Composition for preventing and / or treating infections and use thereof

Abstract

The invention provides a composition for preventing and / or treating an infection in an animal, comprising at least one antigen gene selected from the group consisting of SEQ ID NO: 1 (S273R), SEQ ID NO: 2 (E183L), SEQ ID NO: 3 (K78R), SEQ ID NO: 4 (KP177R), SEQ ID NO: 5 (A104R), SEQ ID NO: 6 (A137R), SEQ ID NO: 8 (B646R), SEQ ID NO: 10 (B602L), SEQ ID NO: 11 (CP2475L), SEQ ID NO: 12 (CP204L), SEQ ID NO: 13 (CP530R), SEQ ID NO: 14 (O61R), SEQ ID NO: 15 (D117L), SEQ ID NO: 16 (H108R The invention also provides methods of preventing and / or treating infections by administering the above compositions to an animal.

Description

[0001] Reference to relevant applications This application claims the benefit of U.S. Provisional Patent Application No. 63 / 540,006, filed September 22, 2023, pursuant to 35 USC § 119(e), which is incorporated herein by reference. Technical Field

[0002] This invention relates to a composition for the prevention and / or treatment of infections (particularly infections caused by African swine fever virus (ASFV)) and its use. Background Technology

[0003] Vaccines are known to prevent serious viral infections by stimulating an immune response. Live or attenuated viruses have been effective in preventing major viral diseases such as polio and smallpox. Similarly, subunit vaccines or protein components of viruses have shown considerable efficacy, as demonstrated by vaccines against human papillomavirus, hepatitis B virus, and SARS-CoV-2. Recently, mRNA vaccines have also been shown to be effective against SARS-CoV-2 and influenza viruses.

[0004] African swine fever (ASF) is a highly contagious infectious disease caused by the African swine fever virus (ASFV), affecting wild and domestic pigs of all breeds and ages. ASFV is a large double-stranded DNA virus that severely impacts pigs in Asia and Europe. Infection can lead to a range of clinical symptoms, from severe disease with 100% mortality to long-term persistent infection. Clinical symptoms of virulent strains include pulmonary edema, severe depression, high fever, anorexia, skin spots, cyanosis, thrombocytopenia, lymphopenia, and hemorrhagic lesions. Developing an ASF vaccine is crucial for preventing the spread of ASF, eradicating ASF, and repopulating pigs after an ASF outbreak.

[0005] To date, most vaccines under development are modified live attenuated vaccines. Recently, two live attenuated vaccines based on gene deletions of the African swine fever virus (ASFV) pathogenic factor have been developed and approved for use in Vietnam. However, debates regarding their safety and efficacy continue. The use of modified live attenuated virus vaccines still carries the risk of reverting to a more virulent strain after release into the field, as well as the risk of releasing the vaccine virus, which could further exacerbate disease transmission and increase disease severity. Attempts have been made to produce safer vaccines, including viral vector vaccines, subunit vaccines, and DNA vaccines, but with inconsistent efficacy results, and no effective vaccine against ASFV has yet been successfully produced. Summary of the Invention

[0006] As stated above, the object of the present invention is to provide a safe and / or effective alternative therapy for treating infections, particularly those caused by African swine fever virus (ASFV), thereby preventing severe clinical symptoms and death.

[0007] This invention provides a composition for the prevention and / or treatment of animal infections, comprising at least one antigen gene selected from the group consisting of: SEQ ID NO: 1 (S273R), SEQ ID NO: 2 (E183L), SEQ ID NO: 3 (K78R), SEQ ID NO: 4 (KP177R), SEQ ID NO: 5 (A104R), SEQ ID NO: 6 (A137R), SEQ ID NO: 7 (A151R), SEQ ID NO: 8 (B646R), SEQ ID NO: 9 (B438L), SEQ ID NO: 10 (B602L), SEQ ID NO: 11 (CP2475L), SEQ ID NO: 12 (CP204L), SEQ ID NO: 13 (CP530R), SEQ ID NO: 14 (O61R), SEQ ID NO: 15 (D117L), SEQ ID NO: 16 (H108R), SEQ ID NO: 17 (E199L), SEQ ID NO: 18 (E120R), SEQ ID NO: 19 (E248R), SEQ ID NO: 20 (MGF_110-4L), SEQ ID NO: 21 (EP402R), SEQ ID NO: 22 (MGF_505-5R), SEQ ID NO: 23 (MGF_360-12L), SEQ ID NO: 24 (DP96R), SEQ ID NO: 25 (A224L), SEQ ID NO: 26 (A179L), SEQ ID NO: 27 (I329L), SEQ ID NO: 28 (I10L), SEQ ID NO: 29 (I215L) and SEQ ID NO: 30(G1211R).

[0008] Preferably, the at least one antigen gene is selected from the group consisting of: SEQ ID NO: 2 (E183L), SEQ ID NO: 4 (KP177R), SEQ ID NO: 8 (B646L), SEQ ID NO: 12 (CP204L), SEQ ID NO: 13 (CP530R), and SEQ ID NO: 21 (EP402R).

[0009] Preferably, the at least one antigen gene is inserted into at least one plasmid vector.

[0010] Preferably, the composition further comprises a pharmaceutically acceptable carrier, more preferably nanoparticles, and even more preferably BioCapZ.

[0011] The present invention also provides a method for preventing and / or treating animal infections, comprising administering an effective amount of any of the above-described compositions to the animal.

[0012] Preferably, the composition is administered orally (OR), intramuscularly (IM), intravenously (IV), subcutaneously (SC), or intradermally, by inhalation (IN), aerosol, bioballistic particle delivery system, or using a gene gun.

[0013] Preferably, the concentration of the composition is from about 10 to about 100 µg / ml.

[0014] Preferably, the animals include pigs.

[0015] Preferably, the infection is caused by a pathogen, more preferably by a DNA virus or an RNA virus.

[0016] Preferably, the DNA virus comprises African swine fever virus.

[0017] Preferably, the RNA virus comprises classical swine fever virus.

[0018] Therefore, the present invention has at least the following advantages: (a) The composition for which protection is sought can be used as a DNA vaccine and has been shown to be safe and provide high protective efficacy against infection.

[0019] (b) The composition for which protection is sought exhibits a therapeutic effect, allowing ASF-infected animals to be treated after vaccination, thereby rendering the vaccinated animals free of ASF virus and without any clinical symptoms.

[0020] (c) This invention can effectively prevent the spread of disease, help eradicate disease, and promote the repopulation of populations after an outbreak of African swine fever. Attached Figure Description

[0021] Figure 1A The diagram illustrates a manufacturing process for an ASF pDNA vaccine and BioCapZ, delivered via different routes, according to one embodiment of the present invention.

[0022] Figure 1BThe diagram illustrates a manufacturing process for an ASF pDNA vaccine and BioCapZ delivered via the same route, according to one embodiment of the present invention.

[0023] Figure 2 The diagram shows the structure of the plasmid vector pcDNA3.1(+) / myc-His A according to one embodiment of the present invention.

[0024] Figure 3 The diagram shows the structure of the plasmid vector pcDNA3.1(+) / myc-His B according to one embodiment of the present invention.

[0025] Figure 4 The diagram shows the structure of the plasmid vector pcDNA3.1+N-eGFP according to one embodiment of the present invention.

[0026] Figure 5 The figure shows the relationship between nanoparticle size (in nm) and strength (%) measured using DLS (Malvern Zetasizer) according to an embodiment of the present invention.

[0027] Figure 6A The figure shows the concentration of dsDNA in a sample according to one embodiment of the present invention.

[0028] Figure 6B The figure shows the concentration of a protein according to one embodiment of the present invention.

[0029] Figure 7A The figure shown is a standard curve established using the Invitrogen PicoGreenQuant-IT dsDNA measurement reagent according to an embodiment of the present invention.

[0030] Figure 7B The image shows the fluorescence intensity of a sample according to one embodiment of the present invention. Detailed Implementation

[0031] The foregoing and other aspects of the invention will now be described in more detail by the embodiments set forth herein. It should be understood that the invention may be embodied in different forms and is not limited to those described herein. These embodiments are presented to ensure that the disclosure herein is comprehensive and to clearly convey the scope of the invention to those skilled in the art. Each embodiment and feature should be understood to be interchangeable and combined with each other embodiment and feature within this application.

[0032] All publications, patent publications, patents, and other references cited in this article are incorporated in their entirety by reference for the purpose of teaching in relation to the sentences and / or paragraphs in which they are presented.

[0033] definition The terminology used in this disclosure is for the purpose of describing particular embodiments only and is not intended to limit the invention. Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention pertains.

[0034] As used herein, the terms “a,” “an,” and “the” are intended to include both singular and plural forms unless the context clearly indicates otherwise.

[0035] As used herein, the terms “comprise(s)”, “comprising”, “include(s)”, “including”, “has / have”, “having”, “contain(s)”, “containing”, or any other variations thereof are intended to cover non-exclusive inclusion, subject to any expressly specified limitations. This means that a composition, mixture, procedure, or method comprising, for example, a list of elements is not limited to those individual elements, but may also cover compositions, mixtures, procedures, or methods not expressly listed.

[0036] As used herein, the term “about” refers to a value that includes inherent variations, such as measurement errors, variations in the methods used to determine the value, or variations between the subjects of study.

[0037] In the claims, the term "or" is used to mean "and / or" unless otherwise expressly stated to indicate alternatives or mutually exclusive options only. However, this disclosure supports the inclusion of alternatives as well as the definition of "and / or".

[0038] As used herein, the terms "infection prevention," "prevention of infection," or any other variations thereof refer to the purpose of the composition to prevent the occurrence of infections in animals. This can be achieved by stimulating the animal's immune system to produce antibodies against the infection. The composition is effective against a variety of infections, including but not limited to African swine fever.

[0039] As used herein, the terms “treatment of infection,” “treatment of infection,” or any variation thereof refer to the potential of the composition to have beneficial effects on animal health, including but not limited to reducing infection-related mortality, improving the quality of life of animals, and extending the lifespan of animals.

[0040] As used herein, the term "plasmid vector" refers to plasmids, shuttle vectors for prokaryotic or eukaryotic systems, and commercial plasmids for genetic engineering in the field.

[0041] In one embodiment, the composition for preventing and / or treating animal infections comprises at least one antigen gene selected from the group consisting of: SEQ ID NO: 1 (S273R), SEQ ID NO: 2 (E183L), SEQ ID NO: 3 (K78R), SEQ ID NO: 4 (KP177R), SEQ ID NO: 5 (A104R), SEQ ID NO: 6 (A137R), SEQ ID NO: 7 (A151R), SEQ ID NO: 8 (B646R), SEQ ID NO: 9 (B438L), SEQ ID NO: 10 (B602L), SEQ ID NO: 11 (CP2475L), SEQ ID NO: 12 (CP204L), SEQ ID NO: 13 (CP530R), SEQ ID NO: 14 (O61R), SEQ ID NO: 15 (D117L), SEQ ID NO: 16 (H108R), SEQ ID NO: SEQ ID NO: 17 (E199L), SEQ ID NO: 18 (E120R), SEQ ID NO: 19 (E248R), SEQ ID NO: 20 (MGF_110-4L), SEQ ID NO: 21 (EP402R), SEQ ID NO: 22 (MGF_505-5R), SEQ ID NO: 23 (MGF_360-12L), SEQ ID NO: 24 (DP96R), SEQ ID NO: 25 (A224L), SEQ ID NO: 26 (A179L), SEQ ID NO: 27 (I329L), SEQ ID NO: 28 (I10L), SEQ ID NO: 29 (I215L), and SEQ ID NO: 30 (G1211R). In one embodiment, a method for preventing and / or treating an animal infection is provided, comprising administering any of the compositions to the animal.

[0042] Genes encoding antigens Sequence listing of SEQ ID NO: 1-30 In one embodiment, the antigen gene of the present invention does not need to be a full-length gene. For example, a sequence fragment of the gene may be sufficient to encode an antigen that provides preventive and / or therapeutic efficacy to the composition. Therefore, such fragments should be considered within the scope of the present invention.

[0043] In one embodiment, at least one antigen gene is inserted into at least one plasmid vector to obtain a plasmid antigen gene, such as plasmid DNAs (pDNA). For example, the antigen gene may be inserted into the same plasmid vector or different plasmid vectors. This includes, but is not limited to, cases where two or more antigen genes are each inserted into a single plasmid vector, or cases where a single antigen gene is inserted into two or more plasmid vectors.

[0044] In one embodiment, the composition of the present invention may further comprise a pharmaceutically acceptable carrier, such as nanoparticles, preferably BioCapZ.

[0045] Nanoparticles can be used to encapsulate target substances and / or deliver target substances to specific cells or tissues in animals. Nanoparticles can improve the efficacy and safety of substance delivery. In one embodiment, an antigen gene or pDNA can be encapsulated in a nanoparticle or administered separately from the nanoparticle via the same or different routes, either simultaneously or at different times (see [link to implementation details]). Figure 1A and Figure 1B ).

[0046] Nanoparticles may include, but are not limited to, BioCapZ. BioCapZ is a transgenic, protein-capped, non-infectious immunostimulatory nanoparticle derived from hepatitis E virus (HEV). It retains the natural structural stability, antigenicity, and cell-binding ability of HEV, aiming to stimulate an immune response against the antigen in a low-toxicity and cost-effective manner. (Baikoghli, MA, Chen, CC, & Cheng, RH (2022). BioCapZ: A Highly Efficient Capsid-Based Nano-Platform for Non-Invasive Theranostics Delivery. Current Practice in Medical Science. Vol. 4, pp. 140-147, the contents of which are incorporated herein by reference in their entirety.)

[0047] In one embodiment, the composition is administered, for example, by oral (OR), intramuscular (IM) injection, intravenous (V) injection, subcutaneous (SC) or intradermal injection, inhalation (IN), aerosol, bioballistic particle delivery system or using a gene gun.

[0048] In one embodiment, the concentration of the composition is about 10 to about 100 µg / ml, about 15 to about 90 µg / ml, about 15 to about 70 µg / ml, about 15 to about 50 µg / ml, about 20 to about 90 µg / ml, about 20 to about 70 µg / ml, about 20 to about 45 µg / ml, about 25 to about 40 µg / ml, about 30 to about 80 µg / ml, about 30 to about 60 µg / ml, about 30 to about 35 µg / ml, about 40 to about 70 µg / ml, or about 50 to about 60 µg / ml. The concentration of the composition may be a concentrated form for storage and diluted to a working solution before administration to animals.

[0049] In one implementation, the animal is a pig (also called a swine). A pig can be any member of the genus *Sus*, including domestic pigs (*Sus scrofa domestica*), wild boars (*Sus scrofa*), and all other pig breeds that may be infected with classical swine fever.

[0050] In one embodiment, the infection may be caused by a pathogen. Preferably, the pathogen may be a DNA virus or an RNA virus. More preferably, the DNA virus is African swine fever virus; the RNA virus is classical swine fever virus.

[0051] In one embodiment, the present invention provides compositions and methods combining at least two antigen genes encoded with target antigens selected from SEQ ID NO: 1-30. These compositions can be delivered together with immune-enhancing nanoparticles (e.g., BioCapZ) to prevent and / or treat infections such as swine fever.

[0052] Example Materials and Methods Methods for manufacturing plasmid DNA vaccines (see steps 1-10) Step 1. Preparation of recombinant plasmid DNA: Recombinant plasmid DNA was constructed by inserting a gene encoding a target antigen into an appropriate plasmid vector. The target antigen was selected from the group consisting of: SEQ ID NO: 2 (E183L), SEQ ID NO: 4 (KP177R), SEQ ID NO: 8 (B646L), SEQ ID NO: 12 (CP204L), SEQ ID NO: 13 (CP530R), and SEQ ID NO: 21 (EP402R). The appropriate plasmid vector was, for example, pcDNA3.1(+) / myc-His A, pcDNA3.1(+) / myc-His B, and pcDNA3.1+N-eGFP, as shown below. Figures 2 to 4 As shown. Plasmid vectors contain essential elements, such as strong promoters and selectable markers.

[0053] Step 2. Transformation into E. coli: To transform plasmid DNA into competent E. coli cells, follow these steps: ●Step i: Thaw 50 µl of competent cells on ice.

[0054] ●Step ii: Add 1-5 µl of sample DNA (i.e., the plasmid DNA from step 1) to a test tube containing competent cells.

[0055] ●Step iii: Gently tap the test tube to mix gently, ensuring that the DNA is evenly distributed without damaging the cells.

[0056] ●Step iv: Incubate on ice for 30 minutes to allow DNA to adhere to the cells.

[0057] ●Step v: Heat shock the cells in a 42°C water bath for 30 seconds. Do not mix or shake during this step.

[0058] ●Step vi: Immediately place on ice for 2 minutes to stabilize the cells.

[0059] ●Step vii: Add 250 µl of SOC medium to the test tube and shake at 225 rpm for 1 hour at 37°C to promote cell recovery and the expression of antibiotic resistance.

[0060] ●Step viii: After transformation, spread the cells from each transformation onto LB agar plates containing antibiotics (e.g., ampicillin).

[0061] ●Step ix: Incubate the culture plate at 37°C overnight to allow colonies to grow.

[0062] Step 3. Select a single colony: Colony selection: Select individual colonies to ensure that a single clone with successfully incorporated plasmid DNA is chosen.

[0063] Step 4. Cell Bank: Establish a cell bank: Build and maintain a cell bank from selected single colonies to ensure a stable source of strains for future production. Store them under appropriate conditions to maintain their viability.

[0064] Step 5. Fermentation: Scale-up culture: Culture cells from a cell bank under controlled conditions in a larger fermenter or bioreactor to increase biomass. Ensure optimal growth conditions, including temperature, pH, and aeration, to maximize plasmid DNA yield.

[0065] Step 6. Centrifugal harvesting: Cell harvesting: After fermentation, bacterial cells are harvested by centrifugation. This separates the cells from the growth medium and concentrates the cell clusters for further processing.

[0066] Step 7. Cell lysis via alkaline lysis: Cell disruption: Alkaline lysis is performed to break down bacterial cells and release plasmid DNA into solution. This method involves using an alkaline buffer followed by neutralization to ensure the plasmid DNA remains intact while removing cell debris.

[0067] Step 8. Purification: DNA purification: Plasmid DNA is purified from the lysate using techniques such as column chromatography or ultrafiltration. This step removes contaminants, including proteins, RNA, and other impurities, to isolate high-purity plasmid DNA.

[0068] Step 9. Aseptic filling: Formulation: Dissolve the purified plasmid DNA in an appropriate buffer solution to prepare the final vaccine formulation. Perform aseptic filling, transferring the vaccine solution to sterile vials or containers under aseptic conditions to prevent contamination.

[0069] Detailed process of steps 3-9: Preparation of bacterial cultures (#Starter1): 1. Preparation of bacterial strains: The initial bacterial strains are stored in glycerol for long-term preservation.

[0070] 2. Streak isolation: Streak the glycerol culture solution onto an LB agar plate containing ampicillin (100 µg / ml) to isolate a single colony.

[0071] 3. Incubation: Place the LB agar plate in a 37°C incubator overnight to allow colonies to grow.

[0072] 4. Inoculation of the starting culture: Select a single colony from an LB agar plate and inoculate it into 5 ml of LB medium containing ampicillin (100 µg / ml).

[0073] 5. Culture growth: Place the culture on a shaker at 37°C and 200 rpm for 16-18 hours to allow for full bacterial growth.

[0074] 6. Prepare bacterial culture (#Starter1): Use this culture to prepare larger volumes of bacterial cells as needed for plasmid DNA preparation.

[0075] 7. Plasmid DNA Extraction: Plasmid DNA was extracted from bacterial cultures using the QIAprep® Spin Miniprep Kit. Plasmid DNA concentration was measured using a Nanodrop spectrophotometer, and microbial growth was assessed by measuring the optical density at 600 nm (OD600).

[0076] 8. Preserve the culture: Store the prepared bacterial culture (#Starter1) at 4°C for future use.

[0077] Preparation of the working cell bank (#Starter 2): 1. Using bacterial culture (#Starter1): Transfer 0.30 ml of #Starter1 culture to 30 mL of LB medium containing ampicillin (100 µg / ml).

[0078] 2. Growth of the culture: Incubate the culture on a shaker at 37°C and 200 rpm for 16-18 hours.

[0079] 3. Store the culture: Store the prepared working cell bank (#Starter 2) at 4°C for future use.

[0080] Preparation of the final culture: 1. Scale-up culture: Transfer 1 ml of #Starter 2 culture to 1,000 ml of LB medium containing ampicillin (100 µg / ml).

[0081] 2. Growth of the final culture: The culture was incubated in a shaker at 37°C and 200 rpm for 16-18 hours to achieve the final bacterial density required for plasmid DNA production.

[0082] Centrifugation and plasmid DNA extraction were performed using the EndoFree® Plasmid Giga Kit: 1. Harvesting cells: Centrifuge the final culture at 8,000 xg for 10 minutes at 4°C. Carefully discard the supernatant and freeze the cell pellet at -20°C.

[0083] 2. Resuspending bacteria: Resuspend the bacterial precipitate in 32 ml of Buffer P1.

[0084] 3. Ligation: Add 32 ml of Buffer P2, invert the test tube 4-6 times to mix thoroughly, then add 32 ml of Buffer P3, invert the test tube vigorously 4-6 times to mix thoroughly, until the solution is completely colorless.

[0085] 4. Filter the lysis buffer: Pour the lysis buffer into a filter and incubate at room temperature for 10 minutes. Vacuum filter and add 50 ml of Buffer FWB2. Add 30 ml of Buffer ER to the filtered lysis buffer, incubate on ice for 30 minutes, and then add 75 ml of Buffer QBT to equilibrate the column.

[0086] 5. Add lysis buffer: Add the filtered lysis buffer to the column and wash with 600 ml of buffer QC.

[0087] 6. Wash plasmid DNA: Wash plasmid DNA with 100 ml of Buffer QN (preheating the buffer to 65°C can help increase yield). Add 70 ml of isopropanol to the wash buffer and mix to precipitate the plasmid DNA.

[0088] 7. Centrifugation and washing: Centrifuge at 15,000 xg for 30 minutes at 4°C, carefully pour off the supernatant, and wash the DNA with 70% ethanol (without endotoxin) at room temperature to precipitate the DNA.

[0089] 8. Final centrifugation and drying: Centrifuge at 15,000 xg for 10 minutes at 4°C and carefully discard the supernatant. Air dry the particles for 10-20 minutes.

[0090] 9. Redissolve the DNA precipitate: Redissolve the DNA precipitate in Buffer TE, measure the concentration with a Nanodrop spectrophotometer, aliquot the sample, and store at -20°C.

[0091] Solution preparation: Solution 1: 1. Thawing plasmid DNA: Thaw plasmid DNA stored at -20℃ at room temperature before use and use it within 30 minutes.

[0092] 2. Solution preparation: Rotate at 4,000 rpm for 5 seconds. Add the carrier to the container, then add the plasmid DNA and stir for 15 minutes. Add the additional vector and stir for another 15 minutes.

[0093] 3. Divide and store: Divide each vial into 2.5 ml portions (one vial is used for 5 injections) and store at -20°C. The total volume of each vial is 2.5 ml.

[0094] Solution 2: 1. Solution preparation: Add the carrier to the container, then add the nano-adjuvant and stir for 15 minutes. Add additional carrier and stir for another 15 minutes.

[0095] 2. Divide and store: Divide each vial into 12 ml portions (one vial is used for 20 injections) and store at -20°C. The total volume of each vial is 12 ml.

[0096] Step 10. Quality Assurance and Quality Control: Quality Control: Comprehensive quality assurance and quality control testing is conducted to ensure that the plasmid DNA vaccine meets all required specifications. This includes assessing DNA concentration, purity, and integrity, as well as performing sterility tests and verifying whether the vaccine elicits the expected immune response in preclinical testing.

[0097] Carrier design Mutations: 1. Deliver at least two plasmids intramuscularly, each plasmid expressing an antigen (e.g., Experimental Example 1).

[0098] 2. Intramuscular delivery of at least one plasmid expressing at least two antigens.

[0099] 3. Intramuscular delivery of at least two plasmids, each expressing one antigen, and an orally administered immunostimulant nanoparticle (e.g., BioCapZ; see below). Figure 1A (Compared with Experimental Example 2).

[0100] 4. Intramuscular delivery of at least one plasmid expressing at least two antigens, and an orally administered immunostimulant nanoparticle (e.g., BioCapZ; see below). Figure 1A (Compared with Experimental Example 2).

[0101] 5. At least two plasmids, each expressing one antigen, are encapsulated / encapsulated separately in immunostimulatory particles (e.g., BioCapZ), but delivered orally together (see [link to product description]). Figure 1B (Compared with Experimental Example 3).

[0102] 6. At least one plasmid expressing at least two antigens, encapsulated / encapsulated in an immunostimulant particle (e.g., BioCapZ), for oral delivery (see [link to product description]). Figure 1B (Compared with Experimental Example 3).

[0103] Experimental Example 1 A vaccine (ASF DNA vaccine) was formulated with codes derived from six different antigens of African swine fever virus type II genotype and its safety and efficacy were tested.

[0104] Specifically, a combination of six plasmids containing antigen genes (E183L, KP177R, B646L, CP204L, CP530R, and EP402R), each expressing a different antigen, was prepared using the aforementioned plasmid DNA vaccine manufacturing method and administered as a vaccine to pigs via intramuscular injection. The efficacy of the vaccine in preventing ASFV infection was evaluated by comparing vaccinated and unvaccinated pigs under controlled conditions and in field trials conducted on farms exposed to the virus.

[0105] The study is divided into three phases: Phase I, Phase II, and Phase III.

[0106] Phase I study: Target The purpose of this study was to evaluate the efficacy and safety of an ASF DNA vaccine in preventing ASFV infection under field conditions. The primary endpoints were the survival rate of vaccinated pigs and the safety of the ASF DNA vaccine, particularly monitoring for any adverse reactions after vaccination.

[0107] Research Location The study consisted of two independent trials conducted during an acute outbreak of ASF at two separate farms located in Nakhon Sri Thammarat and Ratchaburi provinces of Thailand.

[0108] The first trial (Trial 1) was conducted at a single-site production farm in Nakhon Si Thammarat province, with 4,500 sows. The trial took place in one of eight nursery pens, each capable of accommodating approximately 1,800 weaned piglets. All eight nursery pens were enclosed structures with tunnel ventilation.

[0109] The second trial (Trial 2) was conducted at a single-site production farm in Ratchaburi province, which housed 2,500 sows. The trial took place at one of two nursery facilities approximately one kilometer from the main farm. This facility consisted of three open-air buildings, each capable of housing 400 pigs, including ASFV-infected pigs transported from the main farm.

[0110] Experimental Design i. Animals ● Experiment 1 included 25 pigs (20 sows and 5 boars). All pigs were crossbred (Large White x Landrace x Duroc) with an average weight of 25 kg.

[0111] ● Experiment 2 included 30 pigs (15 sows and 15 boars). All pigs were crossbred (Large White x Landrace x Duroc) with an average weight of 25 kg.

[0112] ii. Animal management and feeding ● Experiment 1 was conducted in one of eight nursery buildings, each capable of housing approximately 1,800 weaned piglets. All eight nursery buildings were enclosed structures with tunnel ventilation. After the experiment began, some pigs in the research building tested positive for African swine fever via PCR.

[0113] ●Trial 2 was conducted in one of three open-air facilities. All pigsties were used to house pigs infected with African swine fever, with approximately 400 pigs in each building. The facility had previously experienced a severe African swine fever outbreak, housing approximately 400 pigs, some of which tested positive for African swine fever via PCR.

[0114] ● Unlimited supply of feed and water.

[0115] ● Implement existing standard operating procedures for biosafety on farms.

[0116] iii. Animal selection and administration Before being transferred to the experimental facility, all pigs were visually screened according to the following criteria: ● All animals were outwardly and clinically healthy, with no obvious signs of fever, red skin, serious illness, trauma, or lameness.

[0117] ●For animal welfare reasons, cases of mild clinical symptoms that occur during the study will receive necessary treatment under the supervision of the attending veterinarian.

[0118] ●All other vaccinations and support are provided in accordance with farm practices.

[0119] The ASF DNA vaccine is prepared in buffered saline and mixed with an equal volume of adjuvant (polyethyleneimine) to obtain a final concentration of 28 µg DNA vaccine per ml. Each dose contains either 1 ml (28 µg DNA) or 2 ml (56 µg DNA) of vaccine formulation.

[0120] iv. Animal handling and positioning ● Vaccinated pigs were kept in separate pens but shared the same building's airspace as pigs infected with African swine fever virus. Vaccination treatments differed between the different treatment groups.

[0121] ●The dead animals were dumped in the farm’s existing burial pits.

[0122] v. Treatment group and vaccination Experiment 1: 25 crossbred pigs (n=25), each weighing approximately 25 kg, were randomly divided into three groups as follows: ●Group 1 (n=15; 10 sows and 5 boars): Each pig was immunized by intramuscular injection of 1 ml (28 µg) ASF DNA vaccine weekly for 3 consecutive weeks.

[0123] ●Group 2 (n=5; all sows): Each pig was immunized by intramuscular injection of 2 ml (56 µg) ASF DNA vaccine weekly for 3 consecutive weeks.

[0124] ●Group 3 (n=5; all sows): Unvaccinated.

[0125] Experiment 2: 30 hybrid pigs (n=30), each weighing approximately 25 kg, were randomly divided into the following two groups; ●Group 1 (n=25): Each pig was immunized by intramuscular injection of 1 ml (28 µg) ASF DNA vaccine, and was immunized again with the same dose two weeks later. A second immunization was given four weeks after the first immunization.

[0126] ●Group 2 (n=5): Unvaccinated.

[0127] vi. Experimental unit ●Each individual pig is an experimental unit.

[0128] ● Each pig is tagged and assigned a unique tag number.

[0129] ● All treatment groups are mixed within each pen.

[0130] vii. Blood collection and serological testing, and qRT-PCR ●For Experiment 1, RT-PCR was used to monitor whether pigs were infected with ASF on days 0, 14, 28, and 50 post-vaccination. On day 70 post-vaccination, antibody response against ASF B646L (p72) was measured by ELISA.

[0131] ●For Experiment 2, RT-PCR was used to monitor whether pigs were infected with ASF on days 0, 14, 28 and 50 after vaccination.

[0132] viii. Animal disposal after a confirmed outbreak of African swine fever Animals that died from African swine fever were buried in burial pits.

[0133] ix. Parameters 1. Mortality rate and animal health monitoring (day 0 to day 90) ● Adverse reactions occurring immediately after vaccination until 21 days after vaccination: All adverse events will be recorded, especially swelling / inflammation, pain, redness, abscess, lump, lesion, and fever at the injection site.

[0134] ● Mortality rate: Record labels, date of death, and observed clinical symptoms.

[0135] ●Incidence: Observe the animals for clinical signs and symptoms daily.

[0136] 2. Serological reaction ● All samples should be tested for ASF antibodies using an ELISA test targeting P72.

[0137] 3. Virus testing ● Quantify the presence of the virus using qRT-PCR.

[0138] Evaluation and conclusions of the results i. Mortality rate and vaccine efficacy Experiment 1: ● After vaccination, body temperature remains within the normal range for 7 consecutive days.

[0139] ●Day 0: Some experimental pigs were pre-exposed to ASF, as shown by PCR(+) (see Table 1, PCR0DPV column).

[0140] ●Day 14: Vaccinated pigs showed mixed responses, including positive and negative RT-PCR results in the presence of ASF (see Table 1, PCR14DPV column).

[0141] ●Day 28: According to RT-PCR, all 20 vaccinated pigs tested negative for ASF (see Table 1, PCR28DPV column).

[0142] ● All five tagged, unvaccinated pigs and approximately 1,000 untagged, unvaccinated pigs died.

[0143] ● All vaccinated pigs survived. According to ELISA, the vaccinated pigs also showed anti-P72 antibodies (see Table 1, P72 70DPV column).

[0144] ●The sows later became breeding pigs for farm reproduction.

[0145] ● Castrated pigs are sold at market weight.

[0146] Table 1: Results of Experiment 1 Experiment 2: ● After 90 days, all 25 vaccinated animals in Group 1 were healthy and ASFV negative.

[0147] ● All five unvaccinated pigs in Group 2 died within two weeks of being transferred to the research facility.

[0148] ii. Observe the adverse reactions, clinical symptoms, and safety of the vaccine. No adverse reactions were observed associated with administration of the ASF DNA vaccine.

[0149] Summarize This study was conducted in two independent trials on a small group of pigs to evaluate the working dose and protective efficacy under natural field challenges. The first trial was conducted in an evaporative cooling facility housing approximately 1,800 pigs that tested positive for African swine fever (ASF) virus, some of whom had experienced an acute outbreak of ASF. The first group consisted of 15 vaccinated pigs, each receiving a 1 ml (28 µg) dose of ASF DNA vaccine. The second group consisted of 5 pigs, each receiving a 2 ml (56 µg) dose of ASF DNA vaccine. Both groups received three doses at one-week intervals. The third group of 5 pigs received no vaccine. All groups were housed in the same facility as 1,800 unvaccinated ASF-positive pigs. After 28 days, all 20 vaccinated pigs in both groups survived, while all 5 unvaccinated pigs (including the other 1,800 ASF-positive pigs) died from ASF. Ultimately, the surviving, vaccinated sows became breeding stock, while the castrated pigs were sold at market weight. A second trial was conducted in an open-air pigsty housing 400 pigs transferred from a herd that had experienced an acute outbreak of African swine fever (ASF). RT-PCR revealed that some of these pigs were ASF-positive. Thirty pigs (25 kg each) that tested negative for ASF via RT-PCR were randomly assigned to two groups. The first group, consisting of 25 pigs, received 2 ml (56 µg) of ASF DNA vaccine every two weeks. The second group of 5 pigs was not vaccinated. Both groups were housed in the same open-air facility that had experienced the ASF outbreak. After 90 days, all 25 vaccinated pigs survived, while all 5 unvaccinated pigs died from ASF. No adverse reactions were observed after ASF DNA vaccination in either trial. In conclusion, the results demonstrate that, in addition to safety, ASF DNA vaccination at the recommended dose provides protection against environmental challenges.

[0150] Phase II study: Target: The study was conducted to further verify the safety and efficacy of the ASF DNA vaccine in preventing ASF infection in a large population under field conditions, as well as the potential of the ASF DNA vaccine to treat ASF-infected animals.

[0151] Research Location The study consisted of two independent trials conducted during acute outbreaks at two separate farms located in Nakhon Si Thammarat and Ratchaburi provinces of Thailand.

[0152] The first trial (Trial 1) was conducted at a single-site production farm with 4,500 sows in Nakhon Si Thammarat province during an acute outbreak of ASF. The trial took place in six weaning-to-finishing pig pens, each capable of accommodating approximately 1,000 pigs. All six buildings were enclosed structures with tunnel ventilation.

[0153] The second trial (Trial 2) was conducted at a single-site production farm with 2,500 sows in Ratchaburi province during an acute outbreak of ASF. The trial took place in 14 weaning-to-finishing pig pens, each accommodating approximately 650 pigs. All 14 pens were enclosed and equipped with tunnel ventilation.

[0154] Experimental Design i. Animals ●Species / Breed: Pigs / Hybrids (Large White x Landrace x Duroc) ● Number of animals: 8,032 pigs ●Source: African swine fever virus-negative pig herds from other provinces ●Gender / Age / Weight: Experiment 1 included 5,042 pigs (10-week-old boars and sows, 25 kg).

[0155] Experiment 2 included 2,990 pigs (10-week-old boars and sows, 25 kg).

[0156] ii. Animal management and feeding Housing: All experimental animals were housed in different buildings.

[0157] ●Trial 1 was conducted at a farm in Ratchaburi province during a severe outbreak of the disease. The farm had 14 evaporative cooling houses, each capable of housing 650 pigs. The farm had previously been affected by the ASF outbreak and had been emptied for 2-4 weeks.

[0158] ● Experiment 2 was conducted on a farm in Nakhon Si Thammarat province during the height of the outbreak. There were six evaporative cooling houses, each capable of housing 1,000 pigs, while adjacent buildings housed pigs infected with African swine fever.

[0159] ● Unlimited supply of feed and water.

[0160] ● Implement existing standard operating procedures for biosafety on farms.

[0161] iii. Animal selection and administration Similar to Phase I study.

[0162] iv. Animal handling and positioning ● These animals were housed in different buildings. Vaccination treatments differed between the different treatment groups.

[0163] ●The dead animals were dumped in the farm’s existing burial pits.

[0164] v. Treatment group and vaccination Trial 1: A total of 5,042 pigs weighing approximately 20 kg were introduced from outside and housed in 12 separate buildings. Three batches of 25 kg pigs from two different sources (NH and T) were brought in. Since one building was introduced each week, two vaccination programs were implemented.

[0165] ● Two buildings (700 pigs) were treated with an intramuscular injection of 1 ml of ASF DNA vaccine (28 µg), followed by a second dose of the same vaccine two weeks later.

[0166] ● Nine buildings (3,293 pigs) were given 2 ml of ASF DNA vaccine (56 µg) via intramuscular injection, followed by the same dose of vaccine two weeks later.

[0167] ● Upon arrival at the three buildings (1,049 pigs), 30 pigs (10 pigs per building) were tested. Results showed that 4 pigs tested positive for African swine fever (ASF) by RT-PCR (1-2 positive pigs per building). All pigs, including the ASF-positive pigs, were given an intramuscular injection of 2 ml of ASF DNA vaccine (56 µg), followed by a second vaccination with the same dose two weeks later. No blood samples were collected for analysis; only clinical symptoms and mortality were monitored. Pigs were given two intramuscular injections of 2 ml of ASF DNA vaccine (56 µg) two weeks apart. Pigs residing in the three buildings tested positive for ASF virus by qPCR upon arrival and were vaccinated with the same vaccine as the other ASF-negative pigs.

[0168] ● Monitor pig mortality rates until 22 weeks after the first immunization.

[0169] Trial 2: The second trial was conducted on a separate farm that had recently experienced an African swine fever outbreak. A total of 2,990 African swine fever-negative pigs, with an average weight of 21 kg, were imported from outside and housed in six separate buildings. Since one building was introduced each week, three vaccination programs were implemented.

[0170] ● Five hundred pigs were introduced into the first building and immunized with 1 ml of ASF DNA vaccine (28 µg) via intramuscular injection. However, some pigs in this group developed ASF-related clinical symptoms two weeks after introduction. The group was then further immunized three times with 2 ml of ASF DNA vaccine (56 µg), each two weeks apart. Following the initial booster dose of 2 ml of ASF DNA vaccine (56 µg), ASF-related clinical disease appeared to decrease. The mortality rate was only 4.40% 22 weeks after the first vaccination.

[0171] ●The next four buildings (2,140 pigs) were immunized with a standard 2 ml dose of ASF DNA vaccine (56 µg), and then twice more with the same dose every two weeks.

[0172] ● Four pigs died during transport in the last building, and 30 of them tested positive for African swine fever via RT-PCR. The pigs in the last building were immunized with a standard 2 ml dose of ASF DNA vaccine (56 µg), and then given two more doses of the same vaccine every two weeks.

[0173] vi. testing unit ● Each animal is an experimental unit.

[0174] ● Each pig is tagged and assigned a unique tag number.

[0175] ● Each processing group has a different colored ear tag.

[0176] ● All treatment groups are mixed within each pen.

[0177] vii. Blood collection and serological testing, and qRT-PCR For Experiments 1 and 2, no blood was collected, and no serological tests or RT-PCR were performed. However, RT-PCR was used to screen animals for inclusion in the experiments. Serological testing was not performed.

[0178] viii. Animal disposal after a confirmed outbreak of African swine fever Animals that died from African swine fever were buried in burial pits.

[0179] ix. Parameters Mortality and animal health monitoring (0 to 22 weeks post-vaccination) ● Adverse reactions occurring immediately after vaccination until 21 days after vaccination: All adverse events will be recorded, especially swelling / inflammation, pain, redness, abscess, lump, lesion, and fever at the injection site.

[0180] ● Mortality rate: Record labels, date of death, and observed clinical symptoms.

[0181] ●Incidence: Observe the animals for clinical signs and symptoms daily.

[0182] Evaluation and conclusions of the results i. Mortality rate and vaccine efficacy Experiment 1: A total of 5,042 pigs were introduced from outside and housed in 14 separate buildings. Two vaccination regimens were implemented: 1 ml and 2 ml. In two buildings (700 pigs), 1 ml of ASF DNA vaccine (28 µg) was administered intramuscularly, followed by vaccination with the same dose two weeks later. For the 2 ml dose group, pigs in buildings 9 and 3 (3,293 and 1,049 pigs, respectively) were administered 2 ml of ASF DNA vaccine (56 µg) intramuscularly, followed by vaccination with the same dose two weeks later. At 22 weeks post-vaccination, the mortality rates for the three groups were only 8.28%, 2.04%, and 7.24%, respectively. Notably, four pigs in the last three buildings tested positive for ASF PCR upon arrival (1-2 pigs per building). After vaccination, the mortality rate was only 7.24%. In summary, in Experiment 1, all pigs, including ASF-positive pigs, received 2 ml of ASF DNA vaccine (56 µg) intramuscularly, followed by a second vaccination with the same dose two weeks later. No blood samples were collected for analysis; only clinical signs and mortality rates were monitored in the animals. Twenty-two weeks after the initial immunization, 201 animals contracted ASF and died, while the remaining vaccinated animals (n = 4,841) survived (96%) and tested negative for ASF by RT-PCR. The surviving animals appeared healthy, showed no clinical signs, and were sold to the market once they reached marketable size.

[0183] Experiment 2: A total of 2,990 African swine fever (ASF)-negative pigs were introduced from outside the farm and housed in six separate buildings. Two vaccination regimens were implemented: 1 ml and 2 ml. The first building (500 pigs) was immunized with 1 ml of ASF DNA vaccine (28 µg) intramuscularly. However, some pigs in this group developed ASF-related clinical symptoms two weeks after introduction. This group was then further immunized three times with 2 ml of ASF DNA vaccine (56 µg), each two weeks apart. Following the initial 2 ml ASF DNA vaccine (56 µg) booster vaccination, ASF-related clinical disease appeared to decrease. The mortality rate was only 4.40% 22 weeks after the first vaccination. The next four buildings (2,140 pigs) were immunized with the standard 2 ml ASF DNA vaccine (56 µg), followed by two more doses of the same vaccine every two weeks. The mortality rate was only 1.95% 22 weeks after the first vaccination. Notably, four pigs died during transport in the last building, and 30 of them tested positive for African swine fever (ASF) via RT-PCR. The pigs in the last building were immunized with a standard 2 ml dose of ASF DNA vaccine (56 µg), followed by two more doses of the same vaccine every two weeks. Twenty-two weeks after the initial vaccination, the mortality rate was only 5.43%. All other pigs survived without any clinical symptoms and tested negative for ASF via RT-PCR, including the initial 30 pigs that tested positive for the virus.

[0184] In the farm’s F29 building, all 1,214 unvaccinated pigs died from an outbreak of African swine fever.

[0185] ii. Observe the adverse reactions, clinical symptoms, and safety of the vaccine. No adverse reactions were observed associated with administration of the ASF DNA vaccine.

[0186] Summarize: A preliminary Phase I study involving 55 pigs in two independent trials demonstrated some protective and therapeutic effects of the ASF DNA vaccine. To validate these observations with a larger sample size, two larger independent confirmatory trials were conducted, focusing primarily on clinical symptoms and mortality. The results showed that, in addition to demonstrating safety, administration of the ASF DNA vaccine at the recommended dose also provides protection against environmental challenges.

[0187] Phase III Study: Target: The study aimed to evaluate the therapeutic effect of the ASF DNA vaccine on animals infected with ASF.

[0188] Research location: The trial was conducted in Sa Kaeo Province, Thailand. This study was carried out on a farm that had experienced an African swine fever outbreak, where 2,000 pigs had to be euthanized to prevent the spread of the infection due to a 100% mortality rate among infected pigs. Prior to the study, environmental sampling revealed the presence of the African swine fever virus on the farm.

[0189] Experimental Design i. Animals ●Species: Pig ●Number of animals: 20 pigs ●ASFV negative farm ●Sex / Age / Weight: Castrated boar / 4 weeks old / 7 kg ●Identification method: Ear tag Twenty castrated boars (n=20), each four weeks old, were housed in two separate pens, with ten boars in each pen. The boars were kept in the pen for two weeks, and then exposed orally and nasally to a substance containing Ct with a value of 25 (approximately 10). 6 HAD 50 ASF virus samples were collected at a rate of 2 ml / ml. One week after exposure to ASF virus (challenge), all pigs were vaccinated twice with 2 ml of ASF DNA vaccine (56 µg), with each vaccination two weeks apart. Blood samples were collected weekly from each pig and serological tests were performed using PCR and ELISA to detect the presence of the virus.

[0190] ii. Animal management and feeding Feeding: 20 pigs were brought in and kept in two separate pens, with 10 pigs in each pen.

[0191] ● Unlimited supply of feed and water.

[0192] ● Implement existing standard operating procedures for biosafety on farms.

[0193] iii. Animal selection and administration Similar to Phase I and Phase II studies.

[0194] iv. Animal handling and positioning ● The animals are kept in different pens, with 10 pigs in each pen.

[0195] ● Animals were given two doses of 2 ml ASF DNA vaccine (56 µg) two weeks apart.

[0196] ●The dead animals were dumped in the farm’s existing burial pits.

[0197] v. Treatment group and vaccination All 20 pigs received the same challenge and vaccination regimen, namely two doses of 2 ml ASF DNA vaccine (56 µg) two weeks apart.

[0198] vi. testing unit Similar to Phase II study.

[0199] vii. Blood collection and serological testing, and qRT-PCR Blood was collected before immunization and every two weeks. The presence of antibodies in the blood was analyzed by ELISA, and the presence of ASF virus was analyzed by PCR.

[0200] viii. Animal disposal after a confirmed outbreak of African swine fever Animals that died from African swine fever were buried in pits.

[0201] ix. Parameters 1. Mortality rate and animal health monitoring (day 0 to day 150) ● Adverse reactions occurring immediately after vaccination up to 21 days after vaccination: All adverse events will be recorded, especially swelling / inflammation, pain, redness, abscess, lump, lesion, and fever at the injection site.

[0202] ● Mortality rate: Record labels, date of death, and observed clinical symptoms.

[0203] ●Incidence: Observe the animals for clinical signs and symptoms daily.

[0204] 2. Serological reaction ● All samples should be tested for ASF antibodies using an ELISA test targeting P72.

[0205] 3. Virus testing ● Quantify the presence of the virus using qRT-PCR.

[0206] Evaluation and conclusions of the results i. Mortality rate and vaccine efficacy All 20 heads were pre-exposed to a Ct value of 25 (approximately 10). 6 HAD 50 Pigs that tested negative for ASF virus ( / ml) after vaccination remained healthy.

[0207] ii. Observe the adverse reactions, clinical symptoms, and safety of the vaccine. No adverse reactions were observed associated with administration of the ASF DNA vaccine.

[0208] Summarize An experimental plasmid DNA vaccine mixture encoding six different antigens derived from African swine fever virus (ASF DNA vaccine) was formulated, and its safety and efficacy were tested. At least four independent studies consistently demonstrated the safety and efficacy of this DNA vaccine formulation. An interesting and unexpected feature of this vaccine is its ability to reverse ASF virus infection and its potential to treat animals infected with ASF virus.

[0209] Experimental Example 2 BioCapZ and pDNA were administered concurrently as an ASFV vaccine. Field trials evaluated the enhanced humoral immune response when BioCapZ and anti-ASFV pDNA were administered concurrently.

[0210] The dosage of BioCapZ varies depending on the breed of pig. For sows weighing between 250 and 300 kg, the dosage of BioCapZ is 6 mg per dose, delivered in a total volume of 10 ml. In contrast, for piglets weighing 6 kg, the dosage of BioCapZ is 0.12 mg per dose, delivered in a volume of 0.2 ml.

[0211] The study involved two methods: sow trials and piglet trials.

[0212] In the sow trial, 15 multiparous sows, each weighing between 250 and 300 kg, were randomly selected and equally assigned to the following three treatment groups: ●Group A: Sows were given two intramuscular injections of the DNA vaccine, 2 ml each time, with a two-week interval.

[0213] ●Group B: Sows received the same DNA vaccine regimen as Group A, but were also given an additional 10 ml of BioCapZ via intramuscular injection.

[0214] ●Group C: Sows received two intramuscular injections of 2 ml of DNA vaccine every two weeks, and 10 ml of BioCapZ was added to their feed for 20 consecutive days, starting 3 days before the first vaccination and continuing until 3 days after vaccination.

[0215] Serum samples were collected from all sows on days 0, 14, 28, and 42 post-vaccination. ASFV P30 and P72 antibodies were detected using ELISA (IDVet commercial kit or in-house ELISA), along with any adverse reactions such as fever and daily monitoring of fasting.

[0216] In the piglet trial, 15 piglets weighing 6 kg each were randomly selected and divided into three treatment groups: ●Group A: Piglets were given two intramuscular injections of the DNA vaccine, 2 ml each time, with a two-week interval.

[0217] ●Group B: Piglets received the same DNA vaccine regimen as Group A, but were additionally given an intramuscular injection of 0.20 ml of BioCapZ.

[0218] ●Group C: Piglets were also injected with two 2 ml doses of DNA vaccine every two weeks and given 0.2 ml of BioCapZ orally for 20 consecutive days, starting 3 days before the first vaccination and continuing until 3 days after the booster vaccination.

[0219] Serum samples were collected from all piglets on days 0, 14, 28, and 42 post-vaccination to test for antibodies against ASFV P30 and P72 by ELISA, while monitoring for any adverse reactions such as fever and fasting daily.

[0220] Observation of adverse reactions, clinical signs and safety of vaccines No adverse reactions were observed associated with administration of ASF DNA vaccines with or without BioCapZ. BioCapZ has demonstrated immune-enhancing effects in field trials of DNA vaccines against African swine fever virus (ASFV) with promising results.

[0221] Experimental Example 3 HEVNP packaged in ASFV carrier 1. DLS measurements of complete, disassembled, and reassembled HEVNPs See Figure 5 The figure shows the relationship between nanoparticle size measurements (in nm) and intensity (%) obtained using DLS (Malvern Zetasizer).

[0222] (A) Intact HEVNP: The size of the purified, intact nanoparticles (without any encapsulated payload) is 25 nm. Note that the nanoparticles shown here are not purified; they are derived from the supernatant of the expression cell lines.

[0223] (B) Disassembled HEVNPs: The nanoparticles can be disassembled to facilitate the encapsulation of the payload (pDNA in this study). The disassembly process involves adding a reducing agent (1 mM EGTA and 20 mM DTT) and incubating at room temperature for 30 minutes, followed by spin purification using a desalting column. After disassembly, the nanoparticle size was approximately 10 nm as measured by DLS.

[0224] (C) Reassembled HEVNP: Similarly, the nanoparticles can be reassembled by adding CaCl2 in increments of up to 5 mM per hour for four hours, followed by the spin cleaning procedure described above.

[0225] 2. Add load to the split HEVNP for packaging. The study utilized two plasmid DNAs encoding different antigenic regions of ASFV: B646L (encoding p72, the major capsid protein required for viral entry into cells) and KP177R (encoding p22, a structural protein of ASFV required for viral replication). The plasmid encoding B646L was 3311 bp in size, and the plasmid encoding KP177R was 1889 bp in size.

[0226] For the encapsulation process, 150 µg of plasmid was used to mix plasmid DNA with nanoparticles (HEVNP) at a 1:1 ratio.

[0227] Previous experiments have shown that HEVNP has limitations on the effective encapsulation size if the plasmid DNA is less than 3 kb.

[0228] Perform the following encapsulation: ●pDNA: KP177R (1889 bp) ●pDNA: B646L (3311 bp) ● pDNA concentrated with arsenic (2 mM): B646L 3. DNase (DNase) treatment to remove unincorporated pDNA After encapsulation and reassembly of the nanoparticles, the samples were divided into two groups: ● DNAse treatment (DnoZ): Perform DNAse treatment (37°C for 30 minutes) to digest unincorporated plasmid DNA, eliminating the need for rotation cleaning. ● Use rotation cleanup (DZ) for DNAse treatment: Perform DNAse treatment (37°C for 30 minutes), followed by rotation cleanup to digest unincorporated plasmid DNA.

[0229] 4. Nanodrop measurement of double-stranded DNA / protein cc.

[0230] After DNAse treatment (with or without spin-cleaning), assess the concentrations of double-stranded DNA (dsDNA) (ng / µl) and protein (mg / ml) in the sample. See also Figure 6A and Figure 6B In the two graphs, the X-axis represents the sample (each bar represents each sample type), while the Y-axis indicates the nucleic acid concentration (ng / µl) or protein concentration (mg / ml).

[0231] Example tag description: ●NP = Nanoparticles (complete) ●Disassemb = Nanoparticles that have been separated (without effective loading). ●Rea = Reassembled nanoparticles (without effective loading) ●DNoZ KP NP = Nanoparticles encapsulated with plasmid DNA KP177R as a payload, reassembled and treated with DNAse but not spin-cleaned. ●DNoZ BL NP = Same as DNoZ KP NP above, but with pDNA encoding B646L. ●DNoZ BLAs NP = Same as DNoZ BL NP above, but the B646L plasmid has been prepared using arsenic condensation. ●DZ KP NP = Same as DNoZ KP NP above, but with rotation cleaning performed. ●DZ BL NP = Same as DNoZ BL NP above, but with rotation cleaning performed. ●DZ BLAs NP = Same as DNoZ BLAs NP above, but with rotation cleaning performed. 5. Quantification of dsDNA (pDNA) used for encapsulation efficiency assessment The aim of this study was to quantify the amount of plasmid DNA payload successfully incorporated into nanoparticles. To achieve this, the Invitrogen PicoGreen Quant-IT dsDNA assay kit (model: #P7581) was used.

[0232] For this assay, a standard curve was established using plasmid DNA of known concentration (see [reference]). Figure 7A and Figure 7B ,exist Figure 7B They are labeled A, B, C, and D according to their respective concentrations.

[0233] The researchers then determined the working reagents according to the kit supplier's recommendations and pipetted them into 96-well plates. The required samples were added to each well. After incubation at room temperature for 25 minutes, the fluorescence intensity in each well was measured using a fluorescence microplate reader (e.g., excitation at 480 nm and emission at 520 nm).

[0234] Based on the above experiments, it can be concluded that the payload in HEVNP can be effectively packaged.

[0235] The foregoing representative examples illustrate various features and embodiments of the invention. These examples are illustrative and not restrictive. Those skilled in the art will recognize that the specific embodiments are merely illustrative of the invention, as described more fully in the following claims.

Claims

1. A composition for the prevention and / or treatment of animal infections, comprising at least one antigen gene selected from the group consisting of: SEQ ID NO: 1 (S273R), SEQ ID NO: 2 (E183L), SEQ ID NO: 3 (K78R), SEQ ID NO: 4 (KP177R), SEQ ID NO: 5 (A104R), SEQ ID NO: 6 (A137R), SEQ ID NO: 7 (A151R), SEQ ID NO: 8 (B646R), SEQ ID NO: 9 (B438L), SEQ ID NO: 10 (B602L), SEQ ID NO: 11 (CP2475L), SEQ ID NO: 12 (CP204L), SEQ ID NO: 13 (CP530R), SEQ ID NO: 14 (O61R), SEQ ID NO: 15 (D117L), SEQ ID NO: 16 (H108R), SEQ ID NO: 17 (E199L), SEQ ID NO: 18 (E120R), SEQ ID NO: 19 (E248R), SEQ ID NO: 20 (MGF_110-4L), SEQ ID NO: 21 (EP402R), SEQ ID NO: 22 (MGF_505-5R), SEQ ID NO: 23 (MGF_360-12L), SEQ ID NO: 24 (DP96R), SEQ ID NO: 25 (A224L), SEQ ID NO: 26 (A179L), SEQ ID NO: 27 (I329L), SEQ ID NO: 28 (I10L), SEQ ID NO: 29 (I215L) and SEQ ID NO: 30(G1211R).

2. The composition according to claim 1, wherein the at least one antigen gene is selected from the group consisting of: SEQ ID NO: 2 (E183L), SEQ ID NO: 4 (KP177R), SEQ ID NO: 8 (B646L), SEQ ID NO: 12 (CP204L), SEQ ID NO: 13 (CP530R) and SEQ ID NO: 21 (EP402R).

3. The composition according to claim 1, wherein the at least one antigen gene is inserted into at least one plasmid vector.

4. The composition according to claim 1, further comprising a pharmaceutically acceptable carrier.

5. The composition according to claim 4, wherein the pharmaceutically acceptable carrier comprises nanoparticles.

6. The composition of claim 5, wherein the nanoparticles comprise BioCapZ.

7. The composition according to claim 1, wherein the animal comprises a pig.

8. The composition according to claim 1, wherein the infection is caused by a pathogen.

9. The composition according to claim 8, wherein the pathogen comprises a DNA virus or an RNA virus.

10. The composition of claim 9, wherein the DNA virus comprises African swine fever virus and the RNA virus comprises classical swine fever virus.

11. A method for preventing and / or treating an infection in an animal, comprising administering an effective amount of the composition according to any one of claims 1 to 6.

12. The method of claim 11, wherein the composition is administered orally (OR), intramuscularly (IM), intravenously (IV), subcutaneously (SC), intradermally, inhaled (IN), via aerosol, via a bioballistic particle delivery system, or using a gene gun.

13. The method of claim 11, wherein the concentration of the composition is from about 10 to about 100 µg / ml.

14. The method of claim 11, wherein the animal comprises a pig.

15. The method of claim 11, wherein the infection is caused by a pathogen.

16. The method of claim 15, wherein the pathogen comprises a DNA virus or an RNA virus.

17. The method of claim 16, wherein the DNA virus comprises African swine fever virus.

18. The method of claim 16, wherein the RNA virus comprises classical swine fever virus.

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