COMPOSITION FOR THE PREVENTION AND / OR TREATMENT OF INFECTION AND ITS USE

Abstract

A composition for prophylaxis and / or treatment of an infection in an animal is provided, including al least one antigen gene selected from the group consisting of; SEQ ID NO: 1 (S273R), SEQ ID NO: 2 (EI83L), 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 (D1I7L), 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 l 10-4L), 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). Amethod of preventing and / or treating an infection by administering the above composition to an animal is also provided.

Description

COMPOSITION FOR PROPHYLAXIS AND / OR TREATMENT OF INFECTIONAND USE THEREOFCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Patent Application Serial No. 63 / 540,006, filed September 22, 2023, which is incorporated herein by reference.FIELD OF INVENTION

[0002] The present invention relates to a composition for prophylaxis and / or treatment of infections, specifically those caused by an African swine fever virus (ASFV) and use thereof.BACKGROUND OF THE INVENTION

[0003] Vaccines are know n to prevent serious viral infections by stimulating the immune response. Live or attenuated viruses have effectively prevented major viral diseases such as polio and smallpox. Similarly, subunit vaccines or protein components of viruses have shown comparable efficacy, as demonstrated by vaccines for human papillomavirus, hepatitis B virus, and S ARS-CoV-2. Recently, mRNA vaccines have also proven effective against SARS-CoV-2 and influenza viruses.

[0004] African swine fever (ASF) is a highly contagious and infectious disease caused by the ASF virus (ASFV), affecting both feral and domestic pigs of all breeds and ages. The ASFV is a large double-stranded DNA virus that has severely impacted swine populations in Asia and Europe. The infection can result in a range of clinical outcomes, from severe illness with 100% mortality to long-term persistent infections. Clinical signs of infection with virulent strains include pulmonary edema, severe depression, high fever, anorexia, spotty skin, cyanosis, thrombocytopenia, lymphopenia, and haemorrhagic lesions. Developing a vaccine against ASF is crucial for preventing the spread of the disease, eradicating it, and repopulating herds following an ASF outbreak.

[0005] To date, most of the vaccines being developed are modified live attenuated vaccines. Recently, two live attenuated vaccines based on gene deletions of a pathogenicfactor of ASFV have been developed and approved for use in Vietnam. However, there is ongoing debate regarding their safety and efficacy. The use of modified live attenuated viral vaccines remains to pose risks of reversion to a more virulent strain once released in the field, and of shedding the vaccine virus, which can further exacerbate the spread of the disease and increase the disease severity. There have been attempts to produce more safer vaccines including viral vector vaccines, subunit vaccines and DNA vaccines, but the results on its efficacy have been mixed, and none have yet succeeded in producing an effective vaccine against ASFV.SUMMARY OF THE INVENTION

[0006] As described above, one aim of the present invention is to provide a safe and / or effective alternative therapeutics in treating infections, specifically those caused by African swine fever virus (ASFV), thereby preventing severe clinical manifestations and mortality.

[0007] The present invention provides is a composition for prophylaxis and / or treatment of an infection in an animal, including 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_l 10-4L), 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 includes a pharmaceutical acceptable carrier, more preferably a nanoparticle, and further preferably a BioCapZ.

[0011] The present invention further provides a method of preventing and / or treating an infection in an animal, including administering an effective amount of any one of the aforementioned compositions to the animal.

[0012] Preferably, the composition is administered orally (OR), by intramuscular (IM) injection, by intravenous (IV) injection, by subcutaneous (SC) or intradermal injection, by inhalation (IN), by aerosol, by a biolistic particle delivery7system, or by using a gene gun.

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

[0014] Preferably, the animal includes a swine.

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

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

[0017] Preferably, the RNA virus includes a Classical swine fever.

[0018] Therefore, the present invention at least provides the following advantages:(a) The claimed composition can be used as a DNA vaccine, which has been shown to be safe and to provide high protection efficacy against infection.(b) The claimed composition demonstrates a therapeutic effect, allowing ASF- infected animals to be treated upon vaccination, thereby rendering the vaccinated animals ASF virus-free and devoid of any clinical symptoms.(c) The present invention can effectively prevent the spread of the disease, aid in its eradication, and facilitate the repopulation of herds following an ASF outbreak.BRIEF DESCRIPTION OF THE DRAWINGS

[0019] FIG. 1A illustrates the manufacturing processes of the ASF pDNA vaccine and BioCapZ for deliver}' through different routes according to an embodiment of the present invention.

[0020] FIG. IB illustrates the manufacturing processes of the ASF pDNA vaccine and BioCapZ for delivery7through the same routes according to an embodiment of the present invention.

[0021] FIG. 2 illustrates the structure of the plasmid vector, pcDNA3.1(+) / myc-His A according to an embodiment of the present invention.

[0022] FIG. 3 illustrates the structure of the plasmid vector. pcDNA3.1(+) / myc-His B according to an embodiment of the present invention.

[0023] FIG. 4 illustrates the structure of the plasmid vector, pcDNA3.1+N-eGFP according to an embodiment of the present invention.

[0024] FIG. 5 illustrates illustrates the nanoparticle size measurements (in nm) vs. intensity (%) using DLS (Malvern Zetasizer) according to an embodiment of the present invention.

[0025] FIG. 6A illustrates the concentration of dsDNA in the samples according to an embodiment of the present invention.

[0026] FIG. 6B illustrates the concentration of protein according to an embodiment of the present invention.

[0027] FIG. 7A illustrates a standard curve established using the PicoGreen Quant-IT dsDNA measurement reagent by Invitrogen according to an embodiment of the present invention.

[0028] FIG. 7B illustrates the fluorescence intensity of the samples according to an embodiment of the present invention.DETAILED DESCRIPTION

[0029] The foregoing and other aspects of the present invention will now be described in more detail with respect to embodiments described herein. It should be understood that the invention can be embodied in different forms and is not limited to the embodiments set forth herein. These embodiments are presented to ensure that the disclosure is comprehensive and clearly conveys the scope of the invention to those skilled in the art. Every’ embodiment and feature should be understood to be interchangeable and combinable with every other embodiment and feature within the present application.

[0030] All publications, patent disclosure, patents, and other references cited herein are incorporated by reference in their entirety for the teachings relevant to the sentence and / or paragraph in which the reference is presented.

[0031] Definitions

[0032] The terminology' used in the present disclosure is for the purpose of describing particular embodiments only and is not intended to be limiting to the present invention. Unless otherwise defined, all technical and scientific terms used herein have meanings that are commonly understood by one of ordinary’ skill in the art to which this invention belongs.

[0033] As used herein, the terms “a”, '‘an’’ and '‘the” are meant to include both singular and plural forms, unless the context clearly indicates otherwise.

[0034] As used herein, the terms '‘comprise(s),” ‘'comprising,” “include(s),” “including,” “has / have,” “having,” “contain(s)”, “containing,” or any other variation thereof, are intended to cover a non-exclusive inclusion, subject to any explicitly stated limitations. This means that, for example, a composition, mixture, process or method that includes a list of elements is not limited to those elements alone but may also encompass additional elements that are not expressly listed or are inherent to the composition, mixture, process, or method.

[0035] As used herein, the term “about” refers to a value that includes inherent variations such as measurement error, the method used to determine the value, or variations among study subjects.

[0036] In the claims, the term “or"’ is used to mean “and / or"’ unless explicitly stated otherwise to indicate alternatives only or mutually exclusive options. However, the disclosure supports a definition that includes both alternatives and “and / or.”

[0037] As used herein, the terms “prophylaxis of an infection”, “prevent(ing) an infection,” or any other variation thereof refer to the composition’s aim to prevent the occurrence of an infection in an animal. This may be achieved by stimulating the animal’s immune system to produce antibodies against the infection. The composition may be effective against various infections, including, but not limited to, African swine fever.

[0038] As used herein, the terms “treatment of an infection,” “treating an infection,"’ or any variations thereof refer to the composition's potential to have a beneficial effect on an animal's health, including, but not limited to, reducing the mortality’ associated with the infection, improving the animal’s quality’ of life, and extending the animal’s lifespan.

[0039] As used herein, the term “plasmid vector” refers to plasmids used in prokaryotic or eukary otic systems, shuttle vectors, and commercial plasmids employed in geneticengineering within this field.

[0040] In an embodiment, a composition for prophylaxis and / or treatment of an infection in an animal includes 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 (Al 51 R), 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). In one embodiment, a method of preventing and / or treating infections in an animal is provided, including administering any of the composition to the animal.

[0041] Genes Encoding Antigens

[0042] Sequence Listing of SEQ ID NOs: 1-30

[0043] Tn 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 prophylactic and / or therapeutic efficacy for the composition. Therefore, such fragments should be considered within the scope of the present invention.

[0044] In an embodiment, at least one antigen gene is inserted to at least one plasmidvector to obtain plasmid antigen genes, such as plasmid DNAs (pDNA). For example, the antigen genes may be inserted to either the same plasmid vector or different plasmid vectors. This includes, but is not limited to, scenarios where two or more antigen genes each are inserted to a single plasmid vector, or where a single antigen gene is inserted to two or more plasmid vectors.

[0045] In an embodiment, the composition of the present invention can further comprise a pharmaceutical acceptable carrier, such as nanoparticle, preferably BioCapZ.

[0046] The nanoparticle may be used to encapsulate and / or deliver target substances to specific cells or tissues in the animal. The nanoparticle may improve the efficacy and safety of substance delivery. In an embodiment, the antigen genes or pDNA may be encapsulated in the nanoparticle or administered separately, either simultaneously or at different times from the nanoparticles by the same or different routes (See FIGs. 1A and IB).

[0047] The nanoparticle can include, but is not limited to, BioCapZ. BioCapZ is a genetically modified, protein-capsid-based, non-infectious immunostimulant nanoparticle derived from the Hepatitis E virus (HEV). It retains HEVs natural structural stability, antigenicity, and cell-binding capabilities, and is designed to stimulate an immune response to the antigen with low toxicity and cost-effective manufacturing. (Baikoghli, M. A., Chen, C. C., & Cheng, R. H. (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 herein incorporated by reference in their entirety).

[0048] In an embodiment, the composition is administered such as orally (OR), by intramuscular (IM) injection, by intravenous (IV) injection, by subcutaneous (SC) or intradermal injection, by inhalation (IN), by aerosol, by a biolistic particle delivery system, or by using a gene gun.

[0049] In an embodiment, a concentration of the composition is from about 10 to about 100 pg / ml, about 15 to about 90 pg / ml, about 15 to about 70 pg / ml, about 15 to about 50 pg / ml, about 20 to about 90 pg / ml. about 20 to about 70 pg / ml, about 20 to about 45 pg / ml, about 25 to about 40 pg / ml, about 30 to about 80 pg / ml. about 30 to about 60 pg / ml, about 30 to about 35 pg / ml, about 40 to about 70 pg / ml, or about 50 to about 60 pg / ml. The concentration of the composition may be in a concentrated form for storage and dilution to a working solution before administration to the animal.

[0050] In an embodiment, the animal is a swine (also known as a pig). The swine canbe, for example, any member of the genus Sus, including Sus scrofa domcslica. Sus scrofa, and all other species of Sus that may be infected by swine fever.

[0051] In an embodiment, the infections may be caused by a pathogen. Preferably, the pathogen can be a DNA virus or RNA virus. More preferably, the DNA virus is an African swine fever; and the RNA virus is a Classical swine fever.

[0052] In an embodiment, the present invention provides compositions and methods of combining at least two antigen genes selected from SEQ ID NOs: 1-30, which encode the target antigens. These compositions can be delivered with an immuno-enhancer nanoparticle, such as BioCapZ, to prevent and / or treat infections, such as swine fever.

[0053] Examples

[0054] Material and Methods

[0055] Plasmid DNA Vaccines Manufacturing Method (see Steps 1-10 below)

[0056] Step 1. Preparation of Recombinant Plasmid DNAs:

[0057] Recombinant plasmid DNAs were constructed by inserting the genes encoding the target antigens, 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), into appropriate plasmid vectors, such as pcDNA3.1(+) / myc-His A, pcDNA3. I(+) / myc-His B. and pcDNA3.1+N-eGFP, as illustrated in FIGs. 2-4, respectively. The plasmid vector contains essential elements such as a strong promoter and selectable marker.

[0058] Step 2. Transformation into E. coli:

[0059] To transform plasmid DNA into competent E. coli cells, follow these steps:• Step i: Thaw 50 pl of competent cells on ice.• Step ii: Add 1-5 pl of sample DNA (i.e. plasmid DNA of Step 1) into the tube containing the competent cells.• Step iii: Mix gently by flicking the tube to ensure even distribution of the DNA without damaging the cells.• Step iv: Incubate on ice for 30 minutes to allow the DNA to adsorb to the cells.• Step v: Heat shock the cells for 30 seconds at 42°C in a water bath. Do notmix or shake during this step.• Step vi: Immediately incubate on ice for 2 minutes to stabilize the cells.• Step vii: Add 250 pl of S.O.C. medium to the tube and shake at 225 rpm for 1 hour at 37°C to facilitate cell recovery and expression of antibiotic resistance.• Step viii: After transformation, spread the cells from each transformation onto LB agar plates containing antibiotic (e.g., ampicillin).• Step ix: Incubate the plates overnight at 37°C to allow colonies to grow.

[0060] Step 3. Picking Single-Colony:

[0061] Colony Selection: Pick individual colonies to ensure selection of single clones that have successfully incorporated the plasmid DNA.

[0062] Step 4. Cell Banks:

[0063] Establishing Cell Banks: Create and maintain cell banks from the selected singlecolony to ensure a stable source of the bacterial strain for future production. Store these in appropriate conditions to preserve their viability.

[0064] Step 5. Fermentation:

[0065] Scale-Up Culture: Grow the cells from the cell bank in a larger fermenter or bioreactor under controlled conditions to increase the biomass. Ensure optimal growth conditions, including temperature. pH. and aeration, to maximize plasmid DNA yield.

[0066] Step 6. Harvesting by Centrifuge:

[0067] Cell Harvesting: Once the fermentation process is complete, harvest the bacterial cells by centrifugation. This separates the cells from the growth medium, concentrating the cell mass for further processing.

[0068] Step 7. Cell Lysis by Alkaline Lysis:

[0069] Cell Disruption: Perform alkaline lysis to break open the bacterial cells and release the plasmid DNA into the solution. This method involves using an alkaline buffer followed by neutralization to ensure the plasmid DNA remains intact while cellular debris is removed.

[0070] Step 8. Purification:

[0071] DNA Purification: Purify the plasmid DNA from the lysate using techniques suchas column chromatography or ultrafiltration. This step removes contaminants including proteins, RNA. and other impurities to isolate high-purity plasmid DNA.

[0072] Step 9. Aseptic Fill:

[0073] Formulation: Prepare the final vaccine formulation by dissolving the purified plasmid DNA in a suitable buffer. Perform aseptic filling to transfer the vaccine solution into sterile vials or containers under aseptic conditions to prevent contamination.

[0074] Detailed Processes for Steps 3-9 •'

[0075] Preparation of Stock Bacterial Culture (#S tarter 1):1. Prepare Stock Bacteria: Store the initial bacterial stock in glycerol for long-term preservation.2. Streak for Isolation: Streak the glycerol stock onto LB agar plates containing ampicillin (100 pg / ml) to isolate individual colonies.3. Incubate: Place the LB agar plates in an incubator at 37°C overnight to allow colony growth.4. Inoculate Starter Culture: Select a single colony from the LB agar plate and inoculate it into 5 ml of LB broth containing ampicillin (100 pg / ml).5. Grow the Culture: Incubate the culture in a shaker at 37°C and 200 rpm for 16-18 hours to achieve sufficient bacterial growth.6. Prepare Bacterial Culture (#Starterl): Use this culture to prepare larger volumes of bacterial cells as needed for plasmid DNA preparation.7. Extract Plasmid DNA: Extract plasmid DNA from the bacterial culture using the QIAprep® Spin Miniprep Kit. Measure the plasmid DNA concentration with a Nanodrop spectrophotometer and assess microbial growth by measuring the optical density at 600 nm (OD600).8. Store Culture: Store the prepared bacterial culture (#Starterl) at 4°C for future use.

[0076] Preparation of Working Cell Bank (#Starter 2):1. Use Bacterial Culture (#Starterl): Transfer 0.30 ml of the #Starterl culture to 30 mL of LB broth containing ampicillin (100 pg / ml).2. Grow the Culture: Incubate the culture in a shaker at 37°C and 200 rpm for 16-18 hours.3. Store Culture: Store the prepared working cell bank (#Starter 2) at 4°C for future use.

[0077] Preparation of Final Culture:1. Scale-Up Culture: Transfer 1 ml of the #Starter 2 culture to 1,000 ml of LB broth containing ampicillin (100 pg / ml).2. Grow the Final Culture: Incubate the culture in a shaker at 37°C and 200 rpm for 16-18 hours to achieve the final bacterial density required for plasmid DNA production.

[0078] Centrifugation and Plasmid DNA Extraction by EndoFree® Plasmid Giga Kit:1. Harvest Cells: Centrifuge the final culture at 8,000 x g for 10 minutes at 4°C. Carefully decant the supernatant and freeze the cell pellets at -20°C.2. Resuspend Bacteria: Resuspend the bacterial pellet in 32 ml of Buffer Pl.3. Lysis: Add 32 ml of Buffer P2, mix thoroughly by inverting the tube 4-6 times, then add 32 ml of Buffer P3 and mix thoroughly by vigorously inverting the tube 4-6 times until the solution is completely colorless.4. Filter Lysate: Pour the lysate into the filter and incubate at room temperature for 10 minutes. Filter by vacuum and add 50 ml of Buffer FWB2. Add 30 ml of Buffer ER to the filter lysate, incubate on ice for 30 minutes, then equilibrate the column by applying 75 ml of Buffer QBT.5. Apply Lysate: Apply the filtered lysate to the column and wash with 600 ml of Buffer QC.6. Elute Plasmid DNA: Elute the plasmid DNA with 100 ml of Buffer QN (prewarming the buffer to 65°C may help increase yield). Precipitate the plasmid DNA by adding 70 ml of isopropanol to the eluate and mix.7. Centrifuge and Wash: Centrifuge at 15,000 x g for 30 minutes at 4°C, carefully decant the supernatant, and wash the DNA pellet with endotoxin-free 70% ethanol at room temperature.8. Final Centrifuge and Drying: Centrifuge at 15,000 x g for 10 minutes at 4°C and carefully decant the supernatant. Air dry the pellet for 10-20 minutes.9. Redissolve DNA Pellet: Redissolve the DNA pellet in Buffer TE, measure the concentration with a Nanodrop spectrophotometer, aliquot, and store at -20°C.

[0079] Preparation of Solutions:

[0080] Solution 1:1. Thaw Plasmid DNA: Thaw plasmid DNA stored at -20°C at room temperature before use and use within 30 minutes.2. Prepare Solution: Spin down at 4,000 rpm for 5 seconds. Add vehicle to a container, then add the plasmid DNA and stir for 15 minutes. Add additional vehicle and stir for another 15 minutes.3. Aliquot and Store: Aliquot 2.5 ml per vial (one vial for 5 injections) and store at - 20°C. Total volume per vial is 2.5 ml.

[0081] Solution 2:1. Prepare Solution: Add vehicle to a container, then add Nano Adjuvant and stir for 15 minutes. Add additional vehicle and stir for another 15 minutes.2. Aliquot and Store: Aliquot 12 ml per vial (one vial for 20 injections) and store at -20°C. Total volume per vial is 12 ml.

[0082] Step 10. Quality Assurance and Quality Control:

[0083] Quality Testing: Conduct comprehensive quality assurance and quality control tests to ensure the plasmid DNA vaccine meets all required specifications. This includes assessing DNA concentration, purity, and integrity, as well as performing sterility tests and verifying that the vaccine elicits the intended immune response in preclinical testing.

[0084] Design of Vaccines

[0085] Variations:1. At least two plasmids, each expressing one antigen, delivered intramuscularly (e.g. , Experimental Example 1).2. At least one plasmid expressing at least two antigens, delivered intramuscularly.3. At least two plasmids, each expressing one antigen, delivered intramuscularly, and an oral immunostimulant nanoparticle (e.g, BioCapZ; see FIG. 1A and Experimental Example 2).4. At least one plasmid expressing at least two antigens, delivered intramuscularly, and an oral immunostimulant nanoparticle (e g, BioCapZ; see FIG. 1 and Experimental Example 2).5. At least two plasmids, each expressing one antigen, encapsulated / encapsidated separately in immunostimulant particles (e.g., BioCapZ) but delivered together orally (see FIG. IB and Experimental Example 3).6. At least one plasmid expressing at least two antigens, encapsulated / encapsidated in an immunostimulant particle (e.g., BioCapZ), delivered orally (see FIG. IB and Experimental Example 3).

[0086] Experimental Example 1

[0087] A vaccine encoding six different antigens derived from the African swine fever virus genotype II (DNA ASF vaccine) was formulated and tested for its safety and efficacy.

[0088] In detail, a combination of six plasmids including antigen genes (E183L, KP177R, B646L, CP204L, CP530R, and EP402R), each expressing a different antigen and prepared using the Plasmid DNA Vaccines Manufacturing Method described above, was used as the vaccine to inject intramuscularly into pigs. The efficacy of the vaccine in preventing ASFV infection was evaluated by comparing vaccinated pigs to unvaccinated pigs under controlled conditions and in field trials on farms exposed to the virus.

[0089] The studies were divided into three phases: I, II, and III.

[0090] Phase I Study:

[0091] Objectives

[0092] The study was conducted to evaluate the efficacy and safety of the DNA ASF vaccine for preventing ASFV infection under field conditions. The primary endpoints were the survival rate of vaccinated pigs and the safety of the DNA ASF vaccine, specifically monitoring for any adverse reactions following vaccination.

[0093] Location of Study

[0094] The study, consisting of two independent trials, was conducted during acuteoutbreaks in two separated farms located in Nakhon Sri Thammarat and Ratchaburi provinces, Thailand.

[0095] The first trial (Trial 1) was conducted in a one-site production farm, with an inventory of 4,500 sows, located in Nakhon Sri Thammarat province. The trial was conducted in one of eight nursery buildings, capable of housing approximately 1,800 weaned pigs each. All eight nursery7buildings are closed housed, tunnel ventilated.

[0096] The second trial (Trial 2) was conducted in a one-site production farm, with an inventory7of 2,500 sows, located in Ratchaburi province. The trial was conducted in one of two nursery facilities, located approximately one kilometer from the main farm. The facility has three open-air buildings, capable of housing 400 pigs each in which housed ASFV infected pigs moved from the main farm.

[0097] Experimental Design

[0098] i. Animals• Trial 1 included 25 pigs (20 females and 5 males). All pigs were crossbred (Largewhite x Landrace x Duroc) with an average weight of 25 kg.• Trial 2 included 30 pigs (15 females and 15 males). All pigs were crossbred (Largewhite x Landrace x Duroc) with an average weight of 25 kg.

[0099] ii. Animal Management and Housing• Trial 1 was conducted in one of eight nursery7buildings, capable of housing approximately 1,800 weaned pigs each. All eight nursery buildings are closed housed, tunnel ventilated. Upon the start of the trial, some pigs in the study buildings were tested ASF positive by PCR.• Trial 2 was conducted in one of three open-air facilities. All were used to housed ASF infected pigs, at approximately 400 pigs per building. The facility previously experienced an acute ASF outbreak and had approximately 400 pigs, some of which were tested ASF positive by PCR.• Feed and water were given ad libitum.• Existing farm biosecurity7standard operating procedures were implemented.

[0100] iii. Animal Selection and Medications

[0101] Prior to transferring to the trial facility, all pigs were visually screened accordingto the following criteria:• All animals were apparently and clinically healthy, without obvious signs of fever, redness of skin, severe illnesses, trauma or lameness.• Minor cases of clinical illnesses during the study were given with necessary treatments for animal’s welfare and were administered in supervision with attending veterinarian.• All other vaccinations and supportive were followed in accordance to farm’s practices.

[0102] The ASF DNA vaccine was formulated in buffered saline and mixed with the adjuvant (polyethylene imine) in equal volumes to give a final concentration of 28 pg of DNA vaccine per milliliter. Each dose consisted of 1 ml (28 pg of DNA) or 2 ml (56 pg of DNA) of the vaccine formulation.

[0103] iv. Animal Handling and Positioning• Vaccinated pigs were housed in separated pens, but shared the air space in the same building with ASFV-infected pigs. Vaccination treatments varied from treatment groups.• Dead animals were disposed in the existing burial pit in the farm.

[0104] v. Treatment Groups and Vaccination

[0105] Trial 1: Twenty -five crossbred pigs (n=25), each weighing approximately 25 kg, were randomly assigned to three groups as follows:• Group 1 (n=15; 10 females and 5 males): Each pig was immunized intramuscularly with 1 ml (28 pg) of the DNA ASF vaccine every week for 3 consecutive weeks.• Group 2 (n=5; all females): Each pig was immunized intramuscularly with 2 ml (56 pg) of DNA ASF vaccine every week for 3 consecutive weeks• Group 3 (n=5; all females): no vaccine w as administered.

[0106] Trial 2: Thirty crossbred pigs (n=30), each weighing approximately 25 kg, w ere randomly assigned to tw o groups as following;• Group 1 (n=25): Each pig was immunized intramuscularly with 1 ml (28 pg) of the DNA ASF vaccine, followed by another immunisation with the same dose after two weeks and another one at the fourth week after first vaccination.• Group 2 (n=5): no vaccine was administered.

[0107] vi. Experimental Unit• Each individual pig was the experimental unit.• Each pig was tagged and assigned with individual tag numbers.• All treatment groups were co-mingled within each pen.

[0108] vii. Blood Collection and Serological Testing and qRT-PCR• For Trial 1, RT-PCR was used to monitor presence of ASF infection of the pigs at days 0, 14, 28 and 50 days post-vaccination. Antibody response against ASF B646L (p72) was measured by ELISA at day 70 post-vaccination.• For Trial 2, RT-PCR was used to monitor presence of ASF infection of the pigs at days 0, 14, 28 and 50 days post-vaccination.

[0109] viii. Disposition of animals in case of Confirmed ASF Outbreak

[0110] Animals that died of ASF were buned in the pit.

[0111] ix. Parameters1. Mortalities and Animal Health Monitoring (Day 0 to 90)• Immediate adverse reactions after vaccination up to 21 days post vaccination: all adverse events will be recorded, especially to swelling / inflammation, soreness, redness, abscesses, lumps, lesions, and warmth at the injection site.• Mortalities: recording of tags, date of mortality and clinical signs observed.• Morbidity: daily observance of animals for clinical signs and symptoms.2. Serological Response• All samples shall be tested for the presence of antibodies to ASF by ELISA against P72.3. Virus detection• Presence of virus were quantified by qRT-PCR.

[0112] Evaluation and Conclusion of Results

[0113] i. Mortalities and Vaccine Efficacy

[0114] Trial 1:• Body temperature following vaccination was at normal range for seven consecutive days post-vaccination.• At Day 0: Some of the test pigs were pre-exposed with ASF, as indicated by PCR(+) (see Table 1, column PCRODPV).• At Day 14: Vaccinated pigs had mixed responses, including positive and negative RT-PCR results on the presence of ASF (see Table 1, column PCR14DPV).• At Day 28: All 20 vaccinated pigs tested negative for ASF based on RT- PCR (see Table 1, column PCR28DPV).• All 5 tagged non-vaccinated pigs, along with approximately 1,000 untagged and unvaccinated pigs, died.• All vaccinated pigs survived. Vaccinated pigs also showed presence of anti-P72 antibodies based on ELISA (see Table 1, column P72 70DPV).• Females later became breeders, used for farm repopulation.• Barrows w ere sold at market weight.

[0115] Table 1: Results of Trial 1

[0116] Trial 2:• After 90 days, all 25 vaccinated animals in Group 1 were healthy and ASFV -negative.• All 5 non-vaccinated pigs in Group 2 died within 2 weeks after being transferred into the study facility.

[0117] ii. Observance of adverse reaction, clinical signs and safely of vaccine

[0118] There were no observed adverse reactions associated with administration of the DNA ASF Vaccine.

[0119] Summary

[0120] The pilot study was conducted in two independent trials with a small group of pigs to evaluate its working dose and protective efficacy under a natural field challenge. The first trial was conducted in an evaporative cooling house that was occupied by approximately 1,800 pigs that tested positive for the ASF virus and part of a larger herd that experienced an acute outbreak of ASF. The first group composed of 15 vaccinated pigswas vaccinated with 1 ml dose (28 pg) of DNA ASF vaccine while the second group composed of 5 pigs was vaccinated with 2 ml (56 pg) of DNA ASF vaccine. Both groups were vaccinated three times at an interval of one week with the respective dose. The third group of 5 pigs didn’t receive any vaccine. All groups were housed in the same building as the other 1,800 non-vaccinated ASF positive pigs. After 28 days, all 20 animals in the two groups of vaccinated pigs survived while all 5 unvaccinated pigs including the other 1.800 resident ASF positive pigs died from ASF. Eventually the surviving, vaccinated, females became breeders while the barrows were sold at market weight. The second trial was conducted in an open-air house that was occupied by 400 pigs that have been moved from a herd that experienced an acute ASF outbreak. Some of these pigs were found to be ASF positive by RT-PCR. Thirty of the pigs (25 kg each) that were ASF negative based on RT-PCR were randomly allocated into two groups. The first group included 25 pigs that were immunized with 2 ml (56 pg) of DNA ASF vaccine at an interval of 2 weeks. The second group of 5 pigs was not immunized. Both groups were housed in the same open- air facility that experienced an ASF outbreak. After 90 days, all 25 vaccinated pigs survived, while all 5 non-vaccinated pigs died from ASF. In both trials, no adverse effect was obser ed following the DNA ASF vaccination. In conclusion, the results demonstrated that the immunization of the DNA ASF vaccine at suggested dose conferred protection under a natural field challenge, in addition to safety.

[0121] Phase II Study:

[0122] Objectives

[0123] The study was conducted to further validate the safety and the efficacy of the DNA ASF vaccine to prevent ASF infection in a larger population under a field condition as well as the potential for the DNA ASF vaccine to treat ASF-infected animals.

[0124] Location of Study

[0125] The study, consisting of two independent trials, was conducted during acute outbreaks in two separated farms located in Nakhon Sri Thammarat and Ratchaburi provinces, Thailand.

[0126] The first trial (Trial 1) was conducted, while the farm were experiencing an ASF acute outbreak, in an one-site production farm, with an inventory of 4,500 sows, located inNakhon Sri Thammarat province. The trial was conducted in six wean-to-finish buildings, capable of housing approximately 1,000 pigs each. All six buildings are closed housed, tunnel ventilated.

[0127] The second trial (Trial 2) was conducted, while the farm was experiencing an ASF acute outbreak, in a one-site production farm, with an inventory of 2,500 sows, located in Ratchaburi province. The trial was conducted in fourteen wean-to-finish buildings, capable of housing approximately 650 pigs each. All fourteen buildings are closed housed, tunnel ventilated.

[0128] Experimental Design

[0129] i. Animals• Species / Breed: Porcine / crossbred (Largewhite x Landrace x Duroc)• Number of Animals: 8.032 pigs• Origin: ASFV negative herds in other provinces• Sex / Age / weight:Trial 1 included 5,042 pigs (male and female at 10 weeks of age, 25 kg).Trial 2 included 2, 990 pigs (male and female at 10 weeks of age, 25 kg).

[0130] ii. Animal Management and Housing

[0131] Housing : All Trial animals were housed in different buildings.• Trial 1 was conducted in a farm in Ratchaburi province, during an acute outbreak, with fourteen evaporated cooling houses and housing capacity of 650 pigs per facility, that previously had been affected by the ASF outbreak and has been vacated for 2-4 weeks.• Trial 2 was conducted in a farm in Nakhon Sri Thammarat province, during an acute outbreak, with six evaporated cooling houses and housing capacity of 1,000 pigs per building, with adjacent buildings housing ASF infected pigs.• Feed and water were given ad libitum.• Existing farm biosecurity standard operating procedures were implemented.

[0132] iii. Animal Selection and Medications

[0133] Similar to Phase I Study.

[0134] iv. Animal Handling and PositioningThe animals were housed in different buildings. Vaccination treatments varied from treatment groups.Dead animals were disposed in the existing burial pit in the farm.

[0135] v. Treatment Groups and Vaccination

[0136] Trial 1: A total of 5,042 pigs weighing at approximately 20 kg, were externally brought in, and housed in twelve separate buildings. Three batches of 25 kg pigs from two different sources (NH and T) were brought. Due to weekly introduced of one building each, two vaccination programs were implemented.• Two buildings (700 pigs) were vaccinated intramuscularly with 1 ml of DNA ASF vaccine (28 pg), followed by the same dosage at two weeks later.• Nine buildings (3,293 pigs) were vaccinated intramuscularly with 2 ml of DNA ASF vaccine (56 pg), followed by the same dosage at tw o w eeks later.• Three buildings (1,049 pigs), testing of 30 pigs (10 pigs each) upon arrival, revealed that 4 pigs were found to be ASF positive based on RT-PCR (1-2 positive pigs per building). All of the pigs, including the ASF-positive pigs, were vaccinated intra-muscularly with 2 ml of DNA ASF vaccine (56 pg) followed by a second vaccination with the same amount after two weeks. There w as no blood collection for analysis and the animals w ere just monitored for clinical signs and mortality7. Pigs w ere vaccinated intramuscularly twice with 2 ml DNA ASF vaccine (56 pg) with a two-week interval. Pigs residing in three buildings tested positive for the presence of ASF virus based on qPCR upon arrival and were given the same vaccine as the other ASF-negative pigs.• Pigs w ere monitored for mortality until 22 weeks from the first immunization.

[0137] Trial 2: The second trial w as conducted in a separate farm that also had a recent ASF breakout. A total of 2,990 ASF negative pigs with an average weight of 21 kg were externally introduced to the farm and housed in six separate buildings. Due to weekly introduced of one building each, three vaccination programs were implemented.• The first building, 500 pigs were introduced and immunized intramuscularly with 1 ml of DNA ASF vaccine (28 pg). However, some pigs in the group developed clinical symptoms associated with ASF 2 weeks following the introduction. This group w as then further immunized three more times at two-week intervals with 2 ml of DNA ASF vaccine (56 pg). After the booster with the first vaccination with 2 ml of DNA ASF vaccine (56 pg). clinical diseases associated with ASF seems to reduce. After 22-weeks from the first vaccination, a mortality rate of only 4.40% were observed.• The next 4 buildings (2,140 pigs) were immunized with the standard 2 ml of DNA ASF vaccine (56 pg) and followed by two additional vaccinations with the same amount at two-week intervals.• The last building, 4 pigs died during transportation and 30 pigs were tested positive for ASF by RT-PCR. Pigs in this last building were immunized with the standard 2 ml of DNA ASF vaccine (56 pg) and followed by two additional vaccinations with the same amount at two-week intervals.

[0138] vi. Experimental Unit• Each individual animal was the experimental unit.• Each pig was tagged and assigned with individual tag numbers.• Each treatment group had a separate color of ear tags.• All treatment groups were co-mingled within each pen.

[0139] vii. Blood Collection and Serological Testing and qRT-PCR

[0140] For Trials 1 and 2, there was no collection of blood as well as no serological testing and RT-PCR. However, RT-PCR was used to screen incoming animals admitted to the trial. Serological testing was not conducted.

[0141] viii. Disposition of animals in case of Confirmed ASF Outbreak

[0142] Animals that died of ASF were buried in the pit.

[0143] ix. Parameters

[0144] Mortalities and Animal Health Monitoring (0 to 22 weeks post vaccination)• Immediate adverse reactions after vaccination up to 21 days post vaccination: all adverse events will be recorded, especially to swelling / inflammation, soreness, redness, abscesses, lumps, lesions, and warmth at the injection site• Mortalities: recording of tags, date of mortality and clinical signs observed• Morbidity: daily observance of animals for clinical signs and symptoms

[0145] Evaluation and Conclusion of Results

[0146] i. Mortalities and Vaccine Efficacy

[0147] Trial 1:

[0148] The total of 5,042 pigs were externally brought in and housed in fourteen separate buildings. Two vaccination programs, 1 ml and 2 ml, were implemented. For 1 ml, two buildings (700 pigs) were vaccinated intramuscularly with 1 ml of DNAASF vaccine (28 pg), followed by the same dosage at 2 weeks later. For 2 ml, nine and three buildings (3,293 and 1,049 pigs) were vaccinated intramuscularly with 2 ml of DNAASF vaccine (56 pg), followed by the same dosage at 2 weeks later. After 22-weeks post the first vaccination, mortality rates of only 8.28%, 2.04% and 7.24% were observed in these three groups, respectively. It is noteworthy that 4 pigs in the last three buildings found to be ASF PCR positive based on RT-PCR upon arrival (1-2 positive pigs per building). Following vaccination, a mortality rate of only 7.24% were observed. In summary of the Trial 1, all of the pigs, including the ASF-positive pigs, were vaccinated intra-muscularly with 2 ml of DNAASF vaccine (56 pg) followed by a second vaccination with the same amount after 2 weeks. There was no blood collection for analysis and the animals were just monitored for clinical signs and mortality. After 22 weeks from the first immunization, 201 of the animals were infected with ASF virus and died while the rest of the vaccinated animals (n=4,841) survived (96%) and were ASF-negative based on RT-PCR. The surviving animals appeared healthy, with no clinical symptoms and were sold to the market upon reaching marketable size.

[0149] Trial 2:

[0150] A total of 2,990 ASF negative pigs were externally introduced to the farm and housed in six separate buildings. Two vaccination programs, 1 ml and 2 ml, were implemented. The first building, 500 pigs each, were introduced and immunized intramuscularly with 1 ml of DNAASF vaccine (28 pg). However, some pigs in the group developed clinical symptoms associated with ASF two weeks following the introduction. This group was then further immunized three more times at two-week intervals with 2 ml of DNA ASF vaccine (56 pg). After the booster with the first vaccination with 2 ml of DNAASF vaccine (56 pg), clinical diseases associated with ASF seems to reduce. After 22-weeks from the first vaccination, a mortality rate of only 4.40% were observed. The next four buildings (2,140 pigs) were immunized with the standard 2 ml of DNA ASF vaccine (56 pg) and follow ed by two additional vaccinations with the same amount at two-week intervals. After 22-weeks from the first vaccination, a mortality rate of only 1.95% were observed. It is noteworthy that in the last building, 4 pigs died during transportation and 30 pigs were tested positive for ASF by RT-PCR. Pigs in this last building were immunized with the standard 2 ml of DNA ASF vaccine (56 pg) and followed by two additional vaccinations with the same amount at two-week intervals. After 22-weeks from the first vaccination, a mortality rate of only 5.43% were observed. The other pigs survived with no clinical symptoms and ere ASF negative based on RT-PCR including the 30 pigs that initially tested positive for ASF virus.

[0151] In building F29 of the farm, all 1,214 non-vaccinated pigs died from an ASF outbreak.

[0152] ii. Observance of adverse reaction, clinical signs and safety of vaccine

[0153] There were no observed adverse reactions associated with administration of the DNA ASF Vaccine.

[0154] Summary

[0155] An initial pilot study (Phase I Study) involving two independent trials with 55 pigs demonstrated some protective and therapeutic effects of the DNA ASF vaccine. To validate these observations with a bigger sample size, tw o larger independent confirmatory trials were conducted, focusing primarily on clinical signs and mortality. The results demonstrated that the immunization of the DNA ASF vaccine at suggested dose conferred protection under a natural field challenge, in addition to demonstrating safety.

[0156] Phase III Study:

[0157] Objectives

[0158] The study was conducted to evaluate the therapeutic efficacy of the DNA ASF vaccine in treating ASF infected animals.

[0159] Location of Study

[0160] The trial was conducted in Sa Kaeo province, Thailand. The study was conducted in a farm that has experienced an ASF outbreak where 2,000 pigs had to be euthanised to prevent spread of the infection due to 100% mortality of infected pigs. Prior to conduct of the study, environmental sampling indicated that the ASF virus was present in the farm.

[0161] Experimental Design

[0162] i. Animals• Species: Porcine• Number of Animals: 20 pigs• ASFV negative farm• Sex / Age / weight: castrated male / 4 weeks of age / 7 kg• Identification Method: Ear Tags

[0163] Twenty male castrated pigs (n=20), each 4 weeks old, were brought in and housed in two separated pens of 10 pigs each. The pigs were housed for two weeks and then were exposed oro-nasally to a sample containing ASF virus with Ct-value of 25 (approximate 106 HAD50 / ml). One week after exposure to the ASF virus (challenge), all pigs were vaccinated with 2 ml of DNA ASF vaccine (56 pg) twice with a two-week interval. Blood was collected weekly from each of the pigs and tested for presence of the virus using PCR and serologically by ELISA.

[0164] ii. Animal Management and Housing

[0165] Housing : Twenty pigs were brought in and housed in two separated pens of 10 pigs each.• Feed and water were given ad libitum.• Existing farm biosecurity standard operating procedures were implemented.

[0166] iii. Animal Selection and Medications

[0167] Similar to Phase I and Phase II Studies.

[0168] iv. Animal Handling and Positioning• The animals were housed in different pens at a rate of 10 pigs per pen.• Animals were vaccinated with 2 ml DNA ASF vaccine (56 pg) twice with a two-week interval.• Dead animals were disposed in the existing burial pit in the farm.

[0169] v. Treatment Groups and Vaccination

[0170] All 20 pigs received the same challenge and vaccine regimen of 2 ml ASF DNA vaccine (56 pg) with a two-week interval.

[0171] vi. Experimental Unit

[0172] Similar to Phase II Study.

[0173] vii. Blood Collection and Serological Testing and qRT-PCR

[0174] Blood was collected at time before immunization and every two weeks. The blood was analysed for the presence of antibodies by ELISA and ASF virus by PCR.

[0175] viii. Disposition of animals in case of Confirmed ASF Outbreak

[0176] Animals that died of ASF were buried in the pit.

[0177] ix. Parameters1. Mortalities and Animal Health Monitoring (Day 0 to 150)• Immediate adverse reactions after vaccination up to 21 days post vaccination: all adverse events will be recorded, especially to swelling / inflammation, soreness, redness, abscesses, lumps, lesions, and warmth at the injection site• Mortalities: recording of tags, date of mortality and clinical signs observed.• Morbidity: daily observance of animals for clinical signs and symptoms.2. Serological Response• All samples shall be tested for the presence of antibodies to ASF by ELISA against P72.3. Virus detection• Presence of virus were quantified by qRT-PCR.

[0178] Evaluation and Conclusion of Results

[0179] i. Mortalities and Vaccine Efficacy

[0180] All 20 pigs that have been pre-exposed to the ASF virus at Ct-value of 25 (approximate 106 HAD50 / ml) tested negative for ASF after vaccination and remained healthy.

[0181] ii. Observance of adverse reaction, clinical signs and safety of vaccine

[0182] There were no observed adverse reactions associated with administration of the DNA ASF Vaccine.

[0183] Summary

[0184] An experimental cocktail of plasmid DNA vaccine encoding six different antigens derived from the African swine fever virus (DNA ASF vaccine) was formulated and tested for its safety and efficacy. At least four independent studies consistently showed the safety and efficacy of this DNA vaccine formulation. One of the interesting and unexpected features of this vaccine is that it can reverse the infection of the ASF virus and potentially treat ASF virus-infected animals.

[0185] Experimental Example 2

[0186] The concurrently administered BioCapZ with DNA as ASFV vaccine. Field trials evaluate the enhanced humoral immune response of BioCapZ when simultaneously administered with pDNA against ASFV.

[0187] For administering BioCapZ, the dosage varies by the type of pig. For sows weighing between 250 and 300 kg, the dose is 6 mg of BioCapZ per administration, with a total volume of 10 ml. In contrast, for piglets weighing 6 kg, the dosage is 0.12 mg of BioCapZ per dose, delivered in a 0.2 ml volume.

[0188] The study involved two approaches: a sow trial and a piglet trial.

[0189] In the sow trial, 15 multiparous sows, each weighing between 250 and 300 kg, were randomly selected and equally allocated into three treatment groups as follows:• Group A: sows were vaccinated intramuscularly twice, with 2 ml of DNA vaccine at a two-week interval.• Group B: sows were given the same DNA vaccine regimen as Group A but with an additional 10 ml of BioCapZ administered intramuscularly.• Group C: sows also received two intramuscular doses of 2 ml DNA vaccine at a two-week interval, combined with 10 ml of BioCapZ administered in-feed for 20 consecutive days, starting 3 days before the first vaccination and continuing until 3 days after the second vaccination.

[0190] Serum samples were collected from all sows at days 0, 14, 28, and 42 postvaccination to test for antibodies against ASFV P30 and P72 using ELISA (IDVet commercial kit or in-house ELISA), while any adverse effects such as fever and off-feed were monitored daily.

[0191] In the piglet trial. 15 piglets weighing 6 kg each were randomly selected andequally allocated into three treatment groups as follows:• Group A: piglets were vaccinated intramuscularly twice, with 2 ml of DNA vaccine at two-week intervals.• Group B: piglets were given the same DNA vaccine regimen as Group A but with an additional 0.20 ml of BioCapZ administered intramuscularly.• Group C: piglets were also given two doses of 2 ml of DNA vaccine at two- week intervals, with 0.2 ml of BioCapZ administered orally for 20 consecutive days, starting 3 days before the first vaccination and continuing until 3 days after the booster vaccination.

[0192] Serum samples were collected from all piglets at days 0, 14, 28, and 42 postvaccination to test for antibodies against ASFV P30 and P72 by ELISA, while any adverse effects such as fever and off-feed were monitored daily.

[0193] Observance of adverse reaction, clinical signs and safety of vaccine

[0194] There were no observed adverse reactions associated with administration of the DNA ASF Vaccine with / without BioCapZ. BioCapZ has functioned in immune enhancement for field trial with DNA against African swine fever virus (ASFV) and has gained promising results.

[0195] Experimental Example 3

[0196] HEVNP encapsulation with ASFV vectors

[0197] 1. DLS measurement of intact, diassembled and reassembled HEVNP

[0198] Refer to FIG. 5, which illustrates the nanoparticle size measurements (in nm) vs. intensity (%) using DLS (Malvern Zetasizer).

[0199] (A) Intact HEVNP: The intact nanoparticle (without any payload encapsulated) measures 25 nm when purified. Please note that the nanoparticles shown here have not been purified; they are sourced from the supernatant of the expressing cell line.

[0200] (B) Disassembled HEVNP: This nanoparticle can be disassembled to facilitate the encapsulation of a payload (in this study, plasmid DNA (pDNA)). The disassembly process involves adding reducing agents (1 mM EGTA and 20 mM DTT) and incubating for 30 minutes at room temperature, followed by a spin clean-up using a desalting column.After disassembly, the nanoparticle measures approximately 10 nm in size according to DLS.

[0201] (C) Reassembled HEVNP: Likewise, the nanoparticle can be reassembled by incrementally adding CaC12 in increasing concentrations (up to 5 mM) every hour for four hours, followed by the spin clean-up procedure mentioned above.

[0202] 2. Adding payloads to disassembled HEVNP for encapsulation

[0203] The study utilized two plasmid DNAs encoding various antigenic regions from the ASFV: B646L (that encodes for the p72, major capsid protein and is required for virus entry and KP177R (that encodes for p22, a structural protein from ASFV that is required for replication of the virus). The plasmid encoding B646L is 3311 bp in size, and the plasmid encoding for KP177R is 1889 bp.

[0204] For the encapsulation procedure, plasmid DNA and nanoparticle (HEVNP) were mixed in a 1: 1 ratio, usingl50 pg plasmid.

[0205] Previous trials indicated that HEVNP has a size limit for efficiently encapsulating plasmid DNAs if they are below 3 kb.

[0206] The following encapsulation was performed:• pDNA:KP177R (1889 bp)• pDNA:B646L (3311 bp)• pDNA:B646L condensed using arsenic (2 mM)

[0207] 3. DNAse treatment to remove unincorporated pDNAs

[0208] After encapsulation and reassembly of the nanoparticles, the samples were split into two groups:• DNAse Treatment (DnoZ): DNAse treatment (at 37°C for 30 minutes) was conducted to digest unincorporated plasmid DNA, without spin clean-up• DNAse Treatment with Spin Clean-Up (DZ): DNAse treatment (at 37°C for 30 minutes) was performed, followed by spin clean-up to digest unincorporated plasmid DNA.

[0209] 4. Nanodrop measurement for dsDNA / protein cc.

[0210] Following DNAse treatment (with or without spin clean-up), the concentrationof dsDNA (ng / pl) and protein (mg / ml) in the samples were assessed. Please see FIGs. 6A and 6B, wherein the X-axis in both graphs represents the samples (one bar for each sample type), while the Y-axis indicates either nucleic acid concentration (ng / pl) or protein concentration (mg / ml).

[0211] Sample label explanation:• NP = nanoparticle (intact)• Disassemb = nanoparticle that has been disassembled (no payload)• Rea = nanoparticle that has been reassembled (no pay load)• DNoZ KP NP = nanoparticle encapsulated with the payload of plasmid DNA KP177R. treated with DNAse after reassembly but not spin-cleaned• DNoZ BL NP = same as DNoZ KP NP above, but with pDNA that encodes for B646L• DNoZ BLAs NP = same as DNoZ BL NP above, but the plasmid of B646L has been condensed using arsenic• DZ KP NP = same as DNoZ KP NP above but with spin clean-up• DZ BL NP = same as DNoZ BL NP above but with spin clean-up• DZ BLAs NP = same as DNoZ BLAs NP above but with spin clean-up

[0212] 5. Quantification of dsDNA (pDNA) for encapsulation efficiency assessment

[0213] The study aimed to quantify the amount of plasmid DNA payloads successfully incorporated into the nanoparticle. To achieve this, the PicoGreen Quant-IT dsDNA measurement reagent by Invitrogen (CatNo: #P7581) was used.

[0214] For this assay, a standard curve was established using known concentrations of plasmid DNA (see FIGs. 7A and 7B, labeled A, B, C, D in FIG. 7B with their respective concentrations).

[0215] The study then establishes the working reagent according to the kit supplier's recommendations and pipettes it into a 96-well plate. Desired samples are added to each well. After incubating at room temperature for 25 minutes, fluorescence intensity can be measured in each well using a fluorescent plate reader (e.g., excitation at 480 nm and emission at 520 nm).

[0216] Based on the above experiment, it can be concluded that the payloads can be efficiently encapsulated in HEVNP.

[0217] The representative examples above illustrate various features and embodiments of the present invention. These examples are intended to be illustrative rather than limiting. Those skilled in the art will recognize that the specific examples are only illustrative of the invention as described more fully in the subsequent claims.

Claims

1. A composition for the prevention and / or treatment of 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: 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 (061R), 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, characterized in that 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, characterized in that said at least one antigen gene is integrated 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, characterized in that the pharmaceutically acceptable carrier contains a nanoparticle.

6. The composition according to claim 5, characterized in that said nanoparticle contains BioCapZ.

7. The composition according to claim 1, characterized in that said animal includes a pig.

8. The composition according to claim 1, characterized in that said infection is caused by a pathogen.

9. The composition according to claim 8, characterized in that the pathogen includes a DNA virus or an RNA virus.

10. The composition according to claim 9, characterized in that the DNA virus includes African swine fever, and the RNA virus includes classical swine fever.

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

12. The method according to claim 11, characterized in that the composition is administered orally (p.o.), by intramuscular (i.m.) injection, by intravenous (i.v.) injection, by subcutaneous (s.c.) or intradermal injection, by inhalation (in.), in an aerosol, using a biolistic particle delivery system, or using a gene gun.

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

14. The method according to claim 11, characterized in that said animal includes a pig.

15. The method according to claim 11, characterized in that said infection is caused by a pathogen.

16. The method according to claim 15, characterized in that the pathogen includes a DNA virus or an RNA virus.

17. The method according to claim 16, characterized in that said DNA virus includes African swine fever.

18. The method according to claim 16, characterized in that said RNA virus includes classical swine fever.