection, the virus was washed twice to remove the virus and treated with various concentrations of Co ²⁺ after 24 hours of infection. One or two days after the treatment, the supernatant was used to infect TZMbl reporter cells to obtain the release level of the virus (the background RLU was subtracted from each measurement data). HIV-infected CD4 ⁺ T reservoir cells were lysed to quantify luciferase activity, and all RNA was extracted for qPCR to measure TRABD2A RNA and viral Gag RNA, normalized with glyceraldehyde phosphate dehydrogenase. (Figure 9-4, Extended Data Fig. 15a–d)

When treated with 1,10-phenanthroline, there are similar results as the above Co ²⁺ . The specific test procedure was as follows: HIV-infected CD4 ⁺ T reservoir cells were injected by electroporation with a luciferase expression vector and transfected with HIV-1 _NL4.3 . After 6 hours of transfection, the virus was washed twice to remove the virus and treated with various concentrations of 1,10-phenanthroline after 24 hours of infection. One or two days after the treatment, the supernatant was used to infect TZMbl reporter cells to obtain the release level of the virus (the background RLU was subtracted from each measurement data). HIV-infected CD4 ⁺ T reservoir cells were lysed to quantify luciferase activity, and all RNA was extracted for qPCR to measure TRABD2A RNA and viral Gag RNA, normalized with glyceraldehyde phosphate dehydrogenase. (Figure 9-5, Extended Data Fig. 15e–h)

The level of surface CD69 labeling is detected to rule out the possibility that the virus is released due to activation. No cells were found to be activated by 1,10-phenanthroline, Ni ²⁺ or Co ²⁺ . The specific test procedure was as follows: unstained HIV-infected CD4 ⁺ T reservoir cells and activated CD4 ⁺ T cells were used as a control group. In the present embodiment of HIV FIG Example 9 9-2 treated infected CD4 ⁺ T cell library stored in HIV and Example 9 of the present embodiment FIG. 9-4 treated infected CD4 ⁺ T cells were respectively repository 1 , 10- phenanthroline, Co ²⁺ , or Ni ²⁺ treatment, and then the surface marker CD69 expression level was detected by flow cytometry. (Figure 9-6, Extended Data Fig. 16a–e)

Example 10

Inhibition of human TRABD2A protein activity by metal ions, metal chelators, and related monoclonal antibodies can cause HIV-infected CD4 ⁺ T reservoir cells to release HIV-2 virus and be detected.

VSV-G and HIV-2 _{ST were} combined to infect HIV-infected depot cells to increase HIV-2 _ST cell membrane fusion. It was observed that in the presence of Co ²⁺ or Ni ²⁺ , the HIV-2 virus was released from the HIV-infected reservoir cells into the culture supernatant. The specific test procedure is as follows: HIV-infected depot cells were transfected with HIV-2 _ST (VSV-G). After 6 hours of transfection, the cells were washed twice to remove the injected virus and treated with different concentrations of Co ²⁺ and Ni ²⁺ . After 48 hours of transfection, the supernatant was used to infect TZMbl reporter cells to obtain the release level of the virus (the background RLU was subtracted from each measurement data) (Fig. 10-1, original Extended Data Fig. 20b). All RNA was extracted from HIV-infected depot cells for qPCR to measure TRABD2A RNA, normalized using glyceraldehyde phosphate dehydrogenase. (Figure 10-2, original Extended Data Fig. 20c)

Example 11

Removal of the human TRABD2A protein by siRNA can cause HIV-infected depot cells to infect and release the HIV-2 virus and be detected.

After treatment with small interfering RNA for TRABD2A, HIV-2 virus is released from HIV-infected reservoir cells. The specific test procedure was as follows: On day 0, siRNA against TRABD2A and control control siRNA (siCtrl) were separately injected into HIV-infected reservoir cells by electrical breakdown. On day 1, cells were transfected with HIV-2 _ST (VSV-G) by centrifugation. After 6 hours of transfection, two washes were performed to remove the injected virus. 48 hours after transfection, the medium was used to infect TZMbl reporter cells to obtain the release level of the virus (background RLU was subtracted from each measurement data) (Fig. 11-1, original Extended Data Fig. 20e). All RNA was extracted from HIV-infected depot pool cells for qPCR to measure TRABD2A RNA, normalized using glyceraldehyde phosphate dehydrogenase. (Fig. 11-2, original Extended Data Fig.20f)

Example 12

Removal of human TRABD2A protein by siRNA can cause infection of the reservoir cells in the patient, release of HIV-1 virus, and detection.

Two RNA interference (RNAi) strategies were used to eliminate the TRABD2A protein in the reservoir cells. First, siRNA against TRABD2A was electroporated into reservoir cells and subjected to _{spinoculation} 24 hours later using HIV-1 _NL4.3 . TRABD2A was found to be cleared, and after 48 hours, the HIV-1 virus was released in a large amount into the culture supernatant. At the same time, HIV-1 reverse transcription analysis showed that with the clearance of TRABD2A, the reservoir cells produced a large number of viruses, and the viral Gag transcription level did not change. The specific test procedure was as follows: The depot cells were isolated from three healthy donors, and the siRNAs against TRABD2A and the control control siRNA were separately injected into the reservoir cells. After 24 hours, the cells were transfected with HIV-1 _NL4.3 . After 6 hours, wash twice to remove the incoming virus. One or two days after transfection, the supernatant was used to infect TZMbl reporter cells to obtain the release level of the virus (the background RLU was subtracted from each measurement) (Fig. 12-1, original Fig. 5f) and Detection by reverse transcription activity assay (Fig. 12-2, original Fig. 5g). All RNA was extracted for qPCR to quantify HIV-1 Gag (Fig. 12-3, original Fig. 5h) and TRABD2A RNA (Fig. 12-4, original Fig. 5i), normalized using glyceraldehyde phosphate dehydrogenase.

Second, prior to HIV-1 transfection, siRNA was delivered to HIV-infected depot cells by tandem. After clearance of TRABD2A, HIV-1 virus is released in large quantities from HIV-infected reservoir cells, and viral transcription levels remain unchanged. The specific test procedure was as follows: siRNA against TRABD2A and control control siRNA were separately fed into HIV-infected reservoir cells by tandem method, and the cells were transfected with HIV-1 _{NL4.3 by} centrifugation. After 6 hours of transfection, wash twice to eliminate the input virus. 48 hours after transfection, the supernatant was used to infect TZMbl reporter cells to obtain the release level of the virus (the background RLU was subtracted from each measurement data) (Figure 12-5, former Extended Data Fig. 17b). HIV-infected depot cells were extracted for qPCR to measure TRABD2A RNA (Figure 12-6, former Extended Data Fig. 17c) and Gag RNA (Figure 12-7, former Extended Data Fig. 17d), dehydrogenation using glyceraldehyde phosphate The enzyme is normalized.

Example 13

The use of shRNA to clear human TRABD2A protein can cause HIV-infected depot cells to infect and release HIV-1 virus and be detected.

The level of expression of TRABD2A and the sensitivity to HIV-1 infection after activation of HIV-infected depot cells were examined to determine if activation is reversible. The primate HIV-infected CD4 ⁺ T reservoir cells are first activated and the IL-2 concentration is gradually reduced to return the CD4 ⁺ T cells to a resting state. The expression level of TRABD2A was significantly reduced in the 1-3 days of activation and began to recover on the 5th day after the CD3/CD28 activator was removed, and reached a considerable extent in Article 15. The specific test procedure is as follows: The test procedure is: (1) CD3 CD28 and IL2 (50 U/mL) are used to activate CD4 ⁺ T cells on day 0; (2) Beads are removed on day 3, and IL2 content is decreased to 25 U/ (3) IL2 content decreased to 15 U/mL on day 5; (4) IL2 content decreased to 7.5 U/mL on day 7; (5) IL2 content decreased to 3.75 U/mL on day 9; (6) The IL2 content decreased to 1.5 U/Ml on the 11th day; (7) the IL2 content decreased to 0.75 U/mL on the 13th day; (8) Analysis was performed on the 15th day. The RNA level of TRABD2A after activation of HIV-infected depot cells was detected and normalized by glyceraldehyde phosphate dehydrogenase (Fig. 13-1, former Extended Data Fig. 18b)

The primate CD4 ⁺ T cells were treated using the above strategy to channel lentiviral shRNAs directed against TRABD2A into the cell. The degree of activation of these CD4 ⁺ T cells transduced by shRNAs is determined by the level of CD69 surface marker expression, indicating that these cells are indeed at rest. In this process, the above cells were simultaneously transfected with HIV-1 _NL4.3 , and it was found that HIV-infected CD4 ⁺ T reservoir cells cleared by TRABD2A were able to release HIV-1 virus, and there was no change in Gag transcription level. The specific test procedure is as follows: The test procedure is: (1) CD3CD28 and IL2 (50 U/mL) are used to activate CD4 ⁺ T cells on day 0; (2) shRNAs are transferred into cells on day 3, and magnetic beads are removed, IL2 content The decrease was 25 U/mL; (3) the IL2 content decreased to 15 U/mL on the 5th day; (4) the IL2 content decreased to 7.5 U/mL on the 7th day; (5) the IL2 content decreased to 3.75 U/mL on the 9th day; (6) The IL2 content decreased to 1.5 U/Ml on the 11th day; (7) The IL2 content decreased to 0.75 U/mL on the 13th day; (8) The HIV-1 _NL4.3 transfection was added on the 14th day; (9) Analysis was performed for 16 days. The CD69 surface marker was detected using a flow cytometer (Fig. 13-2, original Extended Data Fig. 18d). Transfection was performed on day 14, and the virus was removed by washing twice at 6 hours after transfection. After 48 hours of transfection, the supernatant was used to infect TZMbl reporter cells to obtain the release level of the virus (background RLU was subtracted from each measurement data) (Fig. 13-3, original Extended Data Fig. 18e). All RNA was extracted for qPCR to measure Gag RNA (Fig. 13-4, original Extended Data Fig. 18f) and TRABD2A RNA (Fig. 13-5, original Extended Data Fig. 18g)

Example 14

Human, primate, and mammalian TRABD2A proteins and 2B are highly effective in inhibiting the infection of HIV-1, HIV-2, SIV, and MLV.

Proteins were expressed in 293T cells and tested for virus infectivity (Infectivity). In addition to the TRABD2A protein of Zebrafish, the infectivity of HIV-1, -2 and SIV is TRABD2A protein and TRABD2B by human, chimpanzee, macaque, mouse and mouse. Highly efficient protein inhibition.

The specific test procedure was as follows: HIV-1 virus pNL4.3, HIV-2 virus HIV-2 _ST and SIV _mac239 and TRABD2A protein and TRABD2B protein expression plasmid (from Shanghai Heyuan Biotech Co., Ltd.) were introduced into HEK293T cells, respectively. The virus supernatant was recovered after 48 hours, and the supernatant was used to infect TZMbl reporter cells to obtain the infection level of the virus, as shown in Figure 14-1.

A similar set of specific test procedures is as follows: HIV-1, HIV-2, SIV plasmids and TRABD2A, 2B plasmids (from Shanghai Heyuan Biotech Co., Ltd.) were introduced into 293T cells, and the virus supernatant was recovered 48 hours later. The supernatant was used to infect TZMbl reporter cells to obtain the level of infection of the virus, as shown in Figure 14-2.

Example 15

Inhibition or clearance of TRABD2A protein for Push & Kill treatment of HIV-infected patients.

Monoclonal antibodies to human TRABD2A were used to inhibit the activity of TRABD2A protein on the surface of the reservoir cells in patients. After that, the reservoir cells in the patient's body can release the HIV virus antigen, which can be specifically recognized by the CD8 cells in the patient and killed.

The specific test procedure is as follows: a monoclonal antibody of human TRABD2A in an amount of 0.5 mg/kg is intravenously injected into a blood vessel for long-term ART treatment. 2-4 injections per week, for 1-2 months, after blood test to detect the cell content of the reservoir cells in the patient. In this way, whether to continue to inject or increase the amount of monoclonal antibody.

Example 16

Isolated peripheral blood cells (PBMCs) from HIV-1 infected patients who have been taking drugs for more than two years and have no detectable viral load (Viral Load<20 copies/mL). After the PBMCs were cultured in vitro and treated with the monoclonal blocking antibody of TRABD2A for 72 hours, it was detected that the HIV storage cells began to be cleared.

The specific steps are as follows: 50-100 mL of the patient's blood is taken, 1-2 mL of the separated plasma is taken, and the viral load in the plasma is measured, and the remaining blood is extracted with Percoll peripheral blood mononuclear cells (PBMC). PBMCs were cultured at 3X 106/mL in RPMI 1640 medium containing 10% FBS serum. TRABD2A and control IgG antibodies were added and incubated for 3-5 days without the addition of any stimulating factors. Then, all CD4 ⁺ T cells in PBMCs were recovered, and the recovered CD4 ⁺ T cells were activated with CD3CD28 and IL2 for 24 hours, and the amount of released HIV-1 virus was detected, as shown in FIG.

Since the human TRABD2A protein in the HIV depot cells has an inhibitory effect on the release of HIV-1 virus, it has specific inhibition to HIV-1 and is involved in the specific degradation of the Gag precursor. Inhibition of TRABD2A activity can trigger viral presentation on the surface of the reservoir cells, causing the reservoir cells to be recognized and cleared by CD8 ⁺ T cells.

Example 17

Isolated peripheral blood cells (PBMCs) from HIV-1 infected patients who have been taking drugs for more than two years and have no detectable viral load (Viral Load<20 copies/mL). PBMCs from which such CD8 ⁺ T immune cells were removed and not removed were cultured in vitro and treated with the monoclonal blocking antibody of TRABD2A for 72 hours, and it was detected that the TRADAD blocking antibody elicited immunoreactive CD8 ⁺ T cells to clear HIV-1 storage cells.

The specific steps are as follows: 50-100 mL of the patient's blood is taken, 1-2 mL of the separated plasma is taken, and the viral load in the plasma is measured, and the remaining blood is extracted with Percoll peripheral blood mononuclear cells (PBMC), and the PBMCs are equally divided. Two copies, one of which was followed by CD8 ⁺ T cells. The cells were cultured at 3×10 6 /mL in RPMI1640 medium containing 10% FBS serum. TRABD2A and control IgG antibodies were added and incubated for 3-5 days without the addition of any stimulating factors. Then, all CD4 ⁺ T cells in PBMCs were recovered, and the recovered CD4 ⁺ T cells were activated with CD3CD28 and IL2 for 24 hours, and the amount of released HIV-1 virus was detected. After the CD8 ⁺ T cells are removed, the reservoir cells cannot be removed. The specific test results are shown in Figure 16.

Example 18

Isolated peripheral blood cells (PBMCs) were cultured in vitro from HIV-1 infected patients who had been taking drugs for more than two years and were not detected in plasma (Viral Load<20 copies/mL), and used TRABD2A and PD. After 72 hours of blocking antibody treatment, these two types of antibodies were detected to elicit immunological CD8 ⁺ T cells to significantly kill HIV-1 pool cells.

The specific steps are as follows: 50-100 mL of the patient's blood is taken, 1-2 mL of the separated plasma is taken, and the viral load in the plasma is measured, and the remaining blood is extracted with Percoll peripheral blood mononuclear cells (PBMC). The PBMC is 3X. 106/mL was cultured in RPMI1640 medium containing 10% FBS serum. TRABD2A, PD-1 or both were combined with antibodies against IgG and cultured for 3-5 days without the addition of any stimulating factors. Then, all CD4 ⁺ T cells in PBMCs were recovered, and the recovered CD4 ⁺ T cells were activated with CD3CD28 and IL2 for 24 hours, and the amount of released HIV-1 virus was detected, as shown in FIG.

Example 19

Resting CD4 ⁺ T and resting CD8 ⁺ T cells isolated from HIV-1 infected patients who have been taking drugs for more than two years and are not detected in plasma (Viral Load<20copies/mL) In vitro culture. CD4 ⁺ T cells were treated with the blocking antibody TRABD2A 72 h, CD4 ⁺ T cells with a Gag polypeptide and the activated CD8 ⁺ T cell populations were cultured for 5 days, and the survival rate of HIV-1 detection cell repository.

The specific steps are as follows: 50-100 mL of the patient's blood is taken, 1-2 mL of the separated plasma is taken, and the viral load in the plasma is measured, and the remaining blood is extracted with Percoll peripheral blood mononuclear cells (PBMC) and purified from PBMC. For CD4 ⁺ and CD8 ⁺ T cells, CD4 ⁺ T cells were cultured in RPMI1640 medium containing 10% FBS serum at 3.5×10 6 /mL, and blocking antibody of TRABD2A and control IgG were added. At the same time, CD8 ⁺ T cells were cultured in RPMI1640 medium containing 10% FBS serum at 3.5X 106/mL, and a Gag polypeptide group and IL2 were added. After culturing CD4 ⁺ and CD8 ⁺ T cells for 3 days, the two were combined, and after 5 days of continuous culture, the recovered CD4 ⁺ T cells were activated with CD3CD28 and IL2 for 24 hours, and the amount of released HIV-1 virus was detected. See Figure 19.

Example 20

Resting CD4 ⁺ T and resting CD8 ⁺ T cells isolated from HIV-1 infected patients who have been taking drugs for more than two years and are not detected in plasma (Viral Load<20copies/mL) In vitro culture. CD4 ⁺ T cells treated with the blocking antibody TRABD2A 72 h, CD4 ⁺ T cells and activated with a Gag polypeptide group and treated with PD-1 blocking antibody CD8 ⁺ T cells co-cultured for 5 days, and the check memory HIV-1 The survival rate of the library cells.

The specific steps are as follows: 50-100 mL of the patient's blood is taken, 1-2 mL of the separated plasma is taken, and the viral load in the plasma is measured, and the remaining blood is extracted with Percoll peripheral blood mononuclear cells (PBMC) and purified from PBMC. For CD4 ⁺ and CD8 ⁺ T cells, CD4 ⁺ T cells were cultured in RPMI1640 medium containing 10% FBS serum at 3.5×10 6 /mL, and blocking antibody of TRABD2A and control IgG were added. Meanwhile, CD8 ⁺ T cells were cultured in RPMI1640 medium containing 10% FBS serum at 3.5×10 6 /mL, and stimulated with Gag polypeptide group and IL2, and treated with PD-1 blocking antibody and control IgG antibody. After culturing CD4 ⁺ and CD8 ⁺ T cells for 3 days, the two were combined, and after 5 days of continuous culture, the recovered CD4 ⁺ T cells were activated with CD3CD28 and IL2 for 24 hours, and the amount of released HIV-1 virus was detected. See Figure 19.

Example 21

From the long-term medication for more than two years, and the viral load (Viral Load<20copies/mL) undetectable in the plasma of HIV-1 infected patients, the CNS system was acquired by accidental trauma, and the isolated Microglia cells and resting CD8+ in the blood T cells were cultured in vitro. After 96 hours of treatment with TRABD2A blocking antibody and Gag polypeptide, the survival rate of Microglia cells capable of releasing HIV-1 was examined, as shown in FIG.

Although the invention is disclosed above in the foregoing embodiments, it is not intended to limit the invention. Those skilled in the art can make some modifications and refinements without departing from the spirit and scope of the invention. Therefore, the scope of the invention is defined by the claims.

Claims (36)

1. Use of a substance for the preparation of an agent and/or a medicament for detecting or eliminating HIV-infected cells, characterized in that the substance is capable of inhibiting human TRABD protein activity or scavenging human TRABD protein.
2. The use according to claim 1, characterized in that the human TRABD protein is TRABD2A.
3. The use according to claim 2, wherein the human TRABD2A protein is TRABD2A-201 protein, and the amino acid sequence of the TRABD2A-201 protein is shown in SEQ ID NO.
4. The use according to claim 2, wherein the human TRABD2A protein is TRABD2A-202 protein, and the amino acid sequence of the TRABD2A-202 protein is shown in SEQ ID NO.
5. The use according to any one of claims 1 to 4, characterized in that the substance is a small molecule compound, a metalloproteinase inhibitor, or a human TRABD protein-specific antibody.
6. The use according to claim 5, characterized in that the small molecule compound is capable of competing with Mn ²⁺ for binding to a human TRABD protein.
7. The use according to claim 6, wherein the small molecule compound is a metal ion compound not containing Mn ²⁺ .
8. The use according to claim 7, wherein the metal ion compound is a metal ion compound containing Ni ²⁺ , Co ²⁺ or Zn ²⁺ .
9. The use according to claim 5, characterized in that the metalloproteinase inhibitor is a divalent metal chelating agent.
10. The use according to claim 9, wherein the divalent metal chelating agent is 1,10-phenanthroline.
11. The use according to any one of claims 1 to 10, characterized in that the substance removes human TRABD protein on the cell surface by an RNAi method.
12. The use according to claim 11, characterized in that the substance is siRNA or shRNA.
13. The use according to claim 12, characterized in that the siRNA transfects the cells.
14. The use according to claim 13, characterized in that the siRNA is transfected into the cells by electroporation or tandem.
15. The use according to claim 12, characterized in that the shRNA transfects the cells.
16. Use of a carrier for the preparation of a reagent and/or a medicament for the detection or removal of HIV-infected cells, characterized in that the carrier comprises the use according to any one of claims 1 to 15 for the preparation of a test or A reagent and/or drug substance that removes HIV-infected cells.
17. A method for detecting or eliminating HIV-infected cells, the method comprising: inhibiting human TRABD protein activity in the cell or clearing a human TRABD protein in the cell, detecting the HIV virus released by the cell to detect or clear the Said cells.
18. The method of claim 17 wherein said cells are treated with any of the agents prepared according to any one of claims 1-15.
19. A method according to claim 17 or 18, wherein said cells are HIV reservoir cells.
20. The method according to claim 17 or 18, wherein the cells are HIV-infected CD4 ⁺ T cells.
21. The method according to claim 17 or 18, wherein the cells are HIV-infected peripheral blood cells.
22. The method of claim 17 or 18, wherein said cells are HIV-infected resting CD4 ⁺ T cells.
23. The method according to claim 17 or 18, wherein the cells are HIV-infected NK cells.
24. The method according to claim 17 or 18, wherein the cells are HIV-infected brain cells.
25. Use of a substance for the preparation of an agent for increasing the ability of a cell to resist HIV and/or SIV infection or for reducing the release of HIV and/or SIV progeny virus by an infected cell, characterized in that the substance is an expression plasmid for a TRABD protein.
26. The use according to claim 25, wherein the expression plasmid of the TRABD protein is an expression plasmid of the TRABD2A protein.
27. The use according to claim 26, characterized in that the expression plasmid for the TRABD2A protein is an expression plasmid for the TRABD2A protein of human, primate or mammalian animals.
28. The use according to claim 25, characterized in that the expression plasmid of the TRABD protein is an expression plasmid of the TRABD2B protein.
29. The use according to claim 28, characterized in that the expression plasmid for the TRABD2B protein is an expression plasmid for the TRABD2B protein of human, primate or mammalian animals.
30. Use according to any of claims 25-29, characterized in that the cells are cells which have been infected with HIV and/or SIV.
31. The use according to claim 30, characterized in that the cells are CD4 ⁺ T cells which have been infected with HIV and/or SIV.
32. Use of a vector for the preparation of an agent for increasing the ability of a cell to resist HIV and/or SIV infection or for reducing the release of HIV and/or SIV progeny virus by an infected cell, characterized in that the vector comprises any one of claims 25-31 A substance used in the preparation for the preparation of the reagent.
33. A method of screening for a substance capable of scavenging or detecting cells infected with HIV and/or SIV, the method comprising screening for a substance capable of inhibiting TRABD protein activity or clearing TRABD protein.
34. 30. The method of claim 33, wherein the substance is capable of inhibiting TRABD protein activity in humans, primates or mammals, or scavenging human, primate or mammalian TRABD proteins.
35. 30. The method of claim 33 or 34, comprising performing a cell assay and/or an animal assay to screen for a substance capable of inhibiting TRABD protein activity or clearing TRABD protein.
36. The method of claim 35, wherein said cellular experiments and/or animal experiments are performed using macaques.

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