Medicine for treating chicken infectious bursal disease

Abstract

The invention discloses a medicine for treating chicken infectious bursal disease, and belongs to the technical field of veterinary biological products. The invention finds that the PD-1 signaling pathway inhibitor BMS-202 can inhibit the duplication of IBDV (infectious bursal disease virus) of different genotypes and relieve weight inhibition and bursal atrophy caused by IBDV for the first time, new chicken infectious bursal disease treatment drugs can be developed based on BMS-202, and the BMS-202 has important significance in prevention and treatment of IBDV.

Description

Technical Field

[0001] The present invention relates to the technical field of veterinary biological products, in particular to a medicine for treating infectious bursal disease in chickens. Background Art

[0002] Infectious bursal disease (IBD) in chickens is an acute, highly contagious, immunosuppressive disease caused by the infectious bursal disease virus (IBDV). It is widespread across all regions where poultry is raised, with higher incidence rates in areas with developed poultry industries. Classical IBDV strains first appeared in my country in the late 1970s. From the late 1980s to the early 1990s, very virulent IBDV (vvIBDV) rapidly became prevalent in my country, posing a serious threat to the poultry industry over the past 30 years. With the development of the poultry industry, the incidence of IBD has increased annually. Furthermore, the spread of the epidemic has even led to the emergence of novel variant IBDV (nvIBDV), with a wide range of serotypes, which complicates disease prevention and control. Existing vaccines are ineffective in protecting against nvIBDV infection, and its prevalence continues to expand, becoming one of the major IBDV strains threatening the poultry industry (Zou Weili et al., 2022). Although nvIBDV does not cause obvious clinical symptoms or mass mortality in chickens, it can cause rapid atrophy of the bursa of Fabricius, a central immune organ, leading to severe immunosuppression in infected chickens. Bo et al. found that the new variant strain significantly suppressed antibody titers in the Newcastle disease vaccine layer of broiler chickens, severely impacting their production performance and increasing their vulnerability to secondary infection with other pathogens (Bo et al., 2023). Furthermore, broiler chickens infected with nvIBDV showed a significant weight loss of approximately 16% compared to controls at 42 days of age, resulting in significant economic losses (Fan et al., 2020). Furthermore, co-infection with nvIBDV and other pathogens can further exacerbate the damage (Xu et al., 2015).

[0003] The prevention of IBDV currently relies primarily on vaccines. Commonly used IBDV vaccines include inactivated vaccines and live attenuated vaccines, of which the B87 vaccine strain is widely used for chicken immunization to prevent IBDV. The B87 strain is a weak IBDV strain with low virulence, which induces an immune response without causing serious pathological effects (Liu Yufeng, 2004). However, some studies have shown that the B87 strain can cause damage to immune organs such as the bursa of Fabricius, thymus, and spleen (Dong Sen et al., 2007). From a clinical perspective, classic IBDV vaccine strains cannot prevent damage caused by nvIBDV, which may be due to the difference in serotypes between the two. Therefore, the discovery of new therapeutic drugs is of great significance for the prevention and treatment of IBDV. Summary of the Invention

[0004] In response to the above-mentioned prior art, the present invention aims to provide a drug for the treatment of infectious bursal disease (IBD). The present invention has discovered that BMS-202, a PD-1 signaling pathway inhibitor, can effectively inhibit IBDV replication and alleviate IBDV-induced organ damage, making it a promising new drug for the treatment of IBD.

[0005] To achieve the above object, the present invention adopts the following technical solutions:

[0006] The first aspect of the present invention provides the use of BMS-202 in the following (1) or (2):

[0007] (1) Preparation of drugs for inhibiting the replication of different types of infectious bursal viruses;

[0008] (2) Prepare medicine for treating infectious bursal disease in chickens.

[0009] In the above application, the infectious bursal virus is vvIBDV, attIBDV and / or nvIBDV.

[0010] Preferably, the vvIBDV is the GX8 / 99 strain; the attIBDV is the B87 strain; and the nvIBDV is the IBDV-SD-1208 strain.

[0011] The present invention, for the first time, has found through in vitro experiments that BMS-202 has significant inhibitory activity against the replication of three different genotypes of infectious bursal disease virus: vvIBDV, attIBDV, and nvIBDV. Therefore, BMS-202 could be developed into a new therapeutic drug for infectious bursal disease in chickens.

[0012] A second aspect of the present invention provides use of BMS-202 in the preparation of a product for alleviating IBDV-induced weight suppression and / or bursal atrophy.

[0013] In the above application, the IBDV is a vvIBDV strain. Preferably, the vvIBDV strain is a GX8 / 99 strain.

[0014] IBDV infection inhibits the weight gain of chickens and causes atrophy of the bursa of Fabricius. The present invention found that BMS-202 can effectively alleviate the weight suppression caused by IBDV infection, promote the weight gain of chickens, and alleviate the atrophy of the bursa of Fabricius caused by IBDV infection.

[0015] A third aspect of the present invention provides a drug for treating infectious bursal disease in chickens, wherein the drug contains BMS-202 as an active ingredient.

[0016] Preferably, the concentration of BMS-202 in the drug is 1-10 μM. More preferably, the concentration of BMS-202 is 10 μM.

[0017] Beneficial effects of the present invention:

[0018] The present invention is the first to discover that BMS-202 can inhibit the replication of IBDV of different genotypes and alleviate weight suppression and bursal atrophy caused by IBDV. New drugs for the treatment of infectious bursal disease in chickens can be developed based on BMS-202, which is of great significance for the prevention and treatment of IBDV. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 : mRNA expression levels of immune checkpoints in the bursa of Fabricius after infection with IBDV of different virulences.

[0020] Figure 2 : Effects of SSGJ-707, pitavastatin calcium, and BMS-202 on DF-1 cell viability; In the figure, A. SSGJ-707; B. pitavastatin calcium; C. BMS-202.

[0021] Figure 3 : Inhibitory effects of different concentrations of SSGJ-707, pitavastatin calcium, and BMS-202 on IBDV GX8 / 99 strain.

[0022] Figure 4 : The inhibitory effect of BMS-202 on IBDV B87 and IBDV-SD-1208 strains.

[0023] Figure 5 : Changes in body weight (A) and bursal index (B) after administration of BMS-202.

[0024] Figure 6 : Changes of bursa autopsy after using BMS-202.

[0025] Figure 7: Pathological histological changes of bursa of Fabricius after using BMS-202; In the figure, A: Pathological histological changes of bursa of Fabricius in blank control group; B: Pathological histological changes of bursa of Fabricius in GX8 / 99 strain infection group; C: Pathological histological changes of bursa of Fabricius after using BMS-202.

[0026] Figure 8 : Cytokine expression levels in the bursa of Fabricius after treatment with BMS-202; In the figure, A: PD-1 mRNA expression level; B: PD-L1 mRNA expression level; C: PD-L2 mRNA expression level; D: LAG3 mRNA expression level; E: CTLA4 mRNA expression level; F: TGF-β2 mRNA expression level; G: IL-10 mRNA expression level; H: IL-1β expression level.

[0027] Figure 9 : The number of viral copies in the bursa of Fabricius after using BMS-202; In the figure, A: The bar graph of the viral load in the bursa of Fabricius after using BMS-202; B: The line graph of the viral load in the bursa of Fabricius after using BMS-202. DETAILED DESCRIPTION

[0028] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present application belongs.

[0029] As mentioned above, there are two major challenges in the prevention and treatment of IBDV. First, the multitude of IBDV genotypes, particularly the emergence of nvIBDV, creates gaps in the protection provided by traditional vaccines, which cannot guarantee that chickens will not be infected with nvIBDV. Second, there are currently no commercially available treatments for IBDV. Once an IBDV infection occurs, treatment relies solely on traditional Chinese medicine or immune-boosting medications.

[0030] In light of this, the present inventors conducted in-depth research into the pathogenicity and therapeutic mechanisms of IBDV. They discovered that the PD-1 signaling pathway inhibitor BMS-202 can inhibit the replication of different IBDV genotypes, addressing the protection gaps of traditional vaccines due to the diverse range of IBDV genotypes. Furthermore, BMS-202 can alleviate the damage caused by IBDV. Therefore, BMS-202 could be developed into a new IBDV treatment, leading to the present invention.

[0031] In order to enable those skilled in the art to more clearly understand the technical solution of the present application, the technical solution of the present application will be described in detail below with reference to specific embodiments.

[0032] The test materials used in the examples of the present invention are all conventional test materials in the field and can be purchased through commercial channels. Experimental methods without detailed conditions were carried out in accordance with conventional test methods or the operating instructions recommended by the supplier.

[0033] The CAS number of BMS-202 used in the present invention is 1675203-84-5.

[0034] The IBDV strain GX8 / 99 used was a very virulent IBDV (vvIBDV), GenBank ID: AY305386.1; described in the literature "Comparison of pathogenicity, immunogenicity and VP5 gene of cell-adapted and chicken-adapted IBDV GX8 / 99" ([D]. Shandong Agricultural University, 2007).

[0035] The IBDV strain B87 used was attIBDV, which is a widely used attenuated vaccine strain; GenBank ID: DQ202329.1.

[0036] The IBDV strain IBDV-SD-1208 used is nvIBDV, which is recorded in the document "Isolation, Identification and Pathogenicity Study of a New IBDV Variant in Shandong Province" ([D]. Shandong Agricultural University, 2023.).

[0037] Example 1: Screening for molecular targets of the mechanism of damage caused by IBDV infection of the bursa of Fabricius

[0038] 1. Test method:

[0039] To screen the molecular targets of IBDV after infection of the bursa of Fabricius, SPF chickens were inoculated with IBDV strains of different virulences (GX8 / 99, B87 and IBDV-SD-1208) by oral inoculation, with 10 6.5 TCID 50 An equal volume of PBS was orally administered as a blank control.

[0040] Then, the real-time fluorescence quantitative PCR method was used to detect the expression levels of PD-1 (XM422723), PD-L1 (XM424811), PD-L2 (XM424812), LAG3 (XM416510) and CTLA-4 (NM001040091) 3 days, 7 days, 14 days and 21 days after vaccination.

[0041] 2. Test results:

[0042] The results showed that the expression levels of immune checkpoint genes in the bursa of Fabricius were all upregulated after infection with different strains. Among them, the expression level of PD-1 continued to increase, and at 3, 14 and 21 dpi, the expression level of vvIBDV and attIBDV groups was significantly increased ( Figure 1 A). PD-L1 expression was also sharply upregulated at 3 dpi after IBDV infection. Although PD-L1 expression decreased after 7 dpi, its expression level remained high ( Figure 1 B). Compared with the blank control group, the expression of PD-L2 was significantly increased after infection with different IBDV strains, especially in the vvIBDV group at 3, 7, and 21 dpi. In contrast, PD-L2 in the nvIBDV group was only upregulated at 21 dpi ( Figure 1 C). Compared with the blank control group, the expression of LAG3 increased sharply at 3 dpi and then decreased, but the expression level was still high ( Figure 1 D). After IBDV infection, the expression of CTLA4 increased, reaching the maximum level at 7 dpi, and then gradually decreased ( Figure 1 E).

[0043] Example 2: Screening for inhibitors that inhibit IBDV replication

[0044] 1. Test method:

[0045] In vitro cell experiments were used to screen for inhibitors of the PD-1 pathway that inhibit IBDV replication. This study used an in vitro cell model system to screen inhibitors targeting the PD-1 signaling pathway for their inhibitory effects on IBDV replication. Three candidate PD-1 inhibitors (SSGJ-707, pitavastatin calcium, and BMS-202) were used for screening. The specific methods are as follows:

[0046] First, the CCK-8 assay was used to determine the safe concentrations of SSGJ-707, pitavastatin calcium, and BMS-202 in DF-1 cells. The working concentrations of the three reagents in the CCK-8 assay were set at 200, 100, 50, 25, 12.5, 6, 3, and 1 μM.

[0047] Subsequently, the inhibitory effect of the inhibitor on IBDV replication was detected on DF-1 cells. DF-1 cells were cultured in six-well plates (high-glucose DMEM, 10% FBS, 5% CO2 incubator). When DF-1 grew to 90% density, IBDV GX8 / 99 strain was inoculated into DF-1 (1.0MOI), and three inhibitors, SSGJ-707, pitavastatin calcium, and BMS-202, were added to the cell culture medium 2 hours after infection. The working concentrations of SSGJ-707, pitavastatin calcium, and BMS-202 were 10, 5, and 1 μM, respectively, and an equal volume of DMSO was added to the control group cells. Culture was continued for 2 days in the presence of inhibitors. After 2 days, the cell supernatant was collected and the TCID of the virus was detected. 50 , calculate the inhibitory effect of adding inhibitors on IBDV GX8 / 99 strain.

[0048] On this basis, IBDV B87 and IBDV-SD-1208 were inoculated into DF-1 cells, and BMS-202 was added 2 h after infection to make the working concentrations 10, 5, and 1 μM, respectively. After 2 days, the cell supernatant was collected and the TCID of the virus was detected. 50 , calculate the inhibitory effect of adding inhibitors on IBDV B87 strain and IBDV-SD-1208 strain.

[0049] 2. Test results:

[0050] The results of cytotoxicity experiments (CCK-8 method) showed that SSGJ-707, pitavastatin calcium, and BMS-202 had no significant effect on the survival rate of DF-1 cells at concentrations not higher than 12.5 μM. Figure 2 ), so the subsequent cell experiments used 10, 5, and 1 μM concentrations as working concentrations for research.

[0051] The results of virus titer detection showed that SSGJ-707 and pitavastatin calcium had no significant effect on the replication of IBDV GX8 / 99 strain; BMS-202 could significantly inhibit the replication of IBDV GX8 / 99 strain in DF-1 cells, and at a concentration of 10 μM, the virus titer (TCID 50 ) reduces by 2.3log( Figure 3 ).

[0052] In addition, BMS-202 at a concentration of 10 μM could inhibit the replication of IBDV B87 and IBDV-SD-1208 on DF-1 ( Figure 4 ).

[0053] The above-mentioned in vitro cell experiments confirmed that BMS-202 has inhibitory effects on various types of IBDV.

[0054] Example 3: Investigation of the therapeutic effect of BMS-202 on IBDV infection

[0055] 1. Test method:

[0056] Fourteen-day-old SPF chickens were used as experimental subjects, and three treatments were designed, namely control, GX8 / 99 and GX8 / 99+BMS-202, with 30 chickens in each group.

[0057] Control group: No challenge, but oral administration of equal volume of DMSO.

[0058] GX8 / 99 group: 14-day-old SPF chickens were infected orally with the highly virulent IBDV strain GX8 / 99, and an equal volume of DMSO was administered by gavage.

[0059] GX8 / 99+BMS-202 group: 14-day-old SPF chickens were infected orally with the highly virulent IBDV strain GX8 / 99. One day after infection, BMS-202 was administered orally at a dose of 20 mg / kg.

[0060] The bursa of Fabricius in the treatment group and the simple challenge group were dissected 3, 7 and 14 days after challenge, with three replicates in each group.

[0061] At 3, 7, and 14 dpi after infection with vvIBDV, the chickens in each treatment group were weighed and the bursa of Fabricius was dissected. The expression levels of immune checkpoint genes and cytokines in the bursa of Fabricius were detected by RT-qPCR. The bursa of Fabricius at 3 dpi after infection with vvIBDV was collected, prepared into sections, and stained with HE.

[0062] Fluorescence quantitative PCR was used to detect the viral load in the bursa of Fabricius in the GX8 / 99 group and the GX8 / 99+BMS-202 group at 3, 7, and 14 dpi after infection with vvIBDV.

[0063] 2. Test results:

[0064] (1) BMS-202 can alleviate IBDV-induced weight suppression and bursal atrophy

[0065] The results showed that the weight of chickens after oral administration of BMS-202 was higher than that of the GX8 / 99 group. At 14 dpi, the weight of the GX8 / 99+BMS-202 group was not only significantly higher than that of the GX8 / 99 group, but also higher than that of the blank control group ( Figure 5 A), further analysis of the bursal index showed that the bursal index of the GX8 / 99+BMS-202 group was higher than that of the GX8 / 99 group ( Figure 5B). The bursa of the GX8 / 99 group was dissected and it was observed that the bursa of the GX8 / 99 group was severely atrophied and bleeding, while the bursa of the GX8 / 99+BMS-202 group was atrophied to some extent but without bleeding ( Figure 6 ).

[0066] At 3 dpi, sections of the bursa of Fabricius in the GX8 / 99+BMS-202 group and the GX8 / 99 group were prepared and stained with HE. The results showed that the interstitial lymphoid follicles in the bursa of Fabricius in the GX8 / 99 group were widened, the cortex was thinned, and partly replaced by reticular cells and connective tissue; the medulla was reticular, with vesicles of varying sizes, severe structural damage, disordered cell structure, and unrecognizable boundaries; lymphocytes were necrotic and detached, and the number was significantly reduced. There were a few hemorrhage spots and nuclear fragments in the cortex, and severe vascular congestion. Although the GX8 / 99+BMS-202 group also had certain pathological damage, there was no bleeding, indicating that oral administration of BMS-202 can alleviate the damage of IBDV infection to the bursa to a certain extent ( Figure 7 ).

[0067] (2) BMS-202 restores cytokine expression in the bursa of Fabricius

[0068] The expression levels of immune checkpoint genes and cytokines in the bursa of Fabricius were detected by RT-qPCR. The results showed that the expression levels of PD-1 and its ligands were lower than those in the GX8 / 99 group after BMS-202 administration. Among them, the expression levels of PD-1, PD-L1 and PD-L2 at 3 dpi were significantly lower than those in the GX8 / 99 group ( Figure 8 A, B, C). At the same time, the expression levels of LAG3 and CTLA4 in the GX8 / 99+BMS-202 group were significantly lower than those in the GX8 / 99 group at 3 dpi ( Figure 8 D, E). In addition, the expression levels of three inhibitory and inflammatory cytokines, TGF-β2, IL-10, and IL-1β, were generally lower than those in the GX8 / 99 group after oral administration of BMS-202. Among them, the expression levels of IL-10 and IL-1β were significantly lower than those in the GX8 / 99 group at 3 dpi ( Figure 8 F, G, H).

[0069] (3) BMS-202 inhibits IBDV replication in the bursa of Fabricius

[0070] Fluorescence quantitative PCR was used to detect the viral load in the bursa of Fabricius after BMS-202 was administered. The results showed that BMS-202 administration reduced the viral load in the bursa of Fabricius, and at 7 dpi, the viral load in the GX8 / 99+BMS-202 group was significantly lower than that in the GX8 / 99 group, indicating that BMS-202 had a certain inhibitory effect on the replication of IBDV ( Figure 9 ).

[0071] The above description is merely a preferred embodiment of the present application and is not intended to limit the present application. Various modifications and variations are possible for those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present application shall be included within the scope of protection of the present application.

Claims

1. Application of BMS-202 in (1) or (2) below: (1) Preparation of drugs for inhibiting the replication of different types of infectious bursal viruses; (2) Prepare medicine for treating infectious bursal disease in chickens.

2. The use according to claim 1, characterized in that The infectious bursal disease virus is vvIBDV, attIBDV and / or nvIBDV.

3. The use according to claim 2, characterized in that The vvIBDV is the GX8 / 99 strain; the attIBDV is the B87 strain; and the nvIBDV is the IBDV-SD-1208 strain.

4. Use of BMS-202 in the preparation of products for alleviating IBDV-induced weight suppression and / or bursal atrophy.

5. The use according to claim 4, characterized in that The IBDV is a vvIBDV strain.

6. The use according to claim 5, characterized in that The vvIBDV strain is GX8 / 99 strain.

7. A drug for treating infectious bursal disease in chickens, characterized in that: The drug has BMS-202 as the active ingredient.

8. The drug according to claim 7, characterized in that In the drug, the concentration of BMS-202 is 1-10 μM.

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