Recombinant viral vector and use thereof

Abstract

Provided is a recombinant viral vector, which comprises a viral vector as a backbone vector. The viral vector comprises five elements of HIV-1-LTR, E1A, IRES, E1B, and DTA, and the HIV-1-LTR element is at the 5' end of the other four elements. The recombinant viral vector can be used to specifically and efficiently transfer DTA into a target cell, and when the target cell is not infected with HIV-1, DTA is not induced and produced, so that the recombinant viral vector does not produce cytotoxicity, and only has cytotoxicity towards cells specifically infected with HIV-1.

Description

A recombinant viral vector and its use

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application is based on the Chinese patent application with application number 202310933875.6 and application date of July 27, 2023, and claims the priority of the Chinese patent application. The entire content of the Chinese patent application is hereby incorporated into this application by introduction. Technical Field

[0003] The present invention belongs to the field of biotechnology, and in particular, relates to a recombinant viral vector and uses thereof, and more particularly, to a viral vector for inhibiting HIV-1. Background Art

[0004] Acquired Immunodeficiency Syndrome (AIDS), abbreviated as AIDS, is caused by infection with the human immunodeficiency virus (HIV).

[0005] To date, HIV-1 vaccine research has failed. An early and significant achievement in HIV-1 drug treatment is cocktail therapy (Highly Active Antiretroviral Therapy, or HAART). Continuous cocktail therapy maintains a low viral load in HIV-1-infected individuals, but it does not eliminate the virus. Patients require long-term medication, which can cause serious and complex side effects in HIV-1-infected individuals and AIDS patients. Long-term use of these drugs can also lead to the development of drug-resistant strains of HIV-1.

[0006] Therefore, in order to overcome the various disadvantages of long-term medication, technologies such as viral vectors as delivery systems have emerged, but they have the following disadvantages: 1. Delivery systems using the retrovirus Mo-MLV as a vector need to be integrated into the host cell genome, which carries the risk of causing gene mutations and activating oncogenes, and has low specificity; 2. Transduction systems using retrovirus as a vector have low infection efficiency for T cell lines; 3. After retrovirus transduction of cells, it still has the ability to replicate, and the resulting progeny viruses can infect non-target cells.

[0007] Therefore, there is a need in the art for a vector delivery system that has high transduction efficiency, selective replication, and can kill HIV-1 infected cells.

[0008] Summary of the Invention

[0009] In view of this, in a first aspect, the present invention provides a recombinant viral vector comprising:

[0010] The viral vector serves as a backbone vector, which includes five elements: HIV-1-LTR, E1A, IRES, E1B, and DTA, wherein the HIV-1-LTR element is at the 5' end of the other four elements.

[0011] The recombinant viral vector of the present invention can specifically and efficiently transduce DTA into target cells. At the same time, it does not induce the production of DTA when not infected with HIV-1, and therefore does not produce cytotoxicity, but only has cytotoxicity to cells specifically infected with HIV-1.

[0012] In some specific embodiments, the arrangement of the five elements on the backbone vector from the 5' end to the 3' end is: HIV-1-LTR, E1A, IRES, E1B, and DTA.

[0013] It should be noted that the elements of the present invention may have various nucleotide or amino acid sequence representations. Specifically, they may be sequences that are 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequences shown in SEQ ID NOs. 1-7 and are capable of performing their corresponding specific functions.

[0014] In some specific embodiments, HIV-1-LTR is the long terminal repeat sequence of HIV-1, which is responsible for regulating the expression of the HIV-1 genome and viral replication, and is triggered by the non-structural protein Tat of HIV-1. In a specific embodiment, its sequence is:

[0015] In some specific embodiments, E1A is an immediate early gene of adenovirus, which is the first gene expressed after adenovirus infection and is the most important transcriptional activator for the expression of other viral genes. In a specific embodiment, its sequence is:

[0016] In some specific embodiments, IRES is an internal ribosome entry site for connecting E1A and DTA. In a specific embodiment, its sequence is:

[0017] In some specific embodiments, E1B is another immediate early gene in the E1 region of adenovirus, which is related to viral replication, transcription of viral late mRNA, and transport of viral RNA. In a specific embodiment, its sequence is:

[0018] In some specific embodiments, DTA is a highly effective biotoxin that can inhibit protein synthesis in cells and cause cell apoptosis. In a specific embodiment, its sequence is:

[0019] In some specific embodiments, the viral vector can be any viral vector as long as it can transduce the target element into the target cell.

[0020] Preferably, the viral vector is an Ad5 / F35 adenoviral vector.

[0021] In a specific embodiment, the sequence of the Ad5 / F35 adenoviral vector is shown in SEQ ID NO.6:

[0022] In a specific embodiment, a recombinant adenoviral vector of the present invention is specifically shown as SEQ ID NO.7:

[0023] In a second aspect, the present invention provides a composition for treating HIV-1 infection, comprising the recombinant viral vector as described above, and a pharmaceutically acceptable excipient.

[0024] In a third aspect, the present invention provides a recombinant virus, including a recombinant virus prepared using the recombinant viral vector described above.

[0025] In some specific embodiments, the recombinant virus is a recombinant adenovirus.

[0026] In a fourth aspect, the recombinant viral vector or composition as described above is used to prepare a composition for treating HIV-1 infection.

[0027] In a fifth aspect, the present invention provides a kit for treating HIV-1 infection, comprising the composition described above.

[0028] In some specific embodiments, the kit further comprises other inhibitors that inhibit HIV-1, such as antibodies known in the art that inhibit HIV-1, and chemical drugs that act on various key proteins of HIV-1 (such as various enzyme inhibitors used in cocktail therapy).

[0029] In a sixth aspect, the present invention provides a method for treating HIV-1 infection, comprising: administering the above composition to an individual. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 is a schematic diagram of the gene structures of Tat-CRAds-DTA and Tat-CRAds-mCherry;

[0031] Figure 2 shows DTA expression in HEK293A cells infected with Tat-CRAds-DTA. HEK293A cells transfected with pTat or pcDNA3.1 were infected with Tat-CRAds-DTA at an MOI of 10, and DTA expression was detected by Western blotting 3 days later.

[0032] Figure 3 shows mCherry expression in HEK293A cells infected with Tat-CRAds-mCherry. HEK293A cells transfected with pTat or pcDNA3.1 were infected with Tat-CRAds-mCherry at an MOI of 10 and observed using phase contrast microscopy (lower panel) and fluorescence microscopy (upper panel). Scale bars represent 50 μm. TZM-bl cells were sequentially infected with HIV-1BaL (200 TCID50) and Tat-CRAds-DTA / mCherry at an MOI of 10, with a 4-hour interval.

[0033] FIG4 shows the cell viability measured by virus inhibition assay 3 days after infection;

[0034] FIG5 shows luciferase activity measured by viral inhibition assay 3 days after infection;

[0035] Figure 6 shows the inhibition rate calculated by the virus inhibition assay using GraphPad Prism 7 software 3 days after infection;

[0036] Figure 7 shows TZM-bl cells infected with VSV (200 TCID50) and Tat-CRAds-DTA / mCherry at an MOI of 10, with a 4-hour interval. Luciferase activity was measured 3 days after infection.

[0037] FIG8 shows that infected cells were harvested and the titer of progeny Tat-CRAds-DTA / mCherry was determined by plaque assay in HEK293A cells. DETAILED DESCRIPTION

[0038] The present invention will be described in detail below in conjunction with specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific embodiments and examples are for illustrating the present invention, rather than for limiting the present invention.

[0039] Example 1. Plasmids and cell lines used in the present invention

[0040] Plasmids pAdeasy-1, pShuttle, pDTA, and pTat were obtained from Addgene. The Ad5 / F35 adenoviral backbone plasmid vector was constructed by exchanging the fiber shaft and apical bulb regions of pAdeasy-1 with those of Ad35. The E1A and E1B gene sequences were amplified from total DNA extracted from HEK293A cells using specific primers and ligated into pcDNA3.1(+). Sequence-verified clones were designated pE1A and pE1B, respectively. All HIV-1-related plasmids and the TZM-bl cell line were obtained from the National Institutes of Health (NIH). HEK293T and HEK293A cell lines were purchased from the American Type Culture Collection (ATCC). Both cell lines were grown in DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin at 37°C in the presence of 5% CO2.

[0041] Example 2. Virus production

[0042] Recombinant adenovirus production

[0043] The shuttle plasmids pShuttle-DTA and pShuttle-mCherry were constructed by inserting the HIV-1-LTR, E1A, IRES, E1B, and DTA or mCherry fragments into pShuttle using a one-step cloning kit. Recombinant Ad5 / F35 adenoviral vectors carrying DTA or mCherry (Ad5 / F35-DTA or Ad5 / F35-mCherry) were generated in BJ5183 cells through homologous recombination between pShuttle-DTA or pShuttle-mCherry and the Ad5 / F35 adenoviral backbone plasmid vector. The specific construction scheme is shown in Figure 1. The recombinant adenoviral vectors Ad5 / F35-DTA or Ad5 / F35-mCherry were digested with Pac I endonuclease to expose their inverted terminal repeats (ITRs) and then used to transfect HEK293A cells, where the deleted viral assembly genes were complemented. Recombinant adenovirus (Tat-CRAds-DTA or Tat-CRAds-mCherry) was produced 10-16 days after transfection, subsequently expanded, purified by CsCl density gradient centrifugation, and stored according to standard protocols. The titer of infectious virus was determined by plaque assay in HEK293A cells.

[0044] HIV-1 production

[0045] HIV-1 laboratory-adapted isolate BaL was produced in HEK293T cells transfected with the corresponding full-length infectious HIV-1 clone in a plasmid. 48 hours after transfection, virus-containing media was harvested and filtered through a 0.45 μm filter, aliquoted, and stored in a -80°C freezer.

[0046] Example 3, SDS-PAGE and Western blotting

[0047] To validate Tat-CRAds-DTA, HEK293A cells were transfected with pTat or pcDNA3.1(+) for 4 hours and then infected with Tat-CRAds-DTA at an MOI of 10. Three days after infection, the cells were harvested, boiled with loading buffer for 10 minutes, and the samples were separated by 10% SDS-PAGE. Thereafter, the proteins were transferred to a polyvinylidene fluoride (PVDF) membrane (0.45 μm). The PVDF membrane was blocked with 5% skim milk and then incubated with an anti-DTA protein polyclonal antibody at room temperature for 1 hour. After washing three times with TBS-Tween wash solution, the membrane was incubated with a goat anti-rabbit antibody conjugated to horseradish peroxidase (HRP) for 1 hour at room temperature. Protein bands were observed after incubation with enhanced chemiluminescence (ECL).

[0048] After infecting HEK293A cells transfected with pTat with purified Tat-CRAds-DTA or Tat-CRAds-mCherry virus, the expression of DTA protein or mCherry was detected, confirming that DTA or mCherry could be successfully expressed under the control of HIV-1 protein Tat (as shown in Figures 2-3).

[0049] Example 4, MTT survival assay

[0050] TZM-bl cells were seeded in 96-well plates (5 × 10 3 cells / well). TZM-bl cells were then infected with HIV-1 (200 TCID50) and Tat-CRAdS-DTA or Tat-CRAdS-mCherry at an MOI of 10, with a 4-hour interval. After 3 days of culture, MTT solution was added to the culture medium to a final concentration of 0.5 mg / mL and incubated in a 37°C CO2 incubator for 4 hours. The supernatant was carefully removed, and DMSO (200 μL) was added and mixed. After incubation for 10 minutes, the absorbance at 570 nm (reference wavelength was 630 nm) was measured using a microplate absorbance reader. Cell viability was calculated using GraphPad Prism software.

[0051] According to survival assay analysis, TZM-bl cells co-infected with HIV-1 and Tat-CRAds-DTA showed significant cytotoxicity, while cells co-infected with HIV-1 and Tat-CRAds-mCherry or cells infected with HIV-1, Tat-CRAds-DTA, or Tat-CRAds-mCherry alone did not show significant cytotoxicity, verifying the HIV-1 infection-dependent cytotoxicity of Tat-CRAds-DTA (Figure 4). In addition, after treatment with Tat-CRAds-DTA or Tat-CRAds-mCherry, the luciferase activity of HIV-1-infected TZM-bl cells was significantly reduced, indicating that the recombinant adenovirus Tat-CRAds can inhibit HIV-1 replication (Figure 5). Further data analysis showed that Tat-CRAds-DTA had a significantly higher inhibitory effect on HIV-1 activation than Tat-CRAds-mCherry, indicating that the specifically expressed biotoxin DTA plays an important role (Figure 6).

[0052] Example 5: HIV-1 and VSV virus inhibition test

[0053] HIV-1 and VSV inhibition assays were performed in TZM-bl cells seeded in 96-well plates. Cells were infected with 200 TCID50 of HIV-1 or VSV and subsequently infected 4 hours later with Tat-CRAds-DTA or Tat-CRAds-mCherry at an MOI of 10. After 3 days of culture, cells were lysed with cell lysis buffer, and luciferase activity in the cell lysates was measured using an analog microplate luminometer. Inhibition percentages were calculated using GraphPad Prism software.

[0054] Using VSV as a control, there was no significant difference in luciferase activity in VSV-infected TZM-bl cells after treatment with Tat-CRAds-DTA or Tat-CRAds-mCherry (Figure 7), indicating that VSV cannot initiate recombinant adenoviral replication and DTA expression. We also performed experiments in which all infected cells were collected to measure the titer of progeny Tat-CRAds by plaque assay in HEK293A. We found that TZM-bl cells co-infected with HIV-1 and Tat-CRAds could produce progeny adenovirus (Figure 8). TZM-bl cells co-infected with VSV and Tat-CRAds did not produce progeny adenovirus.

Claims

1. A recombinant viral vector comprising: The viral vector serves as a backbone vector, which includes five elements, namely HIV-1-LTR, E1A, IRES, E1B, and DTA, wherein the HIV-1-LTR element is at the 5' end of the other four elements.

2. The recombinant viral vector according to claim 1, wherein The elements are arranged on the backbone vector in the order from the 5' end to the 3' end: HIV-1-LTR, E1A, IRES, E1B, and DTA.

3. The recombinant viral vector according to claim 2, wherein The nucleotide sequences of HIV-1-LTR, E1A, IRES, E1B, and DTA are shown in SEQ ID NOs. 1-5.

4. The recombinant viral vector according to claim 2, wherein The vector is an Ad5 / F35 adenovirus vector.

5. The recombinant viral vector according to claim 2, wherein The nucleotide sequence of the vector is shown in SEQ ID NO.

7.

6. A composition for treating HIV-1 infection, comprising the recombinant viral vector according to any one of claims 1 to 5, and a pharmaceutically acceptable excipient.

7. A recombinant virus, comprising a recombinant virus prepared using the recombinant virus vector according to any one of claims 1 to 5.

8. Use of the recombinant viral vector according to any one of claims 1 to 5 or the composition according to claim 6 for preparing a composition for treating HIV-1 infection.

9. A kit for treating HIV-1 infection, comprising the composition according to claim 8.

10. The kit according to claim 9, wherein The kit also includes other inhibitors that inhibit HIV-1 。

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