Use of amphotericin B in the manufacture of a medicament for the prevention and / or treatment of an ASFV infection

Abstract

The application discloses use of Amphotericin B in preparation of a medicine for preventing and / or treating ASFV infection, and belongs to the technical field of biotechnology. During the research, it is accidentally found that adding the compound Amphotericin B into a culture medium for culturing ASFV can reduce the replication level of the ASFV, indicating that the compound Amphotericin B has an effect of inhibiting the replication of the ASFV, and can be used for preparing a medicine or an adjuvant against the ASFV infection, and can be applied to inhibiting the replication of the ASFV, and has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the use of Amphotericin B in the preparation of medicaments for the prevention and / or treatment of ASFV infection. Background Technology

[0002] African swine fever (ASF) is an acute and highly contagious disease of wild and domestic pigs caused by the African swine fever virus (ASFV). Infection with ASF primarily manifests as high fever, loss of appetite, cyanosis, systemic organ failure, and hemorrhage. It is highly contagious and pathogenic, with a mortality rate that can reach 100%. Furthermore, it is widely distributed and frequently breaks out, posing a serious threat to the global pig industry and public health security. Currently, there is no commercially available ASF vaccine, and its pathogenicity and immune mechanisms are not fully understood. Therefore, a safe and effective prevention and control strategy is urgently needed.

[0003] Different vaccines have been tested against ASFV, mainly including inactivated vaccines, live attenuated vaccines, subunit vaccines, and DNA vaccines. Inactivated vaccines, however, cannot stimulate cellular immunity and therefore cannot induce effective neutralizing antibodies for protection. Compared to inactivated vaccines, traditional live attenuated vaccines can stimulate both cellular and humoral immune responses, but their cross-immunity is poor. Strict dosage control is also crucial during vaccination. Live attenuated vaccines may cause side effects such as pneumonia, joint swelling, and skin ulceration, and even death in a few animals. While subunit vaccines have high safety profiles, they face difficulties in identifying antigens that provide good immunogenicity and protective efficacy to the host. Furthermore, the large ASFV genome means that vaccines prepared from a single protein may not produce sufficient antibodies, and altered antigen genes can prevent antigen-antibody binding, thus failing to protect against virulent strains. DNA vaccines can induce neutralizing antibodies or T-cell-specific antiviral immune responses. In summary, gene-deleted vaccines address the issue of immunogenicity but pose biosafety risks. While inactivated vaccines, live vector vaccines, and subunit vaccines have good safety profiles, the core issue of "immune non-protection" remains unresolved. Given the current lack of effective commercial vaccines, the development of anti-ASFV drugs is a viable alternative for controlling ASF outbreaks and spread. However, research on chemical methods to inhibit ASFV replication has not yet been reported. Summary of the Invention

[0004] In view of the above-mentioned deficiencies in the prior art, the present invention provides the use of Amphotericin B in the preparation of medicaments for the prevention and / or treatment of ASFV infection.

[0005] To achieve the above objectives, the technical solution adopted by the present invention to solve its technical problem is as follows: The purpose of this invention is to provide the use of Amphotericin B or its derivatives, isomers or pharmaceutically acceptable salts thereof in the preparation of medicaments for the prevention and / or treatment of ASFV infection, the chemical structure of which is as follows: .

[0006] Another object of the present invention is to provide a medicament for the prevention and / or treatment of ASFV infection, wherein the medicament has Amphotericin B or its derivatives, isomers or pharmaceutically acceptable salts thereof as active ingredients.

[0007] Furthermore, the drug also includes Amphotericin B or its derivatives, isomers or pharmaceutically acceptable salts thereof as pharmaceutically acceptable excipients.

[0008] Furthermore, the drug dosage form is tablet, pill, capsule, powder, granule, drop, or suspension.

[0009] Another object of the present invention is to provide a culture medium for inhibiting ASFV virus activity, comprising a basal culture medium and Amphotericin B.

[0010] Furthermore, the basal medium was 1640 medium with 10% FBS.

[0011] Another object of the present invention is to provide the use of the above-mentioned Amphotericin B or its derivatives, isomers or pharmaceutically acceptable salts thereof in the preparation of ASFV viral vaccine adjuvants. The beneficial effects of this invention are: The present invention found that adding Amphotericin B to the culture medium for ASFV can inhibit ASFV replication, indicating that Amphotericin B has the effect of inhibiting ASFV replication and can be used to prepare antiviral drugs for inhibiting the replication of African swine fever virus. Attached Figure Description

[0012] Figure 1 The figure shows the protein levels of ASFV structural proteins P54 and P72 after treating Pam cells with different concentrations of Amphotericin B. Figure 2 The figure shows the mRNA levels of ASFV structural proteins p30 and p72 after treating Pam cells with different concentrations of Amphotericin B. Figure 3After treating Pam cells with different concentrations of Amphotericin B, TCID 50 Measurement results graph; Figure 4 The figure shows the cytotoxicity assay results after treating Pam cells with different concentrations of Amphotericin B. Detailed Implementation

[0013] The specific embodiments of the present invention are described below to enable those skilled in the art to understand the present invention. However, it should be understood that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, various changes are obvious as long as they are within the spirit and scope of the present invention as defined and determined by the appended claims. All inventions utilizing the concept of the present invention are protected.

[0014] The reagents and virus strains used in this invention are from the following sources: Amphotericin B was purchased from MCE.

[0015] The African swine fever virus strain is the genotype II African swine fever virus strain ASFV CN / GS / 2018 isolate (which has been disclosed in the invention patent application number 202510038950.1), isolated by our team and preserved in the Foot-and-Mouth Disease Epidemiology Team Laboratory of Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences.

[0016] Example 1: Compound Amphotericin B inhibits ASFV replication. 1. ASFV vaccination PAM cells were seeded in 24-well plates using 1640 medium containing 10% FBS. After 24 hours, the supernatant was discarded, and the medium was replaced with 1640 medium containing 2% FBS. ASFV was then inoculated and the plates were incubated at 37°C for 1.5 hours. 2. Preparation of culture medium containing Amphotericin B Amphotericin B was serially diluted using 1640 medium containing 10% FBS. After 1.5 h, the liquid in the 24-well plate was discarded, and 500 µL of the diluted compound-containing medium was added to each well to achieve final concentrations of 10 μm, 5 μm, 2.5 μm, 1.25 μm, 0.625 μm, and 0.3125 μm, with 7 replicates for each gradient. A DMSO control group and a MOCK group containing only cells without ASFV infection were also included.

[0017] 3. ASFV inhibition level measurement 24h samples were collected. (1) The protein levels of ASFV structural proteins P54 and P72 were detected by Western Blot in one well. (2) The mRNA levels of ASFV structural proteins p30 and p72 were detected by qPCR in three replicate wells. (3) Cells and supernatant were collected from three replicate wells, frozen and thawed three times at -80℃, and analyzed by TCID. 50 The virus titer was determined and analyzed.

[0018] The protein level detection results of ASFV structural proteins P54 and P72 are as follows: Figure 1 As shown, during PAM cell culture, the protein levels of P54 and P72 of ASFV decreased in a dose-dependent manner when different concentrations of Amphotericin B were added.

[0019] The mRNA level detection results of ASFV structural proteins p30 and p72 are as follows: Figure 2 As shown, during PAM cell culture, the mRNA level of ASFV P30 decreased in a dose-dependent manner when different concentrations of Amphotericin B were added.

[0020] TCID 50 The measurement results are as follows Figure 3 As shown, the TCID of ASFV changes when different concentrations of Amphotericin B are added. 50 It decreases, and in a dose-dependent manner.

[0021] The above results indicate that Amphotericin B inhibits the expression of African swine fever virus (ASFV) structural proteins P30, P54, and P72, reduces ASFV titer, and inhibits ASFV replication; moreover, the inhibitory effect on ASFV replication is stronger with increasing Amphotericin B content, showing a dose-dependent relationship.

[0022] Example 2: Effect of compound Amphotericin B on PAM cell activity PAM cells were seeded in 96-well plates at a cell suspension of 100 μL / well and incubated at 37°C for 24 h. The culture medium in the wells was discarded, and 1640 medium containing different concentration gradients of Amphotericin B was added, with three replicates for each gradient. After culturing for 24 h, the culture medium in the wells was discarded, and 100 μL of 1640 medium containing 10% CCK8 was added to each well. After incubation for 30 min, the OD value was measured using a microplate reader.

[0023] Cell viability test results as follows Figure 4As shown, when the concentration of Amphotericin B was 100 μM, the viability of PAM cells was 60%, and when the concentration of Amphotericin B was less than or equal to 10 μM, the viability of PAM cells was greater than 80%.

[0024] In summary, this invention, using PAM cells as an example, demonstrates that Amphotericin B can inhibit ASFV replication in PAM cells, indicating that the Amphotericin B described in this invention can inhibit ASF virus replication and can be used to prepare drugs or adjuvants against ASF virus infection; it can also be used to prepare antiviral drugs, showing broad application prospects.

[0025] Finally, it should be noted that the above specific embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to examples, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications and substitutions should be covered within the scope of the claims of the present invention.

Claims

1. Use of Amphotericin B or its derivatives, isomers or pharmaceutically acceptable salts thereof in the preparation of medicaments for the prevention and / or treatment of ASFV infection.

2. A drug for the prevention and / or treatment of ASFV infection, characterized in that, The drug uses Amphotericin B or its derivatives, isomers or pharmaceutically acceptable salts as its active ingredient.

3. The drug according to claim 2, characterized in that, The drug also includes pharmaceutically acceptable excipients such as Amphotericin B or its derivatives, isomers or pharmaceutically acceptable salts thereof.

4. The drug according to claim 2, characterized in that, The drug dosage form is tablet, pill, capsule, powder, granule, drop, or suspension.

5. A culture medium for inhibiting ASFV viral activity, characterized in that, This includes the basal culture medium and Amphotericin B.

6. The culture medium for inhibiting ASFV viral activity according to claim 5, characterized in that, The basal culture medium is 1640 medium containing 10% FBS.

7. Use of Amphotericin B or its derivatives, isomers or pharmaceutically acceptable salts thereof as described in claim 1 in the preparation of an adjuvant for an ASFV virus vaccine.

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