CN115844926A - Application of deacetylase inhibitor and NK cell in preparation of medicine for activating and eliminating latent HIV virus - Google Patents

Abstract

The invention discloses a method for activating dormant memory CD4 by using a deacetylase inhibitor as an HIV activator ⁺ T cells, inducing latent HIV replication; then killing activated HIV and clearing its storage by immune cells, thereby achieving the effect of clearing latent HIV cells and even fully inhibiting AIDS.

Description

Sirtuin inhibitors and NK cells in the production of activated and cleared latent HIV disease Application of toxic drugs

technical field

The invention relates to the technical field of biomedicine, and more specifically, relates to the application of sirtuin inhibitors and HK cells in the preparation of drugs for activating and eliminating latent HIV viruses.

Background technique

Combined antiretroviral therapy (ART), commonly known as cocktail therapy, can effectively control the progression of AIDS. The wide application of ART has prolonged the survival period of AIDS patients and made AIDS a chronic disease. But the patient must take medicine for life, otherwise HIV viremia will rebound within 3 to 4 weeks after stopping the medicine. This is because the virus reservoir in the body of AIDS patients is hidden in the lymphoid tissue, which escapes the killing of ART and the effective control of the body's immune system. Because HIV is latent in dormant memory CD4+ T cells, low levels of viral replication persist in lymphoid tissues, so that AIDS remains difficult to cure. Latent HIV hides from ART and immune cells, and long-lived quiescent memory CD4+ T cells are thought to be the major reservoir of proviruses. Latency Reversal Agents (LRAs) help interrupt HIV dormancy, thereby inducing HIV replication in cells, and then eliminate the cells with HIV replication through the cytopathic effect of the virus, immune clearance or ART. There are many LRAs in reports, including: (1) T cell activators: IL-2, anti-CD3 antibody, etc.; (2) Histone deacetylase inhibitors (HDACi): vordenoxet (SAHA), acetone, etc. Valeric acid (VPA); (3) protein kinase C activator: Prostatin, bryostatin (bryostatin); (4) BET inhibitor (JQ-1), etc. Hypoacetylation is one of the best characterized modifications by histone deacetylases (HDACs) to dissociate acetyl groups from lysine residues in nucleosomal histone tails. This results in condensed chromatin, less accessibility to transcription factors, and therefore gene silencing. HDACs are divided into two types and four types according to cofactor dependence and sequence homology. Classical HDAC types (types I, II, and IV) require zinc ions at the catalytic site to initiate sirtuin activity, whereas sirtuin-type HDACs (type II Class III) require nicotinamide adenine dinucleotide as a cofactor to promote their catalytic activity and share no sequence homology with classical zinc ion-dependent HDACs.

In the fight against latently infected HIV, scientists have been using the Shock and kill (activation-kill) method, but so far there has been no report of using this method to fully suppress AIDS. This method usually first activates the latent HIV in the cells so that it can be easily killed by antiviral drugs after exposure. The method includes two components, one part is the above-mentioned latent reversal agent (LRA), and the other part is the anti-HIV ART chemotherapy drug. Previous activation-killing methods have some effects in in vitro studies, but the effects in clinical studies are not significant.

Contents of the invention

The purpose of the present invention is to aim at the problem of HIV latent infection, to provide a more effective plan for activating and eliminating latent HIV virus and fully suppressing AIDS.

The technical scheme that the present invention takes is:

The first aspect of the present invention provides the application of the composition in the preparation of drugs for activating and eliminating latent HIV virus, wherein the combination includes sirtuin inhibitors and NK cells.

Preferably, the combination is a sirtuin inhibitor and NK cells.

Preferably, the sirtuin inhibitor is NAD+ precursor.

More preferably, the sirtuin inhibitor is nicotinamide.

Preferably, the NK cells are highly active NK cells (HANK cells).

The second aspect of the present invention provides the application of the composition in the preparation of drugs for treating latent AIDS, wherein the combination includes sirtuin inhibitors and NK cells.

Preferably, the combination is a sirtuin inhibitor and NK cells.

Preferably, the sirtuin inhibitor is NAD+ precursor.

More preferably, the sirtuin inhibitor is nicotinamide.

Preferably, the NK cells are highly active NK cells (HANK cells).

The third aspect of the present invention provides the application of sirtuin inhibitors in the preparation of NK cell treatment AIDS synergists.

Preferably, the AIDS is latent AIDS.

Preferably, the sirtuin inhibitor is NAD+ precursor.

More preferably, the sirtuin inhibitor is nicotinamide.

Preferably, the NK cells are highly active NK cells (HANK cells).

The fourth aspect of the present invention provides the use of sirtuin inhibitors in the preparation of potentiators for eliminating latent HIV viruses by NK cells.

Preferably, the sirtuin inhibitor is NAD+ precursor.

More preferably, the sirtuin inhibitor is nicotinamide.

Preferably, the NK cells are HANK cells.

The fifth aspect of the present invention provides a composition comprising sirtuin inhibitors and NK cells.

Preferably, the composition is a sirtuin inhibitor and NK cells.

Preferably, the sirtuin inhibitor is NAD+ precursor.

More preferably, the sirtuin inhibitor is nicotinamide.

Preferably, the NK cells are highly active NK cells (HANK cells).

The beneficial effects of the present invention are:

The invention discloses an HIV activator (HIV latency reversal agent, LRA) that uses sirtuins inhibitors to activate dormant memory CD4 ⁺ T cells in AIDS patients and induce latent HIV replication; once HIV replicates, The memory CD4 ⁺ T cells that serve as its reservoir will produce HIV-specific changes; then immune cells will be used to kill the activated HIV and clear its reservoir, thereby achieving the effect of clearing latent HIV cells and even fully suppressing AIDS. The sirtuins inhibitor of LRA is nicotinamide (NAM), and the immune cells that kill HIV and clear its storage are highly active NK (HANK) cells.

Compared with the ART chemotherapeutics used in the reported activation-killing regimens, the use of HANK cells in the present invention has greater advantages in killing HIV produced after activation and clearing the HIV reservoir. Because ART only has a single function of blocking HIV replication, and HANK cells not only have the function of blocking HIV replication through its secreted IFN-γ, but more importantly, they also have the function of clearing HIV storage.

Description of drawings

Figure 1 Schematic diagram of the activation-killing method for inhibiting HIV.

Fig. 2 The activation effect of NAM on latent HIV infection.

Figure 3 The effect of NAM on the expression level of MIC-A/B on T cells.

Figure 4 Effect of NAM on the growth of primary immune cells.

Figure 5 NAM reverses latent virus in CD4 ⁺ T cells and promotes HANK cells to clear HIV infection.

Detailed ways

In order to understand the technical content of the present invention more clearly, the following embodiments are given in detail in conjunction with the accompanying drawings. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention, wherein the schematic diagram of the activation-killing method for inhibiting HIV is shown in Figure 1 . The experimental method that does not indicate specific condition in the following examples, usually according to conventional conditions, such as Sambrook et al., molecular cloning: the conditions described in the laboratory manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer suggested conditions. Various commonly used chemical reagents used in the examples are all commercially available products.

Materials and methods

1. Materials: J1.1 cell line and primary cells were cultured with RPMI 1640 complete medium supplemented with 10% fetal bovine serum, 0.2mM L-glutamine, and 100 units/ml streptomycin. Several parts of peripheral blood were donated by healthy volunteers, and PBMCs were isolated with lymphocyte separation medium.

2. Isolation of CD4 ⁺ T cells. Using PBMCs as the starting material, the EasySep CD4 ⁺ T-cell Enrichment Kit (Stem Cell Technologies, Vancouver, Canada) was used according to the instructions of the kit.

3. Preparation of HANK cells: PBMCs were used as starting materials to expand and activate NK cells. In this embodiment, the NK cell in vitro culture kit of Shenzhen Hanke Bioengineering Co., Ltd. was used. NK cells were prepared according to the instructions, and IL-2 and membrane chimeric cytokines were added to the X-Vivo15 serum-free culture medium. 37 Cultivate in a 5% CO ₂ incubator for about 14 days; count the cultured NK cells with a hemocytometer, and there are 4.8×10 ⁹ NK cells in 1L of culture medium, and about 1L of culture supernatant.

4. Construction and activation of HIV latently infected CD4 ⁺ T cells. Add 29mM CCL19 (R&D Systems) to the CD4 ⁺ T cells in culture, and continue to culture for 2 days; inoculate the NL4-3 HIV-1 virus packaged with VSV-G according to the amount of 300ng p24/10 ⁶ cells, and wash after 2 hours; Latently infected CD4 ⁺ T (HIVCD4T) cells were generated after 3 days. 10mM NAM was added to activate HIV infection, and untreated control cells were set; cells were harvested at 24h, 48h, and 72h, and NKG2DL on the cell surface and HIV p24 protein in the cells were detected by immunofluorescence.

5. HANK cell anti-HIV test. CD4 ⁺ T cells treated with NAM 3 days after HIV infection were used as target cells (T); while NAM was added to treat HIV-infected CD4 ⁺ T cells, HANK cells labeled with eFluor450 were taken, with or without (CRT) NAN treatment , as effector cells (e ⁺ Enam or e ⁺ E). 48h after NAM treatment, transfer 2×10 ⁵ target cells (T) and 2×10 ⁵ HANK effector cells stimulated with NAM (e ⁺ E) to new culture wells, the ratio of E:T is 1:1; Gently mix, continue to culture for 18 hours, fix, infiltrate, stain with anti-p24-FITC, and analyze e ^- p24 ⁺ cells by flow cytometry; the percentage of inhibition of HIV-infected p24 ⁺ T cells mediated by HANK cells is according to the following formula Calculation: 100X【(e ^- p24 ⁺ -T－e ^- p24 ⁺ -T:E)+(e ^- T－e ^- T:E)】/e ^- p24 ⁺ -T. Where e ^- p24 ⁺ -T is activated HIV latent T cells; (e ^- p24 ⁺ -T－e ^- p24 ⁺ -T:E) is the effect of HANK cells on activated T cells; (e ^- T－e ^- T:E) is the T cells killed by HANK cells in the test group, and is also a part of the activated HIV latent T cells. In some experiments, HANK cells were blocked with anti-NKG2D antibody or control IgG1 for 15 min before co-culture with T cells.

6. NK cell degranulation test. Add anti-CD107a/FITC or isotype control IgG/FITC to NK:T co-culture wells and individual HANK cell culture wells at 48 hours; add Golgi stop (1:1500 final concentration) after reacting for 1 hour, and continue to culture cells for 17 hours. The expression of CD107a on eFluor450+ NK cells was detected by flow cytometry.

Example 1 The activation effect of NAM on latent HIV infection and its effect on the expression of NKG2DLs (MICA/B)

J1.1 is a T cell line latently infected with HIV. The inventors used J1.1 to simultaneously study the viral activation of NAM and its effect on the expression of NKGDL (MICA/B). Adding NAM or the culture solution control (CTR) without NAM to J1.1 cell culture, after 48 hours, the spontaneous p24+ cells in the CRT control group were 8%, and the NAM group promoted HIV activation, and the p24+ cells were as high as 60% (results See attached drawing 2). The basal MICA/B expression level of J1.1 cells is relatively low, and the MICA/B expression level of NAM-treated p24+J1.1 cells is significantly increased (results are shown in Figure 3), indicating that NAM has an activation effect on latent HIV infection effect.

Example 2 Effect of NAM on T cell and NK cell viability in PBMC

PBMC (peripheral blood mononuclear cells) from healthy donors were divided into two groups with PRMI 1640 complete culture medium + IL-2 (200IU/ml), in which NAM was added to group 1, and NAM was not added to the control group (CTR); 37 After 24, 48, and 72 hours of culture in a CO ₂ incubator at ℃, the number of CD4+T, CD8+T, and CD56+NK cells was detected (results are shown in Figure 4). Compared with the control group (CTR) without NAM, NAM not only has no toxicity to T cells and NK cells in PBMC, but also has a certain growth-promoting effect, and there is a certain amount of increase every day.

Example 3 NAM reverses latent virus in CD4+ T cells and promotes HANK cells to clear HIV infection

The inventors used NAM to treat CD4+ T cells latently infected by HIV for 48 hours, and at the same time treated HANK cells with NAM, then transferred the above-mentioned HANK cells to the culture of CD4 ⁺ T cells latently infected by HIV, and continued co-cultivation for 18 hours. The ratio of p24 ⁺ in T cells was measured by cytometer (see accompanying drawing 5 for statistical results), and the results showed that:

(1) J1.1T cells without NAM treatment + medium control (NAM ^- J1.1T + CTR): a p24 value of 12% represents the level of spontaneous HIV proliferation in J1.1T cells.

(2) NAM-treated J1.1T cells + medium control (NAM ⁺ J1.1T + CTR): p24 value of 64% indicated that NAM activated latent HIV proliferation in J1.1T cells.

(3) J1.1T cells activated without NAM + HANK cells without NAM treatment (NAM ^- J1.1T + NAM ^- HANK): p24 value of 6% represents the effect of HANK cells on spontaneous HIV proliferation in J1.1T cells Effect; it shows that HANK cells cleared 50% of the spontaneously proliferating HIV virus in J1.1T cells.

(4) NAM activated J1.1T cells + HANK cells without NAM treatment (NAM ⁺ J1.1T + NAM ^- HANK): p24 value of 35% means that HANK cells eliminated about half of the proliferating cells in J1.1T cells after activation HIV.

(5) NAM-activated J1.1T cells + NAM-treated HANK cells (NAM ⁺ J1.1T + NAM ⁺ HANK): p24 value of 13% means that NAM-treated HANK cells cleared about 80% of activated J1.1T cells proliferating HIV. This experiment is the closest to the actual situation in vivo; that is, when NAM is used to activate latently infected HIV in vivo combined with HANK cell therapy, HANK cells must face the pre-existing NAM.

This result shows that the use of NAM, which is not only non-toxic to immune cells, but also has an enhancing effect, not only activates latently infected HIV, but also enhances the function of immune cells including NK cells, and reverses latent infection and eliminates activation. The resulting HIV is organically linked to provide a safe and effective solution for eliminating latent HIV virus and treating AIDS.

The above-mentioned embodiments are only illustrative examples, and their descriptions are more specific and detailed, but should not be construed as limiting the patent scope of the present invention. It should be pointed out that for those skilled in the art, without departing from the concept of the present invention, several variations and improvements, modifications, substitutions, combinations, and simplifications can be made, all of which should be equivalent replacement methods. These all belong to the protection scope of the present invention.

Claims (10)

1. The application of the composition in the preparation of drugs for activating and eliminating latent HIV virus, wherein the composition comprises sirtuin inhibitors and NK cells. 2. The application of the composition in the preparation of medicaments for treating latent AIDS, wherein the composition comprises sirtuin inhibitors and NK cells. 3. The application of the sirtuin inhibitor in the preparation of HK cell therapy AIDS synergist. 4. The application of the sirtuin inhibitor in the preparation of HK cell elimination latent HIV virus potentiator. 5. The application according to any one of claims 1 to 4, characterized in that the sirtuin inhibitor is a NAD+ precursor, preferably nicotinamide. 6. The application according to any one of claims 1 to 4, characterized in that the NK cells are highly active NK cells. 7. A composition comprising a sirtuin inhibitor and NK cells. 8. The composition of claim 7, wherein the sirtuin inhibitor is a NAD+ precursor. 9. The composition according to claim 8, wherein the sirtuin inhibitor is nicotinamide. 10. The composition according to claim 7, wherein the NK cells are HANK cells.

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