African swine fever virus recombinant adenovirus composition and preparation

Abstract

The application belongs to the technical field of biology, and discloses a recombinant adenovirus composition of African swine fever virus and preparation, wherein the recombinant adenovirus composition of African swine fever includes AdV-p30, AdV-p54, AdV-p72, AdV-pp62, AdV-CD2v, AdV-E199L, AdV-A104R, AdV-B602L, AdV-I177L, AdV-B407L, AdV-DP238L and AdV-C962R; the recombinant adenovirus composition of African swine fever can provide good protection efficiency when a virulent strain of African swine fever virus attacks; at the end of the observation period, the immunized pigs can reduce the virus-carrying rate of tissues and organs after being infected with a virulent strain of African swine fever type II, and no live virus can be isolated from the virus-carrying organs, so that the transmission chain is effectively cut off, and the recombinant adenovirus composition of African swine fever can be used for the prevention and control of African swine fever.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a recombinant adenovirus composition for African swine fever virus and its preparation. Background Technology

[0002] African swine fever (ASF) is an acute and highly contagious disease caused by the African swine fever virus (ASFV) infecting domestic and wild pigs. Pigs of all ages are susceptible. It is characterized by a short incubation period, with mortality rates reaching 100% in the most acute and acute forms. Clinical manifestations include fever, rapid heartbeat, difficulty breathing, serous or mucopurulent discharge from the eyes and nose, cyanosis of the skin, significant hemorrhage in the heart, lungs, bile ducts, lymph nodes, kidneys, and gastrointestinal mucosa, and spleen congestion and enlargement. ASF was first reported in Africa in 1909. However, to date, no safe and effective commercial vaccine for the control of African swine fever has been successfully developed worldwide.

[0003] GP Paulino et al. reported that ASFV p72, p54, and p30 protein subunit vaccines could inhibit viral attachment and internalization, thereby affecting viral replication. JG Neilan et al. prepared a combined subunit vaccine expressing ASFV p30, p54, p72, and p22 proteins. These vaccines effectively induced neutralizing antibodies in pigs after immunization, temporarily delaying disease progression, but did not provide effective immune protection. Under challenge with virulent strains, both the immunized and control groups showed 100% mortality, indicating that neutralizing antibodies against these viral proteins are insufficient for neutralization protection. Compared to subunit vaccines, viral vector vaccines can stably induce antigen-specific humoral and cellular immune responses, making them a key area for ASF prevention and control technology development. Lokhandwala et al. evaluated the immunogenicity and safety of two ASFV multi-antigen mixture live vector vaccines. Immunization of pigs with an adenovirus mixture expressing structural antigens (p32, p54, pp62, and p72) induced IgG, IFN-γ+ T cell, and CTL responses. Immunization of pigs with novel ASFV antigen mixtures A151R, B119L, B602L, EP402R1PRR, B438L, K205R, and A104R delivered by adenovirus vectors yielded the same results, indicating that adenovirus vector vaccines can induce effective antigen-specific immune responses. However, the ASFV infection protection rate, histopathological status, and viral load in tissues and organs remain unclear.

[0004] To address the aforementioned problems, the applicant has invented a recombinant adenovirus combination for African swine fever (ASF). This combination comprises 12 coding genes expressing ASF antigens or epitopes inserted into an adenovirus backbone vector to construct a recombinant adenovirus expressing the stated antigens or epitopes. A systematic evaluation was conducted on the body temperature, clinical symptoms, viral shedding, histopathological state, tissue viral load, and isolation of live virus from infected pigs in immunized and non-immunized animals after challenge with virulent strains. The ASF recombinant adenovirus combination provided by this invention provides immunoprotection against challenge with virulent ASF strains, achieving no viral shedding from the mouth, nose, or anus at the end of the observation period, thus reducing environmental pollution. It enables immunized pigs to reduce the viral load in tissues and organs after infection with virulent type II ASF virus, and prevents the isolation of live virus from infected organs, effectively interrupting the transmission chain and making it suitable for the prevention and control of ASF. Summary of the Invention

[0005] The purpose of this invention is to provide an African swine fever virus protein composition, wherein the protein composition comprises: African swine fever p30, p54, p72, pp62, CD2v, E199L, A104R, B602L, I177L, B407L, DP238L and C962R.

[0006] Another object of the present invention is to provide the application of the above-mentioned protein combination in the preparation of African swine fever vaccines.

[0007] To achieve the above objectives, the present invention adopts the following technical measures:

[0008] This application, based on extensive screening and evaluation studies, first discovered that the non-structural proteins B407L, DP238L, and C962R of African swine fever can induce an immune response with positive serum infected with African swine fever and possess immunogenicity. Furthermore, by effectively mixing these antigens with other antigens, an African swine fever virus protein combination is provided. This protein combination includes: P30, P54, P72, PP62, CD2v, E199L, A104R, B602L, I177L, B407L, DP238L, and C962R. All of these proteins are derived from African swine fever virus and can be either full-length proteins or truncated versions of proteins with the same function.

[0009] The scope of protection of this invention includes:

[0010] An African swine fever virus protein composition, the protein composition comprising: p30, p54, p72, pp62, CD2v, E199L, A104R, B602L, I177L, B407L, DP238L and C962R;

[0011] The P30 protein is shown in SEQ ID NO.16, the P54 protein in SEQ ID NO.17, the P72 protein in SEQ ID NO.18, the PP62 protein in SEQ ID NO.19, the CD2v protein in SEQ ID NO.20, the E199L protein in SEQ ID NO.21, the A104R protein in SEQ ID NO.22, the B602L protein in SEQ ID NO.23, the I177L protein in SEQ ID NO.26, the B407L protein in SEQ ID NO.28, the DP238L protein in SEQ ID NO.29, and the C962R protein in SEQ ID NO.30.

[0012] An African swine fever virus fusion protein composition, the protein composition comprising: African swine fever P30, P54, P72, PP62, CD2v, E199L, A104R, B602L, I177L, B407L, DP238L and C962R respectively fused with protein tags to obtain a fusion protein composition.

[0013] An African swine fever virus gene composition, the gene composition comprising the above-mentioned protein composition or fusion protein composition, and a composition encoding a gene corresponding to the protein.

[0014] Combinations of expression cassettes, recombinant vectors, recombinant microorganisms, or ex vivo recombinant cells containing the above-mentioned coding genes.

[0015] The use of protein compositions, fusion protein compositions, compositions encoding genes, expression cassettes of compositions encoding genes, recombinant vectors, recombinant microorganisms or ex vivo recombinant cell compositions having such compositions in the preparation of African swine fever vaccines.

[0016] The recombinant microbial composition described above is a viral composition containing encoding genes.

[0017] The viral compositions described above include, but are not limited to: human adenovirus type 5, human adenovirus type 26 (Ad26), chimpanzee adenovirus (ChAds), poxvirus, porcine pseudorabies virus, porcine reproductive and respiratory syndrome virus, porcine adenovirus, porcine encephalitis virus, classical swine fever virus, rabies virus, retrovirus, paramyxovirus, or other viral or bacterial vector systems that can produce transient, stable, or persistent expression of exogenous genes in mammals without causing any abnormal clinical symptoms.

[0018] The gene composition described above preferably contains genes encoding P30, P54, P72, PP62, CD2v, E199L, A104R, B602L, I177L, B407L, DP238L, and C962R, as shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.11, SEQ ID NO.13, SEQ ID NO.14, and SEQ ID NO.15, respectively.

[0019] Preferably, the viral compositions described above are constructed using the AdMax adenovirus system, and the resulting recombinant viral combinations are: AdV-p30, AdV-p54, AdV-p72, AdV-pp62, AdV-CD2v, AdV-E199L, AdV-A104R, AdV-B602L, AdV-I177L, AdV-B407L, AdV-DP238L, and AdV-C962R.

[0020] Preferably, the recombinant virus combination described above is prepared by mixing the recombinant viruses in a titer ratio of 1~10:1~10:1~10:1~10:1~10:1~10:1~10:1~10:1~10:1~10:1~10:1~10:1~10:1~10:1~10:1~10:1~10.

[0021] The African swine fever virus described above is type II African swine fever virus.

[0022] Compared with the prior art, the beneficial effects of the present invention are:

[0023] (1) The present invention provides an African swine fever recombinant adenovirus composition, wherein the African swine fever recombinant adenovirus composition comprises AdV-p30, AdV-p54, AdV-p72, AdV-pp62, AdV-CD2v, AdV-E199L, AdV-A104R, AdV-B602L, AdV-I177L, AdV-B407L, AdV-DP238L and AdV-C962R; the combination of the present invention has excellent immunization effect.

[0024] (2) When pigs are immunized with the African swine fever recombinant adenovirus combination described in this invention, they can provide good protection efficiency when challenged with virulent African swine fever strains.

[0025] (3) The African swine fever recombinant adenovirus combination provided by the present invention can achieve no viral shedding in the mouth, nose and anus at the end of the observation period, reduce environmental pollution, effectively cut off the transmission chain, and avoid causing infection in other pigs.

[0026] (4) The African swine fever recombinant adenovirus combination provided by the present invention immunized pigs and challenged them. On the 28th day, the viral load in the organs and tissues was 18.3%, and no live virus could be isolated.

[0027] (5) The African swine fever recombinant adenovirus combination described in this invention has good safety. Attached Figure Description

[0028] Figure 1 Roadmap for constructing expression vectors for recombinant intermediate transfer plasmid pAdV-mCMV-X-3×Flag.

[0029] Figure 2 Viral packaging route diagram of recombinant adenovirus AdV-X.

[0030] Figure 3 The condition of each organ after dissection of surviving pigs in the immunization group;

[0031] Where A represents the heart, B the liver, C the lungs, D the kidneys, E the spleen, F the mesenteric lymph nodes, G the inguinal lymph nodes, H the submandibular lymph nodes, and I the tonsils.

[0032] Figure 4 The condition of various organs after dissection of a dying pig in the control group;

[0033] Where A represents the heart, B the liver, C the lungs, D the kidneys, E the spleen, F the mesenteric lymph nodes, G the inguinal lymph nodes, H the submandibular lymph nodes, and I the tonsils.

[0034] Figure 5 Pathological sections of organs from surviving pigs in the immune group after challenge with the virus;

[0035] Where A represents the heart, B the liver, C the lungs, D the kidneys, E the spleen, F the mesenteric lymph nodes, G the inguinal lymph nodes, H the submandibular lymph nodes, and I the tonsils.

[0036] Figure 6 Pathological sections of organs from dying pigs in the control group after viral challenge;

[0037] In the image, A represents the heart, B the liver, C the lungs, D the kidneys, E the spleen, F the mesenteric lymph nodes, G the inguinal lymph nodes, H the submandibular lymph nodes, and I the tonsils. The arrows indicate the locations of different degrees of cell necrosis, nuclear lysis, inflammatory cell infiltration, and multifocal hemorrhage in the tissues and organs on the pathological slides. Detailed Implementation

[0038] To make the technical means, creative features, objectives, and effects of this invention easier to understand, the following detailed embodiments further illustrate this invention. However, the scope of protection of this invention is not limited to the embodiments described below.

[0039] The experiments described in the following examples were conducted with biosafety and African swine fever laboratory activity permits. All experiments related to live African swine fever virus were performed in a biosafety level 3 (ABSL-3) laboratory.

[0040] The experimental cells, viruses, and plasmids described in the following examples are from:

[0041] HEK293A cells were preserved in our laboratory. The Admax packaging system was purchased from Heyuan Biotechnology (Shanghai) Co., Ltd., and the clonal strain E. coli DH5α was preserved in our laboratory.

[0042] Primary porcine alveolar macrophages (PAM) were prepared in our laboratory (prepared by rinsing and separating porcine lungs).

[0043] Cell digestion with trypsin containing 0.25% EDTA and penicillin-streptomycin solution (100×) was purchased from Gino Biosciences; Opti-MEM for transfection was purchased from Gibco; Lipofectamine 2000 for cell transfection was purchased from Invitrogen; DNA gel extraction kit and plasmid extraction kit were purchased from Beijing Tiangen Biotech Co., Ltd.; Trizol extraction lysis buffer was purchased from Invitrogen; T4 ligase, restriction endonuclease, and Reverse Transcriptase XL (AMV) were purchased from Takara Bio Engineering (Dalian) Co., Ltd.; Trans2K Plus DNA Marker was purchased from Beijing Dongsheng Biotechnology Co., Ltd.; ECL chromogenic kit was purchased from Thermo Fisher Scientific; and protease inhibitor PMSF (Servicebio) and protein marker were purchased from Thermo Fisher Scientific.

[0044] LB liquid medium containing resistance: 5g yeast extract powder, 10g tryptone soya agar, and 10g sodium chloride (NaCl) are dissolved in 1000ml of water for injection, sterilized at high temperature, cooled to room temperature, and then ampicillin is added to a final concentration of 100μg / ml for later use.

[0045] LB solid medium containing resistance: 5g yeast extract powder, 10g tryptone soya agar, 10g sodium chloride (NaCl), 15g agar powder, dissolved in 1000ml water for injection, sterilized at high temperature, cooled to about 45℃, added ampicillin to a final concentration of 100μg / ml, poured into petri dishes for later use.

[0046] African swine fever virus SY-1 was isolated, identified, and preserved by Huazhong Agricultural University, with a viral titer of 1×10⁻⁶. 5 HAD 50 / mL, publication number CN117586898A, accession number CCTCC NO:V202154.

[0047] Example 1:

[0048] African swine fever virus protein composition:

[0049] This invention utilizes Western blot serological proteomics analysis and mass spectrometry to analyze the interacting proteins in African swine fever (ASF) infected positive serum and ASF whole virus lysate. It is the first time that ASF B407L, DP238L, and C962R can induce an immune response with ASF infected positive serum and are immunogenic. Furthermore, the invention is the first to use these proteins in the preparation of an ASF vaccine.

[0050] The present invention protects an African swine fever virus protein composition, which includes: African swine fever P30, P54, P72, PP62, CD2v, E199L, A104R, B602L, I177L, B407L, DP238L and C962R proteins.

[0051] In addition, the applicant also tested the immunization effect of a mixture of other immunogenic proteins, E120R, P17 and / or EP153R, with the above-mentioned proteins as a vaccine.

[0052] The gene sequence of the P30 protein described above is shown in SEQ ID NO.1, and the protein is shown in SEQ ID NO.16; the gene sequence of the P54 protein is shown in SEQ ID NO.2, and the protein is shown in SEQ ID NO.17; the gene sequence of the P72 protein is shown in SEQ ID NO.3, and the protein is shown in SEQ ID NO.18; the gene sequence of the PP62 protein is shown in SEQ ID NO.4, and the protein is shown in SEQ ID NO.19; the gene sequence of the CD2v protein is shown in SEQ ID NO.5, and the protein is shown in SEQ ID NO.20; the gene sequence of the E199L protein is shown in SEQ ID NO.6, and the protein is shown in SEQ ID NO.21; the gene sequence of the A104R protein is shown in SEQ ID NO.7, and the protein is shown in SEQ ID NO.22; the gene sequence of the B602L protein is shown in SEQ ID NO.8, and the protein is shown in SEQ ID NO.23; the gene sequence of the E120R protein is shown in SEQ ID NO.9, and the protein is shown in SEQ ID NO.16. The gene sequence of the P17 protein is shown in SEQ ID NO. 24; the protein sequence of the I177L protein is shown in SEQ ID NO. 11; the protein sequence of the EP153R protein is shown in SEQ ID NO. 12; the protein sequence of the B407L protein is shown in SEQ ID NO. 13; the protein sequence of the DP238L protein is shown in SEQ ID NO. 14; the protein sequence of the C962R protein is shown in SEQ ID NO. 15; and the protein sequence of the C962R protein is shown in SEQ ID NO. 30.

[0053] This invention takes the preparation of recombinant adenovirus combinations using the genes corresponding to the above-mentioned proteins as an example to demonstrate the immunogenicity of the protein combinations that need to be protected in this invention. Other methods, such as direct protein synthesis, prokaryotic or eukaryotic expression, and protein expression methods in the prior art, can all achieve the technical effects of this invention.

[0054] The prepared recombinant adenovirus for African swine fever is as follows:

[0055] The following are examples of adenovirus vectors: AdV-P30, AdV-P54, AdV-P72, AdV-PP62, AdV-CD2v, AdV-E199L, AdV-A104R, AdV-B602L, AdV-E120R, AdV-P17, AdV-I177L, AdV-EP153R, AdV-B407L, AdV-DP238L, and AdV-C962R. The encoding genes for each protein are inserted into the adenovirus backbone vector. The specific procedure is as follows:

[0056] 1. Construction of recombinant intermediate transfer plasmid

[0057] 1.1 Based on the ASFV strain sequences published in GeneBank, the coding genes for the proteins P30, P54, P72, PP62, CD2v, E199L, A104R, B602L, E120R, P17, I177L, EP153R, B407L, DP238L, and C962R of African swine fever virus were sequenced and optimized. The optimized genes were synthesized by Wuhan Aoke Biotechnology Co., Ltd., and their sequences are shown in SEQ ID NO. 1~15 above.

[0058] 1.2 Construction of the recombinant intermediate transfer plasmid pADV-mCMV-X-3×Flag ( Figure 1 (where X represents the gene corresponding to different antigen proteins):

[0059] The transfer vector pADV-mCMV-MCS-3×Flag was linearized using EcoRI and BamHI. Simultaneously, the codon-optimized African swine fever virus DNA fragments P30, P54, P72, PP62, CD2v, E199L, A104R, B602L, E120R, P17, I177L, EP153R, B407L, DP238L, and C962R were amplified and then recovered via gel electrophoresis. The purified African swine fever virus P30... Gene fragments including P54, P72, PP62, CD2v, E199L, A104R, B602L, E120R, P17, I177L, EP153R, B407L, DP238L, and C962R were cloned into the linearized transfer vector pADV-mCMV-MCS-3×Flag via homologous recombination, yielding the recombinant intermediate transfer plasmids pADV-mCMV-P30-3×Flag and pA DV-mCMV-P54-3×Flag, pADV-mCMV-P72-3×Flag, pADV-mCMV-PP62-3×Flag, pADV-mCMV-CD2v-3 ×Flag, pADV-mCMV-E199L-3×Flag, pADV-mCMV-A104R-3×Flag, pADV-mCMV-B602L-3×Flag, pADV -mCMV-E120R-3×Flag, pADV-mCMV-P17-3×Flag, pADV-mCMV-I177L-3×Flag, pADV-mCMV-EP153 R-3×Flag, pADV-mCMV-B407L-3×Flag, pADV-mCMV-DP238L-3×Flag, pADV-mCMV-C962R-3×Flag.

[0060] 2. Packaging of recombinant adenovirus Adv-X ( Figure 2 (X represents the gene corresponding to different antigen proteins)

[0061] The synthesized recombinant intermediate transfer plasmids pADV-mCMV-P30-3×Flag, pADV-mCMV-P54-3×Flag, pADV-mCMV-P72-3×Flag, pADV-mCMV-PP62-3×Flag, pADV-mCMV-CD2v-3×Flag, pADV-mCMV-E199L-3×Flag, pADV-mCMV-A104R-3×Flag, pADV-mCMV-B602L-3×Flag, pADV-mCMV-E120R-3×Flag, pADV-mCMV-P17-3×Flag, pADV-mCMV-I177L-3×Flag, and pADV- mCMV-EP153R-3×Flag, pADV-mCMV-B407L-3×Flag, pADV-mCMV-DP238L-3×Flag, and pADV-mCMV-C962R-3×Flag, along with the backbone plasmid pBHGLoxdeLE13cre, were co-transfected into HEK293A cells for viral packaging to obtain recombinant adenoviruses AdV-P30, AdV-P54, AdV-P72, AdV-PP62, AdV-CD2v, AdV-E199L, AdV-A104R, AdV-B602L, AdV-E120R, AdV-P17, AdV-I177L, AdV-EP153R, AdV-B407L, AdV-DP238L, and AdV-C962R.

[0062] 3. Recombinant adenovirus PCR identification: The harvested virus solution was placed at -80℃ and repeatedly frozen and thawed 3 times. After centrifugation at 6000 r / min for 10 min, the supernatant was collected. This step was repeated until the 20th generation. Using the genomic DNA extracted from the harvested supernatant as a template, PCR reactions were performed using specific primers corresponding to the African swine fever virus P30, P54, P72, PP62, CD2v, E199L, A104R, B602L, E120R, P17, I177L, EP153R, B407L, DP238L, C962R genes and the adenovirus vector, respectively. The target bands were amplified. The results showed that the PCR amplified a single band in all cases, and the size was consistent with the expected size.

[0063] 4. Identification of the expression of exogenous genes from recombinant adenovirus

[0064] Using Western blotting, the results showed a single band at the target protein location, with the size as expected, indicating successful expression of the target gene.

[0065] 5. Recombinant adenovirus viral load (TCID) 50 ) Measurement

[0066] According to 1×10 5 HEK293A cells were cultured in 96-well cell culture plates at a cell density of 1 cell / well. The following day, different strains of the virus to be tested were inoculated into the plates. Ten [cells / wells] were cultured in wells 1-11. -1 ~10 -11 Dilute the samples, with columns A through H representing replicates of the same concentration. Incubate at 37°C with 5% CO2 for 10 days and record the number of wells showing cytopathic effect (CPE). Calculate the TCID of the virus under test using the Reed-Muench method. 50 The results are shown in Table 1.

[0067] Table 1. Detection of recombinant adenovirus viral content

[0068] .

[0069] Example 2

[0070] Screening for the optimal antigen combination of recombinant adenovirus from African swine fever

[0071] Different combinations of African swine fever candidate antigens were used to screen the optimal combination through immunoprotective efficacy tests.

[0072] 1. Comparison of different combinations of recombinant adenoviruses for African swine fever

[0073] The African swine fever antigen recombinant adenovirus prepared in Example 1 was concentrated and then combined in different titer ratios for immunization. The total content of recombinant adenovirus in each combination was higher than 10. 10.0 TCID 50 / ml. Through extensive combination screening experiments, it was found that the protective efficacy of different combinations varied significantly. In this embodiment, five combinations from Table 2 are used for demonstration and illustration.

[0074] Table 2 Different combinations of recombinant adenoviruses for African swine fever

[0075] .

[0076] 2. Virus challenge protection test

[0077] Experimental animals: Healthy piglets aged 25-28 days, purchased from pig farms where ASFV was not prevalent, and both the nucleic acid and antibody tests for African swine fever virus were negative.

[0078] 2.1 Immunization and Challenge: Healthy pigs aged 25–28 days were randomly divided into groups of 5. The immunized group was injected intramuscularly with 2.0 ml of recombinant African swine fever adenovirus (ASF) combination, while the control group was not vaccinated. A second immunization was administered 14 days after the first vaccination, at the same dose. 14 days after the second immunization, both the immunized group and the non-immunized control group were orally administered 2000 mg of AAD. 50African swine fever virus fluid (CCTCCNO:V202154) was used for challenge, and the pigs were observed for 28 days after the challenge. Body temperature was measured every other day after challenge, clinical symptoms were observed and recorded, oral, nasal, and anal swabs were collected for African swine fever virus nucleic acid testing, and mortality was recorded.

[0079] 3. Results

[0080] 3.1 Body temperature monitoring and observation of clinical symptoms

[0081] Pigs in the immunized group that did not develop the disease had normal body temperature, no abnormal clinical symptoms, and survived during the observation period. Pigs in the immunized group that developed the disease began to show elevated body temperature from the 9th day after the challenge, which lasted for 3-5 days, and also showed clinical symptoms such as depression, loss of appetite, difficulty breathing, and diarrhea, and died within 14-20 days after the challenge.

[0082] The pigs in the control group developed symptoms of depression, loss of appetite, fever and persistent high body temperature starting from the 7th day after the virus challenge, accompanied by respiratory distress and diarrhea. All of them died by the 18th day.

[0083] 3.2 Nucleic acid detection of African swine fever virus after challenge

[0084] Occasionally, swabs from pigs in the immunized group that did not develop the disease tested positive, but all of them tested negative by the 19th. On the 9th, swabs from pigs in the immunized group that developed the disease tested positive for nucleic acid, and by the 15th, all of the oral, nasal, and anal swabs from the immunized pigs tested positive for nucleic acid, until they died.

[0085] The control group tested positive for viral nucleic acid in oral, nasal, and anal swabs from day 7, and all oral, nasal, and anal swabs tested positive on day 13, until death.

[0086] Table 3. Statistics on protection results against different recombinant adenovirus combinations.

[0087] .

[0088] Based on the results of challenge protection experiments with different recombinant adenovirus combinations, the survival rate of pigs immunized with the V4 combination after challenge was 100%. Furthermore, when any one protein in the V4 combination was mixed at a mass ratio of 1:10, and the remaining proteins were mixed at a mass ratio of 1:10, or when any one protein was mixed at a mass ratio of 1:10, and the remaining proteins were mixed at a mass ratio of 1:1, and the same immunization challenge experiment was conducted, the survival rate was 100%, indicating that AdV-p30, AdV-p54, and AdV-p72... AdV-pp62, AdV-CD2v, AdV-E199L, AdV-A104R, AdV-B602L, AdV-I177L, AdV-B407L, AdV-DP238L, and AdV-C962R, when mixed at a titer ratio of 1~10:1~10:1~10:1~10:1~10:1~10:1~10:1~10:1~10:1~10:1~10:1~10:1~10:1~10:1~10:1~10:1~10, can all achieve 100% survival.

[0089] Therefore, the V4 combination was chosen to further evaluate its immune protection effect.

[0090] Example 3:

[0091] Evaluation of the systemic immunoprotective effect of recombinant adenovirus combination against African swine fever

[0092] Experimental animals: Healthy piglets aged 25-28 days, purchased from pig farms where ASFV was not prevalent, and both the nucleic acid and antibody tests for African swine fever virus were negative.

[0093] 1. Immunity and Infection

[0094] Five piglets were randomly selected from ten healthy piglets aged 25-28 days to serve as the immunization group (i.e., challenged after immunization), and the remaining five served as the control group (i.e., challenged without immunization). A second immunization was administered 14 days after the first immunization. Blood samples were collected from all experimental pigs 28 days after the first immunization, and serum antibody levels for 12 different antigens were detected using the African swine fever virus ELISA antibody detection method. A challenge efficacy test was conducted 28 days after the first immunization. The challenge method involved oral administration of 1.0 ml of African swine fever virus SY-1 strain (virus content 2000 HAD). 50 (CCTCC NO:V202154), continuous clinical observation for 28 days.

[0095] 2. Detection Indicators

[0096] Body temperature monitoring and clinical symptom observation: Body temperature was measured every morning for three days prior to challenge, using the average temperature of the three days prior to challenge (two days before, one day before, and the day of challenge) as the baseline temperature. Body temperature was measured every morning 24 hours after challenge to analyze changes before and after challenge. Clinical symptoms and mortality were observed and recorded daily in the experimental pigs, including lethargy, decreased or absent appetite, diarrhea, difficulty breathing, and petechiae.

[0097] African swine fever virus nucleic acid detection after challenge: During the 28-day observation period after challenge, African swine fever virus nucleic acid was detected in oral, nasal, and anal swabs of the test pigs every 3 days.

[0098] Observation of organs and tissue sections: After the 28-day observation period, surviving pigs in the immunized group were dissected to observe whether there were any lesions in the heart, liver, spleen, lungs, kidneys, mesenteric lymph nodes, submandibular lymph nodes, inguinal lymph nodes, and tonsils. Control pigs were dissected when near death, and the lesions in the piglets' organs were recorded. Simultaneously, samples were taken from major organs and fixed to prepare tissue sections, and the lesions were statistically analyzed.

[0099] Organ and tissue viral load and virus isolation detection: After the 28-day observation period, surviving pigs in the immunized group were dissected, and African swine fever virus nucleic acid was detected in animal tissues (heart, liver, spleen, lung, kidney, bone marrow, mesenteric lymph nodes, submandibular lymph nodes, inguinal lymph nodes, hilar lymph nodes, gastrohepatic lymph nodes, and tonsils) using real-time quantitative PCR. If the tissue sample tested positive, the positive tissue homogenate was inoculated into PAM cells. Five days later, the cell supernatant was collected and blindly passaged in PAM cells for three generations. The African swine fever virus nucleic acid was then detected using real-time quantitative PCR.

[0100] 3 Results

[0101] 3.1 Antibody detection: 14 days after the second immunization, all immunized groups produced corresponding antibodies, OD... 630nm The readings were all between 0.440 and 0.620, with the non-immunized group having an OD value of [missing value]. 630nm The readings were all between 0.060 and 0.120.

[0102] 3.2 Temperature and Clinical Symptom Detection: One immunized pig exhibited a slight increase in body temperature, not exceeding 40.3℃, which returned to normal after 2 days. During this period, no clinical symptoms caused by African swine fever virus infection were observed. The remaining immunized pigs had body temperatures fluctuating within the normal range, and none showed clinical symptoms caused by African swine fever virus infection. Control pigs began to show elevated body temperatures on day 8 after challenge, gradually exceeding the basal body temperature by 1.5℃ from day 10 onwards, lasting for 3-4 days. On day 6 after challenge, they began to show clinical symptoms of lethargy, followed by decreased appetite, difficulty breathing, and diarrhea. The last pig died on day 18. At the end of the 28-day observation period, all experimental pigs in the immunized group survived, while all pigs in the control group either became ill or died.

[0103] Table 4. Body temperature monitoring of experimental pigs challenged after combined immunization with recombinant adenovirus for African swine fever.

[0104] .

[0105] Continued from Table 4:

[0106] .

[0107] Note: " / " indicates death.

[0108] Table 5. Observation results of clinical symptoms in pigs challenged after combined immunization with recombinant adenovirus for African swine fever.

[0109] .

[0110] 3.3 African swine fever virus nucleic acid detection: In the immunized group, swab samples tested positive occasionally before day 25, with Ct values ​​above 37. By day 25, all swab nucleic acid tests were negative. In the control group, oral, nasal, and anal swabs tested positive for viral nucleic acid starting from day 7. By day 10, all oral, nasal, and anal swabs tested positive until death.

[0111] Table 6. Viral content in oral swabs of test pigs challenged after immunization.

[0112] .

[0113] Note: "NoCt": negative for viral nucleic acid; " / ": dead.

[0114] Table 7. Viral content in nasal swabs from experimental pigs challenged after immunization.

[0115] .

[0116] Note: "NoCt": negative for viral nucleic acid; " / ": dead.

[0117] Table 8. Virus content in anal swabs from experimental pigs challenged after immunization.

[0118] .

[0119] Note: "NoCt": negative for viral nucleic acid; " / ": dead.

[0120] 3.4 Observation results of organ lesions: All organs in the immunized group were normal, with no obvious abnormalities. In the control group, the spleen of the dying pigs was significantly enlarged, darkened in color, and brittle in texture. The heart, lungs, kidneys, and liver were congested and enlarged, and the lymph nodes and tonsils were congested. Figure 3-4 ).

[0121] 3.5 Pathological sections: Pathological sections of the immunoassay group showed that the overall structure of the heart, liver, spleen, lungs, kidneys, mesenteric lymph nodes, inguinal lymph nodes, submandibular lymph nodes, and tonsils was basically normal. The control group showed varying degrees of cell necrosis, nuclear lysis, inflammatory cell infiltration, and multifocal hemorrhage in the major organs. Figure 5-6 ).

[0122] 3.6 Virus Carrying in Organs and Virus Isolation: After the observation period, surviving pigs in the immunized group were dissected to detect the virus carrying status in their organs. The heart, tonsils, lungs, spleen, bone marrow, submandibular lymph nodes, inguinal lymph nodes, mesenteric lymph nodes, and hilar lymph nodes were all positive, with a positive rate of 18.3%. The virus was mainly concentrated in the lymph nodes, tonsils, spleen, and bone marrow, accounting for 8.3%, 3.3%, 3.3%, and 1.7% of the total number of organs, respectively. Further virus isolation from the positive tissues did not yield African swine fever virus.

[0123] The above results indicate that: (1) the recombinant adenovirus combination for African swine fever provided by the present invention can produce a good immune response; (2) after immunizing pigs with the recombinant adenovirus combination for African swine fever provided by the present invention, it can provide good protection efficiency when challenged with virulent strains of African swine fever; (3) the recombinant adenovirus combination for African swine fever provided by the present invention can achieve no viral shedding at the mouth, nose, or anus at the end of the observation period; (4) the recombinant adenovirus combination for African swine fever provided by the present invention can protect 100% of the damage to tissues and organs caused by viral infection; (5) after immunizing pigs with the recombinant adenovirus combination for African swine fever provided by the present invention, the viral load in tissues and organs on day 28 was 18.3%, and no live virus could be isolated; (6) the recombinant adenovirus combination for African swine fever provided by the present invention has good safety.

[0124] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, 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 present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. An African swine fever virus protein composition, said protein composition comprising: African swine fever (ASF) strains include P30, P54, P72, PP62, CD2v, E199L, A104R, B602L, I177L, B407L, DP238L, and C962R. The P30 protein is shown in SEQ ID NO.16, the P54 protein in SEQ ID NO.17, the P72 protein in SEQ ID NO.18, the PP62 protein in SEQ ID NO.19, the CD2v protein in SEQ ID NO.20, the E199L protein in SEQ ID NO.21, the A104R protein in SEQ ID NO.22, the B602L protein in SEQ ID NO.23, the I177L protein in SEQ ID NO.26, the B407L protein in SEQ ID NO.28, the DP238L protein in SEQ ID NO.29, and the C962R protein in SEQ ID NO.

30.

2. An African swine fever virus fusion protein composition, wherein the fusion protein composition is obtained by fusing each protein in the protein composition of claim 1 with a protein tag.

3. An African swine fever virus gene composition, said gene composition comprising a composition encoding a protein corresponding to the protein in the protein composition of claim 1 or the fusion protein composition of claim 2.

4. An expression cassette, recombinant vector, recombinant microorganism, or ex vivo recombinant cell composition having the gene composition of claim 3.

5. The use of the protein composition of claim 1, the fusion protein composition of claim 2, the composition encoding a gene of the protein composition of claim 1 or the fusion protein composition of claim 2, and the use of an expression cassette, recombinant vector, recombinant microorganism or ex vivo recombinant cell having the gene composition of claim 3 in the preparation of an African swine fever vaccine.

6. The composition according to claim 4 is a viral composition having the gene composition of claim 3.

7. The composition according to claim 6, wherein the virus used in the viral composition is: human adenovirus type 5, human adenovirus type 26 (Ad26), chimpanzee adenovirus (ChAds), poxvirus, porcine pseudorabies virus, porcine reproductive and respiratory syndrome virus, porcine adenovirus, porcine encephalitis virus, classical swine fever virus, rabies virus, retrovirus and / or paramyxovirus.

8. The gene composition according to claim 3, characterized in that: The genes encoding P30, P54, P72, PP62, CD2v, E199L, A104R, B602L, I177L, B407L, DP238L, and C962R are shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.11, SEQ ID NO.13, SEQ ID NO.14, and SEQ ID NO.15, respectively.

9. The viral composition according to claim 6, wherein the viral composition is constructed using the AdMax adenovirus system to obtain a recombinant viral combination of: AdV-p30, AdV-p54, AdV-p72, AdV-pp62, AdV-CD2v, AdV-E199L, AdV-A104R, AdV-B602L, AdV-I177L, AdV-B407L, AdV-DP238L, and AdV-C962R.

10. The viral composition according to claim 9, wherein the recombinant virus is mixed in a titer ratio of 1~10:1~10:1~10:1~10:1~10:1~10:1~10:1~10:1~10:1~10:1~10:1~10:1~10:1~10:1~10:1~10:1~10.

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