African swine fever virus vaccine compositions and methods of making and using the same

Abstract

A safe and effective live-attenuated virus (LAV) vaccine candidate VNUA-ASFV-LAVL3 is presented herein. It exhibits significant attenuation of virulence in pigs across different doses (103, 104, and 105 TCID50). VNUA-ASFV-LAVL3 is efficiently cleared from the blood by 14-17 days post-infection, even at the highest dose (105 TCID50). Importantly, the attenuation observed in vivo does not compromise the ability of VNUA-ASFV-LAVL3 to induce protective immunity. Vaccination with VNUA-ASFV-LAVL3 elicits robust humoral and cellular immune responses in pigs, achieving 100% protection against a lethal wild-type ASFV (genotype II) challenge at all tested doses (103, 104, and 105 TCID50). Furthermore, a single vaccination (104 TCID50) provides protection for up to 2 months. In some forms, VNUA-ASFV-LAVL3 is represented by SEQ ID NO. 1 or sequences having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity thereto.

Description

[0001] AFRICAN SWINE FEVER VIRUS VACCINE COMPOSITIONS AND METHODS OF MAKING AND USING THE SAME

[0002] GOVERNMENT LICENSE RIGHTS

[0003] [1] This invention was made with government support under grant number 1021491 awarded by the USDA National Institute of Food and Agriculture; under grant numbers 58-8064- 8-011, 58-8064-9-007, 58-3020-9-020, and 59-0208-9-222 awarded by the USDA ARS NonAssistance Cooperative Agreements; under award number 2022-67015-36516 awarded by the USDA NIFA; and under subaward number 25-6226-0633-002 awarded by the USDA NIFA . The government has certain rights in the invention.

[0004] SEQUENCE LISTING

[0005] [2] This application contains a sequence listing, the contents of the electronic sequence listing (KSURF 40487PRO Sequence Listing.xml; Size: 544,908 bytes; Date of Creation: July 30, 2024) is hereby incorporated by reference in its entirety.

[0006] BACKGROUND

[0007] [3] African Swine Fever Virus (“ASFV”) is a lethal and highly contagious transboundary disease with the potential for rapid international spread. The clinical signs and gross pathological lesions of African Swine Fever “ASF” in swine may vary depending on the virus isolate, infection route, dose, and host characteristics. Acute ASF presents with high fever (up to 42°C), lethargy, anorexia, and inactivity. The causative agent, ASF virus (ASFV), is a large, enveloped virus containing a double-stranded DNA (dsDNA) genome of approximately 170-190 kilobase pairs (kbp) and contains over 150 open reading frames (ORFs) depending on the virus strain. A total of 24 ASFV genotypes (I-XXIV) have been described based on the ASFV p72 major capsid protein gene (B646L).

[0008] [4] ASF was first reported in Africa in 1921 and emerged for the first time in Europe in 1957 [3,4], In 2007, a highly pathogenic strain emerged in the Caucasus region of the Republic of Georgia and swiftly spreading to neighboring countries such as the Russian Federation, Armenia, Ukraine, and Azerbaijan. From 2014 to 2018, ASFV re-emerged in the European Union, originating from eastern nations to Lithuania, Poland, Latvia, and Estonia, subsequently spreading to Hungary, the Czech Republic, and Romania. The situation escalated globally when ASF reached China in 2018 and spread swiftly across Asia, including Vietnam. In 2021, it re-emerged in the Western Hemisphere on the Caribbean Island of Hispaniola (Dominican Republic and Haiti) after nearly 40-year. ASF has also spread westward across Europe in recent years. After a 40-year absence, the disease re-emerged in Italy in January 2022. The outbreak has continued to spread within the country. In 2023, ASF was confirmed for the first time on domestic pig farms in Bosnia and Herzegovina, Greece, and Croatia. The disease has now been reported on every continent except Antarctica, despite control efforts in some regions.

[0009] [5] The highly virulent ASFV genotype II that emerged in the Caucasus region in 2007 is responsible for the contemporary pandemic in Europe / Asia, and the outbreaks in Caribbean countries (Dominican Republic and Haiti). The first outbreak of ASF in Vietnam has been reported in early 2019 and quickly spread across the entire country with more than 8 million piglets depopulated, which equal to nearly 25 percent of the total pig population in 2020. At present, ASFV genotype II becomes endemic and ASF outbreak is continuing to occur frequently in Vietnam, raising the greatest concerns not only for the government but also for the pig industries.

[0010] [6] The absence of a safe and effective vaccine remains a critical hurdle in controlling ASF. Despite substantial research efforts in recent years, developing a protective ASF vaccine has proven challenging. While inactivated vaccines are safe, their efficacy in preventing infection in pigs is limited, as demonstrated by numerous clinical trials evaluating various ASFV strains, inactivation methods, adjuvants, and vaccination regimens. Subunit vaccines, targeting individual or multiple ASF antigens, have also shown insufficient protection. Live-attenuated vaccines (LAVs) have shown greater promise, offering improved immune protection compared to inactivated and subunit approaches. These vaccines reduce virulence through natural passage or artificial cell passage or genetic modification, while preserving immunogenicity. Since the ASF outbreak, researchers worldwide have developed several LAVs, such as cell-adapted strains (L'60BM89, ASFV-G / VP110, VNUA-ASFV-LAVL2) and naturally attenuated isolates (NH / P68 and Lvl7 / WB / Riel). Advancements in ASFV genomics have facilitated the creation of gene- deleted LAV candidates. These LAVs achieve attenuation by removing specific virulence genes while maintaining protective immunity. Examples include single-gene deletions (ASFV-G- AI177L, ASFV-G-AA137R, SY18AI226R) and multiple-gene deletions (HLJ / 18-7GD, ASFV-G- AMGF, ASFV-GA9GL / ACD2v, ASFV-G-AH77LALVR). While the 7-gene deletion and H77L gene deletion strains have progressed to clinical trials in China and Vietnam, respectively, commercialization of ideal LAVs faces challenges related to large-scale production, vaccine stability, safety, and the development of companion diagnostics for differentiating infected from vaccinated animals (DIVA).

[0011] [7] What is needed is a composition and accompanying methods of making and administering the composition that reduces the severity of or incidence of clinical and postmortem signs of ASFV infection.

[0012] SUMMARY OF THE DISCLOSURE

[0013] [8] The present disclosure overcomes the problems inherent in the art and provides immunogenic compositions or vaccines against ASFV, methods of making and using such immunogenic compositions or vaccines, and methods of administering such immunogenic compositions or vaccines.

[0014] [9] In one aspect, the present disclosure generally provides an efficacious African Swine Fever Virus (ASFV) immunogenic composition or vaccine.

[0015]

[0010] In another aspect, the present disclosure generally provides methods for making and / or producing an efficacious African Swine Fever Virus immunogenic composition or vaccine.

[0016]

[0011] In another aspect, the present disclosure generally provides an immunogenic composition or vaccine that reduces the severity of or the incidence of infection by ASFV.

[0017]

[0012] In another aspect, the present disclosure generally provides methods for reducing the incidence and / or severity of clinical and postmortem signs of ASFV infection.

[0018]

[0013] In one aspect of the disclosure, an immunogenic composition for African Swine Fever Virus (ASFV) is provided. In general, the composition comprises an antigen selected from the group consisting of an amino acid sequence, a nucleic acid sequence, a modified live virus, or an inactivated or killed virus. In some forms, the modified live virus, killed virus, or inactivated virus has a nucleic acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or even 100% sequence homology or sequence identity with SEQ ID NO. 1 or 2. In some preferred forms, the modified live virus, killed virus, or inactivated virus has a nucleic acid sequence having 100% sequence homology or sequence identity with SEQ ID NO. 1. As used herein, the names VNUA- ASFV-LAVL3 and LAVL3 are considered interchangeable and refer to a virus of the nucleic acid sequence specified in SEQ. ID. NO. 1. SEQ. ID NO. 2 is provided as a reference sequence for VNUA-ASFV-05L1 (GenBank accession number MW465755.1). SEQ. ID NO. 3 is provided as the sequence corresponding to VNUA-ASFV-L2, a parental virus from which VNUA-ASFV- LAVL3 has been experimentally derived, and which was itself experimentally derived from VNUA-ASFV-05L1. In some forms, the modified live virus, killed virus, or inactivated virus invokes or induces an immunogenic response in an animal receiving at least one administration thereof. In some forms, the immunogenic response reduces the incidence of or severity of clinical signs of infection caused by or associated with ASFV. In some forms, the ASFV is genotype II. In some forms, the reduction in incidence of or severity of clinical signs is at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or even 100% in comparison to an animal or a group of animals that did not receive at least one administration of the immunogenic composition of the disclosure. In some forms, the immunogenic composition of the present disclosure exhibits no hemadsorption activity in pig cells. In some forms, the immunogenic composition of the present disclosure exhibits no hemadsorption activity in 3D4 / 21 cells.

[0019]

[0014] In one aspect of the disclosure, by 28 days after administration of at least one dose of the immunogenic composition of the disclosure, a blocking percentage of ASFV-specific antibodies reaches no less than 80%. In some forms, the blocking percentage of ASFV-specific antibodies 28 days is no less than 83.6%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%. 98%, 99, 100%, 101%, 102%, 103%, 103.7%, 104%, 105%, or even higher. In some forms, the blocking percentage of ASFV-specific antibodies 56 days is no less than 80% 83.6%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%. 98%, 99, 100%, 101%, 102%, 103%, 103.7%, 104%, 105%, or even higher.

[0020]

[0015] In one aspect of the disclosure, an ELISPOT assay conducted 28 days after administering at least one dose of the immunogenic composition of the disclosure exhibits a spot count exceeding at least 150, 160, 170, 180, 190, or 200.

[0021]

[0016] The composition according to the disclosure may be administered or applied systemically through an intravenous, intravascular, intramuscular, intranasal, intraarterial, intraperitoneal, oral, subcutaneous, transdermal, or intrathecal route. Preferably, the composition is administered or applied orally or intramuscularly. For oral administration, an edible bait is most preferred as this will also permit immunization of pigs, wild boars, and feral pigs. Depending on the desired duration and effectiveness of the treatment, the compositions according to the disclosure may be administered once or several times, also intermittently, for instance on a daily basis for several days, weeks or months, and in different dosages.

[0022] BRIEF DESCRIPTION OF THE DRAWINGS

[0023]

[0017] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0024]

[0018] FIG. 1A shows growth characteristics and hemadsorption activity of VNUA-ASFV- LAVL3 (passage 50) and its parental virus VNUA-ASFV-L2, specifically growth curves in PAMs.

[0025]

[0019] FIG. IB shows growth characteristics and hemadsorption activity of VNUA-ASFV- LAVL3 (passage 50) and its parental virus VNUA-ASFV-L2, specifically growth curves in 3D4 / 21 cells.

[0026]

[0020] FIG. 1C shows microscopy indicating that VNUA-ASFV-L2-infected 3D4 / 21 cells exhibit hemadsorption 72 hours post-infection (hpi), magnification 200X. Arrows indicate HAD rosettes.

[0027]

[0021] FIG. ID shows microscopy indicating VNUA-ASFV-LAVL3-infected 3D4 / 21 cells showing no hemadsorption activity, 72 hpi, magnification 200X.

[0028]

[0022] FIG. 2 is a graphical panel highlighting the deletion of known genes in MGF regions of VNUA-ASFV-LAVL3 strain. This panel displays a schematic representation of the ASFV genome organization. Different shades represent major ORF categories. The dashed lines highlight the deleted region in the VNUA-ASFV-LAVL3 strain.

[0029]

[0023] FIGS. 3A-3F show rectal temperature and viremia in pigs inoculated with VNUA- ASFV-LAVL3 or parental VNUA-ASFV-L2 virus. Daily rectal temperature of control pigs and pigs inoculated with (A) 103TCID5o, (C) 104TCIDso, and (E) 105TCID50of VNUA-ASFV- LAVL3. Viremia of control pigs and pigs inoculated with (B) 103TCID50, (D) 104TCID50, and (F) Hf TCIDso of VNUA-ASFV-LAVL3. Legend: “C”, control pig; “LAVL3”, VNUA-ASFV- LAVL3; “number in the middle”, individual pig identification; “numbers at the end”, inoculated virus dose.

[0030]

[0024] FIG. 4A shows rectal temperature and viremia in pigs following challenge with virulent wild-type VNUA-ASFV-05L1 strain (genotype II), specifically daily rectal temperature of vaccinated and nonvaccinated control pigs after challenged with 103HAD50 of VNUA-ASFV- 05L1 strain.

[0031]

[0025] FIG. 4B shows rectal temperature and viremia in pigs following challenge with virulent wild-type VNUA-ASFV-05L1 strain (genotype II), specifically viremia of vaccinated and non-vaccinated control pigs after challenged with 103HAD50 of VNUA-ASFV-05L1.

[0032]

[0026] FIG. 5 shows pathological lesion findings in organs of vaccinated and non-vaccinated pigs post-challenge at necropsy. Row “A” shows organs from unvaccinated control pigs at 9 DPC. Row “B” shows organs from pigs vaccinated with VNUA-ASFV-LAVL3 strain at 28 DPC.

[0033]

[0027] FIG. 6A shows humoral and cellular immune responses of VNUA-ASFV-LAVL3- vaccinated pigs and non-vaccinated control pigs, specifically showing ASFV-specific antibodies in serum samples of pigs vaccinated with 103TCID50 of VNUA-ASFV-LAVL3 and control pigs.

[0034]

[0028] FIG. 6B shows humoral and cellular immune responses of VNUA-ASFV-LAVL3- vaccinated pigs and non-vaccinated control pigs, specifically showing ASFV-specific antibodies in serum samples of pigs vaccinated with 104 TCID50 of VNUA-ASFV-LAVL3 and control pigs.

[0035]

[0029] FIG. 6C shows humoral and cellular immune responses of VNUA-ASFV-LAVL3- vaccinated pigs and non-vaccinated control pigs, specifically showing ASFV-specific antibodies in serum samples of pigs vaccinated with 105TCID50 of VNUA-ASFV-LAVL3 and control pigs.

[0036]

[0030] FIG. 6D shows humoral and cellular immune responses of VNUA-ASFV-LAVL3- vaccinated pigs and non-vaccinated control pigs, specifically showing ELISPOT testing of ASFV- specific IFN-y-producing PBMCs at 28 DPV, ****p < 0.0001.

[0037]

[0031] FIG. 7 shows an exemplary timeline for study of the VNUA ASFV-LAVL3 vaccine in 3 and 6 week-old pigs, respectively.

[0038]

[0032] FIG. 8A shows a curve of the body temperatures of 3-week-old pigs vaccinated with the VNUA ASFV-LAVL3 vaccine as part of a VNUA-ASFV-05L1 challenge.

[0033] FIG. 8B shows a curve of the body temperatures of 3-week-old sentinel pigs as part of a VNUA-ASFV-05L1 challenge.

[0039]

[0034] FIG. 8C shows a curve of the body temperatures of 3 -week-old control pigs as part of a VNUA-ASFV-05L1 challenge.

[0040]

[0035] FIG. 9A shows a curve of the body temperatures of 6-week-old pigs vaccinated with the VNUA ASFV-LAVL3 vaccine as part of a VNUA-ASFV-05L1 challenge.

[0041]

[0036] FIG. 9B shows a curve of the body temperatures of 6-week-old sentinel pigs as part of a VNUA-ASFV-05L1 challenge.

[0042]

[0037] FIG. 9C shows a curve of the body temperatures of 6-week-old control pigs as part of a VNUA-ASFV-05L1 challenge.

[0043]

[0038] FIG. 9D shows a curve of the body temperatures of control pigs as part of a VNUA- ASFV-05L1 challenge.

[0044]

[0039] FIG. 10A shows a curve of the weight gains of 3 -week-old pigs vaccinated with the VNUA ASFV-LAVL3 vaccine as part of a VNUA-ASFV-05L1 challenge.

[0045]

[0040] FIG. 10B shows a curve of the weight gains of 3-week-old sentinel pigs as part of a VNUA-ASFV-05L1 challenge.

[0046]

[0041] FIG. 10C shows a curve of the weight gains of 3-week-old control pigs as part of a VNUA-ASFV-05L1 challenge.

[0047]

[0042] FIG. 11 A shows a curve of the weight gains of 6-week-old pigs vaccinated with the VNUA ASFV-LAVL3 vaccine as part of a VNUA-ASFV-05L1 challenge.

[0048]

[0043] FIG. 1 IB shows a curve of the weight gains of 6-week-old sentinel pigs as part of a VNUA-ASFV-05L1 challenge.

[0049]

[0044] FIG. 11C shows a curve of the weight gains of 6-week-old control pigs as part of a VNUA-ASFV-05L1 challenge.

[0050]

[0045] FIG. 12A shows a curve detailing changes in viremia over time for VNUA-ASFV- LAVL3 -vaccinated 3-week-old pigs as part of a VNUA-ASFV-05L1 challenge. Viremia was measured using RT-PCR as a multiple of the level for hemadsorption in 50% of the population (HAD50).

[0046] FIG. 12B shows a curve detailing changes in viremia over time for sentinel 3-week- old pigs as part of a VNUA-ASFV-05L1 challenge.

[0051]

[0047] FIG. 12C shows a curve detailing changes in viremia over time for control 3 -week- old pigs as part of a VNUA-ASFV-05L1 challenge.

[0052]

[0048] FIG. 13 A shows a curve detailing changes in viremia over time for VNUA-ASFV- LAVL3 -vaccinated 6-week-old pigs as part of a VNUA-ASFV-05L1 challenge. Viremia was measured using RT-PCR as a multiple of the level for hemadsorption in 50% of the population (HAD50).

[0053]

[0049] FIG. 13B shows a curve detailing changes in viremia over time for sentinel 6-week- old pigs as part of a VNUA-ASFV-05L1 challenge.

[0054]

[0050] FIG. 13C shows a curve detailing changes in viremia over time for control 6-week- old pigs as part of a VNUA-ASFV-05L1 challenge.

[0055]

[0051] FIG. 14A shows results from an ELISPOT analysis at 28 days post-vaccination, showing the number of formed spots for the 3 -week-old vaccinated pigs, sentinel pigs, and control pigs, respectively, as well as just the media.

[0056]

[0052] FIG. 14B shows results from an ELISPOT analysis at 28 days post-vaccination, showing the number of formed spots for the 6-week-old vaccinated pigs, sentinel pigs, and control pigs, respectively, as well as just the media.

[0057]

[0053] FIG. 15 shows the survival rate of 3 and 6-week-old pigs in the vaccinated, sentinel, and control groups. The 3-week old pig population showed full survival in the vaccinated and sentinel groups, while the 6-week old pig population showed full survival in the vaccinated group. All control pigs did not survive.

[0058]

[0054] FIG. 16A shows a whole-genome sequence (WGS) comparison of the VNUA-ASFV- LAVL3 genome, including BLAST alignment results against reference ASFV genome LAVL2.

[0059]

[0055] FIG. 16B shows a map of differences in the genome of VNUA-ASFV-05L1 and the VNUA ASFV-LAVL3 vaccine, specifically with respect to known coding sequences.

[0060]

[0056] FIG. 17A provides a detailed gene deletion profile of VNUA ASFV-LAVL3 compared to the wild type strain and other attenuated strains of VNUA-ASFV-05L1.

[0057] FIG. 17B provides a detailed gene deletion profile of VNUA ASFV-LAVL3 after 120 passages (denoted by P120) compared to the wild type strain and other attenuated strains of VNUA-ASFV-05L1.

[0061]

[0058] FIG. 17C provides a detailed gene deletion profile of VNUA ASFV-LAVL3 particularly showing the stability of the line after 120, 125 and 130 passages.

[0062]

[0059] FIG. 18A compares the average cytokine response in terms of IFN-y in pg / mL of control pigs against pigs vaccinated with 103, 104, and 105HAD50 / dose of VNUA-ASFV-LAVL3- P128 as a function of days post-vaccination, all pigs being challenged with VNUA-ASFV-05L1.

[0063]

[0060] FIG. 18B compares the average cytokine response in terms of IL-10 in pg / mL of control pigs against pigs vaccinated with 103, 104, and 105HAD50 / dose of VNUA-ASFV-LAVL3- P128 as a function of days post-vaccination, all pigs being challenged with VNUA-ASFV-05L1.

[0064]

[0061] FIG. 19A shows the antibody response in the serum of six sows vaccinated with VNUA-ASFV-LAVL3 vaccine strain at collaborative research farm A.

[0065]

[0062] FIG. 19B shows the antibody response in the serum of five sows vaccinated with different vaccination cycles of VNUA- ASF V-LAVL3 vaccine strains at collaborative research farm B.

[0066]

[0063] FIG. 19C shows the antibody response in the sow colostrum (n=5) vaccinated with different vaccination cycles of VNUA- ASF V-LAVL3 vaccine strains at collaborative research farm B.

[0067]

[0064] FIG. 19D shows antibody response in the serum of piglets (n=6) from the sow (n=3) vaccinated with different vaccination cycles of VNUA-ASFV-LAVL3 vaccine strain at collaborative research farm B.

[0068]

[0065] FIG. 20A shows the survival rate of 4-5-week-old pigs vaccinated with one or two doses of VNUA- ASF V-LAVL3 vaccine post-challenge with the virulent hybrid / recombination VNUA-ASFV-G12.

[0069]

[0066] FIG. 20B shows the survival rate of 6-7-week-old pigs vaccinated with one or two doses of VNUA-ASFV-LAVL3 vaccine post-challenge with the virulent hybrid / recombination VNUA-ASFV-G12. DETAILED DESCRIPTION

[0070]

[0067] The following detailed description and examples set forth preferred materials and procedures used in accordance with the present disclosure. It is to be understood, however, that this description and these examples are provided by way of illustration only, and nothing therein shall be deemed to be a limitation upon the overall scope of the present disclosure.

[0071]

[0068] “A”, “an”, and “the” include the singular and plural forms thereof unless the context clearly indicates otherwise.

[0072]

[0069] “Comprising”, “comprises”, “comprise”, “including”, “includes”, “include”, “having”, “has”, and “with” are all defined as being inclusive of the specified components as well as other unspecified components and can be used interchangeably.

[0073]

[0070] The terms “pig” and “piglet” are used interchangeably herein.

[0074]

[0071] An “immunogenic or immunological composition” refers to a composition of matter that comprises at least one antigen which elicits an immunological response in the host of a cellular and / or antibody-mediated immune response to the composition or vaccine of interest. Usually, an “immunological response” includes but is not limited to one or more of the following effects: the production or activation of antibodies, B cells, helper T cells, suppressor T cells, and / or cytotoxic T cells and / or y5 T cells, directed specifically to an antigen or antigens included in the composition or vaccine of interest. Preferably, the host will display either a therapeutic or protective immunological response such that resistance to new infection will be enhanced and / or the clinical severity of the disease reduced. Such protection will be demonstrated by either a reduction in the severity or prevalence of, up to and including a lack of symptoms normally displayed by an infected host, a quicker recovery time and / or a lowered viral titer in the infected host.

[0075]

[0072] A “reduction” or “decrease” in terms of incidence of symptoms, severity of symptoms, or transmissibility of infection is understood to encompass a comparison to a subject or group of subjects that has not received an administration of a composition of the present disclosure. Preferably, the reduction is at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or even 100% in comparison to an animal that has not received an administration or dose of a composition described herein. It is understood that this percentage of reduction can be in terms of the number of symptoms, incidence of symptoms, severity of symptoms, and transmissibility of infection.

[0076] Further, this can apply to individual animals or groups of animals.

[0077]

[0073] The term “transfected into a viral vector” means, and is used as a synonym for “introducing” or “cloning” a heterologous DNA sequence encoding a desired antigen into a viral vector, such as for example into a single-cycle replicon adenovirus, an attenuated bovine parainfluenza virus type 3 genotype c (BPIV3c), or a conventional vector such as a baculovirus vector. A “transfer vector” means a DNA molecule, that includes at least one origin of replication, the heterologous ASFV DNA sequence that encodes a desired antigen, in the present case of ASFV, DNA sequences which allow the cloning of said heterologous ASFV DNA sequence into the viral vector will be included. Preferably the sequences which allow cloning of the heterologous DNA sequence into the viral vector are flanking the heterologous DNA. Even more preferably, those flanking sequences are at least homologous in parts with sequences of the viral vector. The sequence homology then allows recombination of both molecules, the viral vector, and the transfer vector to generate a recombinant viral construct containing the heterologous DNA sequence encoding a desired antigen.

[0078]

[0074] “Adjuvants” as used herein, can include aluminum hydroxide and aluminum phosphate, saponins e.g., Quil A, QS-21 (Cambridge Biotech Inc., Cambridge MA), GPI-0100 (Galenica Pharmaceuticals, Inc., Birmingham, AL), water-in-oil emulsion, oil-in-water emulsion, water-in-oil-in-water emulsion. The emulsion can be based in particular on light liquid paraffin oil (European Pharmacopea type); isoprenoid oil such as squalene or squalene oil resulting from the oligomerization of alkenes, in particular of isobutene or decene; esters of acids or of alcohols containing a linear alkyl group, more particularly plant oils, ethyl oleate, propylene glycol di- (caprylate / caprate), glyceryl tri -(capryl ate / caprate) or propylene glycol dioleate; esters of branched fatty acids or alcohols, in particular isostearic acid esters. The oil is used in combination with emulsifiers to form the emulsion. The emulsifiers are preferably nonionic surfactants, in particular esters of sorbitan, of mannide (e.g. anhydromannitol oleate), of glycol, of polyglycerol, of propylene glycol and of oleic, isostearic, ricinoleic or hydroxystearic acid, which are optionally ethoxylated, and polyoxypropylene-polyoxyethylene copolymer blocks, in particular the Pluronic products, especially L121. See Hunter et al., The Theory and Practical Application of Adjuvants (Ed. Stewart-Tull, D. E. S.). JohnWiley and Sons, NY, pp51-94 (1995) and Todd et al., Vaccine 15:564-570 (1997).

[0075] For example, it is possible to use the SPT emulsion described on page 147 of “Vaccine Design, The Subunit and Adjuvant Approach” edited by M. Powell and M. Newman, Plenum Press, 1995, and the emulsion MF59 described on page 183 of this same book.

[0079]

[0076] A further instance of an adjuvant is a compound chosen from the polymers of acrylic or methacrylic acid and the copolymers of maleic anhydride and alkenyl derivative. Advantageous adjuvant compounds are the polymers of acrylic or methacrylic acid which are cross-linked, especially with polyalkenyl ethers of sugars or polyalcohols. These compounds are known by the term carbomer (Phameuropa Vol. 8, No. 2, June 1996). Persons skilled in the art can also refer to U. S. Patent No. 2,909,462 which describes such acrylic polymers cross-linked with a poly hydroxylated compound having at least 3 hydroxyl groups, preferably not more than 8, the hydrogen atoms of at least three hydroxyls being replaced by unsaturated aliphatic radicals having at least 2 carbon atoms. The preferred radicals are those containing from 2 to 4 carbon atoms, e.g. vinyls, allyls and other ethylenically unsaturated groups. The unsaturated radicals may themselves contain other substituents, such as methyl. The products sold under the name Carbopol; (BF Goodrich, Ohio, USA) are particularly appropriate. They are cross-linked with an allyl sucrose or with allyl pentaerythritol. Among then, there may be mentioned Carbopol 974P, 934P and 971P. Among the copolymers of maleic anhydride and alkenyl derivative, the copolymers EMA (Monsanto) which are copolymers of maleic anhydride and ethylene. The dissolution of these polymers in water leads to an acid solution that will be neutralized, preferably to physiological pH, in order to give the adjuvant solution into which the immunogenic, immunological or vaccine composition itself will be incorporated.

[0080]

[0077] Further suitable adjuvants include, but are not limited to, the RIBI adjuvant system (Ribi Inc.), Block co-polymer (CytRx, Atlanta GA), SAF-M (Chiron, Emeryville CA), monophosphoryl lipid A, Avridine lipid-amine adjuvant, heat-labile enterotoxin from E. coli (recombinant or otherwise), cholera toxin, IMS 1314 or muramyl dipeptide among many others.

[0081]

[0078] Preferably, the adjuvant is added in an amount of about 100 pg to about 10 mg per dose. Even more preferably, the adjuvant is added in an amount of about 100 pg to about 10 mg per dose. Even more preferably, the adjuvant is added in an amount of about 500 pg to about 5 mg per dose. Even more preferably, the adjuvant is added in an amount of about 750 pg to about 2.5 mg per dose. Most preferably, the adjuvant is added in an amount of about 1 mg per dose.

[0079] In some forms, the immunogenic composition further comprises an immune stimulant. Preferably, said immune stimulant shall be given at least twice. Preferably, at least 3, more preferably at least 5, and even more preferably at least 7 days are between the first and the second or any further administration of the immune stimulant. Preferably, the immune stimulant is given at least 10 days, preferably 15, even more preferably 20, and still even more preferably at least 22 days beyond the initial administration of the immunogenic composition. It is understood that any immune stimulant known to a person skilled in the art can also be used. “Immune stimulant” as used herein, means any agent or composition that can trigger a general immune response, preferably without initiating or increasing a specific immune response, for example the immune response against a specific pathogen. It is further instructed to administer the immune stimulant in a suitable dose.

[0082]

[0080] In some forms, the immunogenic composition is included in a kit comprising the immunogenic composition as described herein and a set of instructions for use.

[0083]

[0081] In some forms, different forms of the immunogenic composition are combined. In some forms, such a composition would include at least two members selected from the group consisting of modified live virus, killed virus, and inactivated virus.

[0084]

[0082] “Sequence Identity” as it is known in the art refers to a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, namely a reference sequence and a given sequence to be compared with the reference sequence. Sequence identity is determined by comparing the given sequence to the reference sequence after the sequences have been optimally aligned to produce the highest degree of sequence similarity, as determined by the match between strings of such sequences. Upon such alignment, sequence identity is ascertained on a position-by-position basis, e.g., the sequences are “identical” at a particular position if at that position, the nucleotides or amino acid residues are identical. The total number of such position identities is then divided by the total number of nucleotides or residues in the reference sequence to give % sequence identity. Sequence identity can be readily calculated by known methods, including but not limited to, those described in Computational Molecular Biology, Lesk, A. N., ed., Oxford University Press, New York (1988), Biocomputing: Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I, Griffin, A.M., and Griffin, H. G., eds., Humana Press, New Jersey (1994); Sequence Analysis in Molecular Biology, von Heinge, G., Academic Press (1987); Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M. Stockton Press, New York (1991); and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073 (1988), the teachings of which are incorporated herein by reference. Preferred methods to determine the sequence identity are designed to give the largest match between the sequences tested. Methods to determine sequence identity are codified in publicly available computer programs which determine sequence identity between given sequences. Examples of such programs include, but are not limited to, the GCG program package (Devereux, J., et al., Nucleic Acids Research, 12( 1 ): 387 (1984)), BLASTP, BLASTN and FASTA (Altschul, S. F. et al., J. Molec. Biol., 215:403-410 (1990). The BLASTX program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S. et al., NCVI NLM NIH Bethesda, MD 20894, Altschul, S. F. et al., J. Molec. Biol., 215:403-410 (1990), the teachings of which are incorporated herein by reference). These programs optimally align sequences using default gap weights in order to produce the highest level of sequence identity between the given and reference sequences. As an illustration, by a polynucleotide having a nucleotide sequence having at least, for example, 85%, preferably 90%, even more preferably 95% “sequence identity” to a reference nucleotide sequence, it is intended that the nucleotide sequence of the given polynucleotide is identical to the reference sequence except that the given polynucleotide sequence may include up to 15, preferably up to 10, even more preferably up to 5 point mutations per each 100 nucleotides of the reference nucleotide sequence. In other words, in a polynucleotide having a nucleotide sequence having at least 85%, preferably 90%, even more preferably 95% identity relative to the reference nucleotide sequence, up to 15%, preferably 10%, even more preferably 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 15%, preferably 10%, even more preferably 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. These mutations of the reference sequence may occur at the 5’ or 3’ terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence. Analogously, by a polypeptide having a given amino acid sequence having at least, for example, 85%, preferably 90%, even more preferably 95% sequence identity to a reference amino acid sequence, it is intended that the given amino acid sequence of the polypeptide is identical to the reference sequence except that the given polypeptide sequence may include up to 15, preferably up to 10, even more preferably up to 5 amino acid alterations per each 100 amino acids of the reference amino acid sequence. In other words, to obtain a given polypeptide sequence having at least 85%, preferably 90%, even more preferably 95% sequence identity with a reference amino acid sequence, up to 15%, preferably up to 10%, even more preferably up to 5% of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or a number of amino acids up to 15%, preferably up to 10%, even more preferably up to 5% of the total number of amino acid residues in the reference sequence may be inserted into the reference sequence. These alterations of the reference sequence may occur at the amino or the carboxyl terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in the one or more contiguous groups within the reference sequence. Preferably, residue positions which are not identical differ by conservative amino acid substitutions. However, conservative substitutions are not included as a match when determining sequence identity.

[0085]

[0083] “Sequence homology”, as used herein, refers to a method of determining the relatedness of two sequences. To determine sequence homology, two or more sequences are optimally aligned, and gaps are introduced if necessary. However, in contrast to “sequence identity”, conservative amino acid substitutions are counted as a match when determining sequence homology. In other words, to obtain a polypeptide or polynucleotide having 95% sequence homology with a reference sequence, 85%, preferably 90%, even more preferably 95% of the amino acid residues or nucleotides in the reference sequence must match or comprise a conservative substitution with another amino acid or nucleotide, or a number of amino acids or nucleotides up to 15%, preferably up to 10%, even more preferably up to 5% of the total amino acid residues or nucleotides, not including conservative substitutions, in the reference sequence may be inserted into the reference sequence. Preferably the homologous sequence comprises at least a stretch of 50, even more preferably 100, even more preferably 250, even more preferably 500 nucleotides.

[0086]

[0084] A “conservative substitution” refers to the substitution of an amino acid residue or nucleotide with another amino acid residue or nucleotide having similar characteristics or properties including size, hydrophobicity, etc., such that the overall functionality does not change significantly.

[0085] It will be found that the immunogenic compositions comprising any of the disclosed vaccine candidates as provided herewith are very effective in reducing the severity of or incidence of clinical signs associated with ASFV infections up to and including the prevention of such signs. Further, such immunogenic compositions reduce the transmissibility of ASFV.

[0087] The immunogenic compositions described herein can further include one or more other immunomodulatory agents such as, e. g., interleukins, interferons, or other cytokines. The immunogenic compositions can also include Gentamicin and Merthiolate. In another preferred embodiment, the present disclosure contemplates vaccine compositions comprising from about lug / ml to about 60 pg / ml of antibiotics, and more preferably less than about 30 pg / ml of antibiotics.

[0088]

[0086] The terms “immunogenic protein”, “immunogenic polypeptide” or “immunogenic amino acid sequence” as used herein refer to any amino acid sequence which elicits an immune response in a host against a pathogen comprising said immunogenic protein, immunogenic polypeptide or immunogenic amino acid sequence. An “immunogenic protein”, “immunogenic polypeptide” or “immunogenic amino acid sequence” as used herein, includes the full-length sequence of any proteins, analogs thereof, or immunogenic fragments thereof. By “immunogenic fragment” is meant a fragment of a protein which includes one or more epitopes and thus elicits the immunological response against the relevant pathogen. Such fragments can be identified using any number of epitope mapping techniques, well known in the art. See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66 (Glenn E. Morris, Ed., 1996) Humana Press, Totowa, New Jersey. For example, linear epitopes may be determined by e.g., concurrently synthesizing large numbers of peptides on solid supports, the peptides corresponding to portions of the protein molecule, and reacting the peptides with antibodies while the peptides are still attached to the supports. Such techniques are known in the art and described in, e.g., U.S. Patent No. 4,708,871; Geysen et al. (1984) Proc. Natl. Acad. Sci. USA 81:3998-4002; Geysen et al. (1986) Molec. Immunol. 23:709-715. Similarly, conformational epitopes are readily identified by determining spatial conformation of amino acids such as by, e.g., x-ray crystallography and 2- dimensional nuclear magnetic resonance. See, e.g., Epitope Mapping Protocols, supra. Synthetic antigens are also included within the definition, for example, polyepitopes, flanking epitopes, and other recombinant or synthetically derived antigens. See, e.g., Bergmann et al. (1993) Eur. J. Immunol. 23:2777-2781; Bergmann et al. (1996), J. Immunol. 157:3242-3249; Suhrbier, A. (1997), Immunol, and Cell Biol. 75:402-408; Gardner et al., (1998) 12th World AIDS Conference, Geneva, Switzerland, June 28-July 3, 1998. It is understood that immunogenic proteins of the present disclosure include the RBD sequence, the F-Tm sequence, the SPD sequence, and the CD40L sequence.

[0089]

[0087] In the present description, the terms polypeptide, peptide and protein are interchangeable.

[0090]

[0088] Additionally, any composition or vaccine candidate of the disclosure can include one or more pharmaceutical-acceptable or veterinary-acceptable carriers. As used herein, “a pharmaceutical-acceptable carrier” or “veterinary-acceptable carrier” includes any and all solvents, dispersion media, coatings, stabilizing agents, diluents, preservatives, antibacterial and antifungal agents, isotonic agents, adsorption delaying agents, and the like. In some forms, the pharmaceutical or veterinary acceptable carrier is selected from the group consisting of a solvent, a dispersion media, a coating, a stabilizing agent, a preservative, an antimicrobrial agent, an antifungal agent, an isotonic agent, and an adsorption delaying agent, and any combination thereof.

[0091]

[0089] It is understood that the immunogenic compositions described herein can be administered to any animal susceptible to ASFV including, but not limited to swine, pigs, warthogs, bush hogs, and boars. Further, administration of any of the immunogenic compositions described herein will reduce the incidence of, severity of, and transmission of ASFV.

[0092]

[0090] Isolated” means altered “by the hand of man” from its natural state, i.e., if it occurs in nature, it has been changed or removed from its original environment, or both. For example, a polynucleotide or polypeptide naturally present in a living organism is not “isolated,” but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is “isolated”, as the term is employed herein.

[0093]

[0091] “Modified polypeptide” of a polypeptide according to the disclosure is understood as designating a polypeptide obtained by genetic recombination or by chemical synthesis as will be described below, having at least one modification with respect to the normal sequence. These modifications will especially be able to bear an amino acids at the origin of a specificity, of pathogenicity and / or of virulence, or at the origin of the structural conformation, and of the capacity of membrane insertion of the polypeptide according to the disclosure. It will thus be possible to create polypeptides of equivalent, increased or decreased activity, and of equivalent, narrower, or wider specificity. Among the modified polypeptides, it is necessary to mention the polypeptides in which up to 5 amino acids can be modified, truncated at the N- or C-terminal end, or even deleted or added.

[0094]

[0092] As is indicated, the modifications of the polypeptide will especially have as objective: to render it capable of modulating, of inhibiting or of inducing the expression of ASFV gene and / or capable of modulating the replication cycle of ASFV in the cell and / or the host organism, of allowing its incorporation into vaccine compositions, and / or of modifying its bioavailability as a compound for therapeutic use.

[0095]

[0093] The methods allowing said modulations on eukaryotic or prokaryotic cells to be demonstrated are well known to the person skilled in the art. It is likewise well understood that it will be possible to use the nucleotide sequences coding for said modified polypeptides for said modulations, for example through vectors according to the disclosure and described below, in order, for example, to prevent or to treat the pathologies linked to the infection.

[0096]

[0094] The preceding modified polypeptides can be obtained by using combinatorial chemistry, in which it is possible to systematically vary parts of the polypeptide before testing them on models, cell cultures or microorganisms for example, to select the compounds which are most active or have the properties sought.

[0097]

[0095] Chemical synthesis likewise has the advantage of being able to use unnatural amino acids, or nonpeptide bonds. Thus, in order to improve the duration of life of the polypeptides according to the disclosure, it may be of interest to use unnatural amino acids, for example in D form, or else amino acid analogs, especially sulfur-containing forms, for example.

[0098]

[0096] Finally, it will be possible to integrate the structure of the polypeptides according to the disclosure, its specific or modified homologous forms, into chemical structures of polypeptide type or others. Thus, it may be of interest to provide at the N- and C-terminal ends compounds not recognized by the proteases.

[0099]

[0097] The present description provides for the manufacture and use of one or more live attenuated vaccines derived from wild type virus. In some forms, the immunogenic composition provides protective immunity to an animal receiving at least one administration thereof. In some forms, the modified live virus is attenuated. In some forms, the attenuation is accomplished through serial passaging. In some forms, the virus is passaged at least 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 times. In some forms, the virus is passaged less than 250, 240, 230, 220, 210, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, or 100 times. In some preferred forms, the virus is passaged 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, or 130 times. In some forms, the virus has at least one mutation in comparison to reference SEQ ID NO. 2 that is provided in Table 1.

[0100]

[0098] In some forms, the modified live, killed, or inactivated virus is missing at least one gene in comparison to a wild type virulent ASFV. In other forms, this gene is disrupted or modified such that it does not encode any corresponding functional protein. In some forms, the missing, disrupted, or modified gene is selected from the group consisting of 285L, MGF 110-8L, MGF 100-1R, MGF 100-9L, MGF 110-1 IL, MGF 110-14L, MGF 110-12L, MGF 110-13L, MGF 360- 4L, MGF360-6L, MGF 300-2R, MGF 505-2R, MGF 360-18R, and any combination thereof. In some preferred forms, the modified live, killed, or inactivated virus includes more than one missing, disrupted, or modified gene. In some forms, the modified live, killed, or inactivated virus includes at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27 missing, disrupted, or modified genes. In some preferred forms, the missing, disrupted, or modified genes is individually and respectively selected from the group consisting of 285L, MGF 110-8L, MGF 100-1R, MGF 100-9L, MGF 110-1 IL, MGF 110-14L, MGF 110-12L, MGF 110-13L, MGF 360-4L, MGF360-6L, MGF 300-2R, MGF 505-2R, MGF 360-18R, and any combination thereof. In some preferred forms, the at least one missing, disrupted, or modified gene is an MGF gene. In some preferred forms, the at least one missing, disrupted, or modified gene is an L gene. In some preferred forms, the at least one missing, disrupted, or modified gene includes at least one MGF gene and one L gene. In some preferred forms, the at least one missing, disrupted, or modified gene is in an uncharacterized gene or sequence. In some preferred forms, the at least one missing, disrupted, or modified gene includes at least one MGF gene and at least one uncharacterized gene or sequence. In some preferred forms, the at least one missing, disrupted, or modified gene includes at least one uncharacterized gene or sequence and at least one L gene. In some preferred forms, the at least one missing, disrupted, or modified gene includes at least one MGF gene, one, L gene, and one uncharacterized gene or sequence. In some preferred forms, the at least one missing, disrupted, or modified gene includes at least one MGF 360 gene and / or at least one MGF 110 gene, and / or at least one MGF 300 gene, and / or at least one MGF 100 gene. In some preferred forms, the at least one missing, disrupted, or modified gene includes MGF 360- 6L, MGF 360-4L, and at least one L gene. In some preferred forms, the at least one missing, disrupted, or modified gene includes at least one MGF 360 gene, at least one L gene, and at least one uncharacterized gene or sequence. In some preferred forms, the at least one missing, disrupted, or modified gene includes MGF 360-6L, X69R, 285L, and at least one uncharacterized gene or sequence. In some preferred forms, the at least one missing, disrupted, or modified gene includes MGF1 10-13L, MGF 360-4L, and at least one uncharacterized gene or sequence. In some preferred forms, the missing, disrupted, or modified gene is selected from the group consisting of MGF 360- 4L, MGF 100-1R, MGF 110-7L, and any combination thereof. In some of the preferred forms of this paragraph, the modified live, killed, or inactivated virus further includes at least one substitution resulting in an amino acid change.

[0101]

[0099] In some forms, the missing, disrupted, or modified gene is a disrupted, substituted, or deleted form of the 285L, X69R, A104R, F1055L, CD2v, M1249L, C45L, B962L, B385R, B407L, G1340L, G1211R, CP2475L, CP530R, NP1450L, D250R, D1133L, S183L, S273R, P1192R, H233R, H240R, E248R, or I177L gene from the VNUA-ASFV-05L1 strain. In some preferred forms, the missing, disrupted, or modified gene is selected from the group consisting of F1O55L, A104R, and X69R, and any combination thereof. In some preferred forms, the missing, disrupted, or modified genes is individually and respectively selected from the group consisting of the F1055L, CD2v, M1249L, C45L, B962L, B385R, B407L, G1340L, G1211R, CP2475L, CP530R, or NP1450L gene from the VNUA-ASFV-05L1 strain. In some preferred forms, the at least one missing, disrupted, or modified gene includes at least one MGF gene, one L gene, and one gene individually and respectively selected from the group consisting of the F1055L, CD2v, M1249L, C45L, B962L, B385R, B407L, G1340L, G1211R, CP2475L, CP530R, or NP1450L gene from the VNUA-ASFV-05L1 strain. In some preferred forms, the at least one missing, disrupted, or modified gene includes at least one MGF gene and the F1055L gene. In some preferred forms, the at least one missing, disrupted, or modified gene includes at least one MGF gene and the X69R gene. In some preferred forms, the at least one missing, disrupted, or modified gene includes at least one MGF gene and the 285L gene. In some preferred forms, the at least one missing, disrupted, or modified gene includes at least one MGF gene, the X69R gene, and the 285L gene. In some preferred forms, the at least one missing, disrupted, or modified gene includes at least one MGF gene, the X69R gene, and the Fl O55L gene. In some preferred forms, the at least one missing, disrupted, or modified gene includes at least one MGF gene, the F1055L gene, and the 285L gene. In some preferred forms, the at least one missing, disrupted, or modified gene includes at least one MGF gene, the X69R gene, the F1055L gene, and the 285L gene. In some preferred forms, the at least one missing, disrupted, or modified gene includes at least one MGF gene, one L gene, and the F1O55L gene. In some preferred forms, the at least one missing, disrupted, or modified gene includes at least one MGF gene, one L gene, and the X69R gene. In some preferred forms, the at least one missing, disrupted, or modified gene includes at least one MGF gene, one L gene, and the 285L gene. In some preferred forms, the at least one missing, disrupted, or modified gene includes at least one MGF gene, one L gene, the X69R gene, and the 285L gene. In some preferred forms, the at least one missing, disrupted, or modified gene includes at least one MGF gene, one L gene, the X69R gene, and the Fl 055L gene. In some preferred forms, the at least one missing, disrupted, or modified gene includes at least one MGF gene, one L gene, the F1O55L gene, and the 285L gene. In some preferred forms, the at least one missing, disrupted, or modified gene includes at least one MGF gene, one L gene, the X69R gene, the F1055L gene, and the 285L gene. In some preferred forms, the at least one missing, disrupted, or modified gene includes at least one MGF gene, one uncharacterized gene, and the Fl 055L gene. In some preferred forms, the at least one missing, disrupted, or modified gene includes at least one MGF gene, one uncharacterized gene, and the X69R gene. In some preferred forms, the at least one missing, disrupted, or modified gene includes at least one MGF gene, one uncharacterized gene, and the 285L gene. In some preferred forms, the at least one missing, disrupted, or modified gene includes at least one MGF gene, one uncharacterized gene, the X69R gene, and the 285L gene. In some preferred forms, the at least one missing, disrupted, or modified gene includes at least one MGF gene, one uncharacterized gene, the X69R gene, and the F1055L gene. In some preferred forms, the at least one missing, disrupted, or modified gene includes at least one MGF gene, one uncharacterized gene, the F1055L gene, and the 285L gene. In some preferred forms, the at least one missing, disrupted, or modified gene includes at least one MGF gene, one uncharacterized gene, the X69R gene, the F1055L gene, and the 285L gene. In some of the preferred forms of this paragraph, the modified live, killed, or inactivated virus further includes at least one substitution resulting in an amino acid change.

[0100] In various exemplary embodiments, the missing, disrupted, or modified gene is a disrupted, substituted, or deleted form of an uncharacterized region of the VNUA-ASFV-05L1 strain genome. In various exemplary embodiments, the at least one missing, disrupted, or modified uncharacterized regions of the VNUA-ASFV-05L1 strain genome is selected from the group consisting of base pair (bp) positions or regions 6,343 to 6,344, 9,004 to 9,131, 9,516 to 9,732, 10,108 to 10,265, 11,139 to 11,428, 11,789 to 11,877, 12,244 to 12,432, 12,793 to 12,878, 13,703 to 13,882, 15,047 to 15,862, 16,991 to 17,868, 23,952 to 23,962, 30,324, 30,882 to 30,915, 33,291, 41,114, 42,931 to 42,947, 43,100 to 43,133, 53,169, 61,819, and 174,320. In some preferred forms, the missing, disrupted, or modified gene is selected from the group of base pair ranges consisting of bps 6,343 to 6,344, 9,004 to 9,131, 9516 to 9,732, 10,108 to 10,265, 11,139 to 11,428, 11,789 to 11,877, 12,244 to 12,432, 12,793 to 12,878, 13,703 to 13,882, 15,047 to 15,862, and 16,991 to 17,868, and any combination thereof. In some preferred forms, the at least one missing, disrupted, or modified gene includes at least one MGF gene and the uncharacterized region from bps 15,047 to 15,862. In some preferred forms, the at least one missing, disrupted, or modified gene includes at least one MGF gene and the uncharacterized region from bps 16,991 to 17,868. In some preferred forms, the at least one missing, disrupted, or modified gene includes at least one MGF gene, one L gene, and the uncharacterized region from bps 15,047 to 15,862. In some preferred forms, the at least one missing, disrupted, or modified gene includes at least one MGF gene, one L gene, and the uncharacterized region from bps 16,991 to 17,868. In some preferred forms, the at least one missing, disrupted, or modified gene includes at least one MGF gene, the X69R gene, and the uncharacterized region from bps 15,047 to 15,862. In some preferred forms, the at least one missing, disrupted, or modified gene includes at least one MGF gene, the X69R gene, and the uncharacterized region from bps 16,991 to 17,868. In some preferred forms, the at least one missing, disrupted, or modified gene includes at least one MGF gene, the F1055L gene, and the uncharacterized region from bps 15,047 to 15,862. In some preferred forms, the at least one missing, disrupted, or modified gene includes at least one MGF gene, the F1055L gene, and the uncharacterized region from bps 16,991 to 17,868. In some of the preferred forms of this paragraph, the modified live, killed, or inactivated virus further includes at least one substitution resulting in an amino acid change.

[0102]

[0101] In various exemplary embodiments, the live attenuated vaccines of the present description are derived from passage of the VNUA-ASFV-05L1 (genotype II) strain in at least two different cell lines. In some preferred forms, the at least two different cell lines includes PAM cells and the 3D4 / 21 cell line. In some preferred forms, the virus is passaged first in the PAM cells and subsequently in a 3D4 / 21 cell line. 3D4 / 21 cell line is a single cell clone of 3D4 parent, a continuous cell line derived from porcine alveolar macrophages. In various exemplary embodiments, the VNUA-ASFV-05L1 strain is passaged for at least 5 passages, at least 10 passages, at least 15 passages, at least 20 passages, at least 25 passages, at least 30 passages, at least 35 passages, at least 40 passages, at least 45 passages, at least 50 passages, at least 55 passages, at least 60 passages, or preferentially at least 65 passages. Subsequently, in various exemplary embodiments, the VNUA-ASFV-05L1 strain is passaged in 3D4 / 21 cells for at least 5 passages, at least 10 passages, at least 15 passages, at least 20 passages, at least 25 passages, at least 30 passages, at least 35 passages, at least 40 passages, at least 45 passages, at least 50 passages, and preferentially at least 55 passages.

[0103]

[0102] In various exemplary embodiments, the live attenuated vaccine of the present description is an attenuated virus whose genome harbors deletions and mutations of the genome of the VNUA-ASFV-05L1 (genotype II) strain. In various exemplary embodiments, these deletions and mutations are such that the live attenuated virus shows comparable growth kinetics to that of the VNUA-ASFV-05L1 (genotype II) strain, with less than 1.5 logio deviation in growth kinetics at a given point in time, less than 1.0 logio deviation in growth kinetics at a given point in time, and preferentially less than 0.5 logio deviation in growth kinetics at a given point in time, as measured in HADso / mL.

[0104]

[0103] In various exemplary embodiments, deletions and mutations in known MGF gene regions of the VNUA-ASFV-05L1 (genotype II) strain found in the live attenuated vaccine of the present description are such that ASFV replication is substantially unaffected. In various exemplary embodiments, deletions and mutations in known MGF gene regions of the VNUA- ASFV-05L1 (genotype II) strain found in the live attenuated vaccine of the present description are such that the live attenuated vaccine does not demonstrate a return to virulence. In various exemplary embodiments, in doses of 102, 103, 104, or 105HAD50, the live attenuated vaccines of the present description are eliminated from the blood by 50 days post injection, or by 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, or 29 days post injection, or preferentially 28 days post injection. In various exemplary embodiments, in doses of 102, 103, 104, or 105HAD50, the live attenuated vaccines of the present description are eliminated from oral fluids by 50 days post injection, or by 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, or 18 days post injection, or preferentially 17 days post injection.

[0105]

[0104] In various exemplary embodiments, the live attenuated vaccines of the present description are the live attenuated vaccines of the present description are capable of inducing a higher ASFV-specific IgG antibody response than in non-vaccinated pigs. In various exemplary embodiments, the live attenuated vaccines of the present description are the live attenuated vaccines of the present description are capable of inducing a higher ASFV-specific IFN-y- producing cells than in non-vaccinated pigs.

[0106]

[0105] In various exemplary embodiments, the live attenuated vaccine of the present description is stable when derived from the passage of VNUA-ASFV-05L1 (genotype I, genotype II, or a genotype I-II hybrid) in a 3D4 / 21 cell line. In various exemplary embodiments, it is stable when derived from passage of VNUA-ASFV-05L1 (genotype I, genotype II, or a genotype I-II hybrid) in a 3D4 / 21 cell line after at least 100 passages. In various exemplary embodiments, it is stable when derived from passage of VNUA-ASFV-05L1 (genotype I, genotype II, or a genotype I-II hybrid) in a 3D4 / 21 cell line after 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130 passages. In various exemplary embodiments, the live attenuated vaccine of the present description, when derived from passage of VNUA-ASFV-05L1 (genotype I, genotype II, or a genotype I-II hybrid) through a 3D4 / 21 cell line, features the same at least one missing, disrupted, or modified gene in comparison to wild type virulent ASFV as in the embodiments described above or the examples described below. For example, in some forms, the missing, disrupted, or modified gene is selected from the group consisting of 285L, MGF 110-8L, MGF 100-1R, MGF 100-9L, MGF 110-1 IL, MGF 110-14L, MGF 110-12L, MGF 110-13L, MGF 360-4L, MGF360-6L, MGF 300-2R, MGF 505-2R, MGF 360-18R, and any combination thereof. In some preferred forms, the modified live, killed, or inactivated virus includes more than one missing, disrupted, or modified gene. In some forms, the modified live, killed, or inactivated virus includes at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27 missing, disrupted, or modified genes. In some preferred forms, the missing, disrupted, or modified genes is individually and respectively selected from the group consisting of 285L, MGF 110-8L, MGF 100- 1R, MGF 100-9L, MGF 110-1 IL, MGF 110-14L, MGF 1 10-12L, MGF 110-13L, MGF 360-4L, MGF360-6L, MGF 300-2R, MGF 505-2R, MGF 360-18R, and any combination thereof.

[0107] EXAMPLES

[0108]

[0106] The following examples illustrate embodiments of the disclosure. Nothing in these examples should be limiting to the disclosure as these are representative in nature.

[0109]

[0107] Animals: All animal care and protocols were reviewed and approved by Institutional Animal Care and Use Committee at Vietnam National University of Agriculture (VNUA-2021 / 01) and at Kansas State University (IACUC#4845). All animal experiments were conducted strictly adhering to the IACUC protocols. Piglets (6-7 weeks old, female) that tested negative for ASFV and ASFV-antibody were obtained from clean pig farms and used for experiments in this study. Pigs were fed a standard commercial diet. In Vietnam, the pigs were housed in the Animal Biosafety Research Facility of the Faculty of Veterinary Medicine, Vietnam National University of Agriculture. In the United States, the pigs were housed under laboratory biosafety level III agriculture (BSL3-Ag) conditions at the Biosecurity Research Institute (BRI), Kansas State University (KSU).

[0110]

[0108] Cells and Virus: Primary pulmonary alveolar macrophages (PAMs) were prepared as previously described and maintained in a medium containing Dulbecco’s modified Eagle medium (DMEM, Life Technologies, Grand Island, NY, USA) supplemented with 10% heat- inactivated fetal bovine serum (FBS, Thermo Scientific, Waltham, MA, USA) and 1% antimycotic (Life Technologies, Grand Island, NY, USA) at 37 °C in 5% CO2 incubator.

[0111]

[0109] 3D4 / 21 (immortalized porcine alveolar macrophage cell line, ATCC, CRL-2843) cells were cultured in RPMI 1640 medium (Life Technologies, Grand Island, NY, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Gibco, Thermo Scientific, Waltham, MA, USA), 1% antimycotic (Life Technologies, Grand Island, NY, USA), 1% MEM Non-Essential Amino Acids Solution (Thermo Scientific, Waltham, MA, USA) at 37°C in 5% CO2 incubator.

[0112] [HO] The wild-type VNUA-ASFV-05L1 strain (genotype II) was isolated from the spleen of a domestic pig with typical acute ASF during an ASF outbreak in Northern Vietnam in 2020. Virulent VNUA-ASFV-L2 strain was generated by serially passaging wild-type VNUA- ASFV-05L1 in PAMs (70 passages). Live attenuated vaccine candidate VNUA-ASFV-LAVL3 was generated by serially passaging VNUA-ASFV-L2 in 3D4 / 21 cells (50 passages). All viruses were maintained in BSL-3 laboratories of Vietnam National University and Kansas State University.

[0113]

[0111] Virus Passage and Virus Replication Evaluation in vitro: Serial passages of the ASFVs were conducted as previously described with slight modifications. Briefly, monolayers of 3D4 / 21 cells at 90% confluency were infected with ASFVs at the multiplicity of infection (MOI) of 0.5. After 2 hours (hrs) of adsorption at 37°C, the inoculums were removed. Cells were rinsed twice with phosphate-buffered saline (PBS, pH 7.2, Thermo Scientific, Waltham, MA, USA), replaced with fresh culture media supplemented with 0.5% dimethyl sulfoxide (DMSO, Millipore Sigma, Lenexa, KS, USA), and incubated for 4 days. Culture supernatant was then harvested, titrated, and passaged onto fresh monolayers at MOI of 0.5. Virus titers in the supernatants at each passage were titrated in PAMs. Briefly, PAMs were pre-seeded (80-100% confluent) and incubated with 10-fold dilutions of the harvested supernatants. For hemadsorption (HAD) testing, 2% porcine red blood cells were added after 2 hrs incubation. After four days culture, the presence of ASFV was assessed by HAD and cytopathic effects (CPE) in tissue culture under an inverted microscope. HADso and TCID50 titers were calculated using the method of Reed and Muench.

[0114]

[0112] For testing the in vitro replication characteristics of ASFVs, monolayers of PAMs and 3D4 / 21 cells at 90% confluency in 24-well culture plates were infected with the viruses at MOI of 0.5. After 2 hrs incubation, the inoculums were removed. Cells were washed and replaced with fresh culture media. Cultures (including cells and culture medium) were collected at 0, 24, 36, 48, 60, 72, 84, and 96 hrs post-infection (HPI). The collected cultures were subjected to three freeze-thaw cycles. After centrifuging the cell debris, ASFVs titers in the supernatant were tested and calculated as described above. All experiments were performed in duplicate.

[0115]

[0113] ASFV Genome Next-Generation Sequencing: Next-generation sequencing of ASFV genome was performed as described previously. Briefly, ASFV DNA was extracted with the QIAamp DNA minikit (Qiagen, Hilden, Germany). The sequencing library was constructed following the manufacturer’s instruction of the NEBNext Ultra DNA Library Prep Kit (New England Biolabs, Ipswich, MA, USA), and sequencing was performed with Illumina NovaSeq 150PE sequencing platform (Illumina, San Diego, CA, USA). After sequencing, primer sequences were removed from raw Illumina reads using bbduk of the BBTools Packages. QC reads were assembled de novo using SPAdes, polished using Pilon version 1.23, and implemented in Unicycler. All contigs were subjected to BLASTN against the NCBI nucleotide database. Open reading frames (ORFs) were predicted using Prodigal and annotated using Prokka 1.14.6. The single contig with BLASTN hit similarity to an ASFV was aligned with a number of reference genomes using MAFFT v7.450. The pairwise comparison of average nucleotide identity (ANI) between ASFV genomes was done by ANI Calculator. Other tools for genomic visualization and classifying multigene family (MGF) proteins in ASFV were geneCo and MGFC, respectively. All bioinformatic tools were run with default parameter settings.

[0116]

[0114] Safety Testing of LAV in Pigs: To evaluate the safety and efficacy of VNUA- ASFV-LAVL3, three groups of pigs (n=5 / group) were intramuscularly (i.m.) inoculated with 103, 104, and 105TCIDso / dose of VNUA-ASFV-LAVL3, respectively. Two control groups (n=3 / group) received (i.m.) 102HAD50 of virulent VNUA-ASFV-L2 or DMEM. At 28 days postinoculation (DPI), all pigs were challenged (i.m.) with 103HAD50 of the wild-type VNUA-ASFV- 05L1 strain for efficacy testing. Blood, oral fluids, rectal swabs, and serum samples were collected at various time points (0-28 DPI and 0-28 days post-challenge, DPC). Clinical signs (anorexia, depression, fever, purple skin discoloration, staggering gait, diarrhea, and cough), body temperature, and survival rate were monitored daily throughout the experiment by qualified personnel. Dead pigs were necropsied for ASFV pathological lesions. Tissue samples (1.5 g) from inner part of brain, tonsil, kidney, liver, spleen, lung, heart, lymph nodes, stomach, bladder, and bone marrow were aseptically collected for virus detection.

[0117]

[0115] To assess the duration of protection afforded by VNUA-ASFV-LAVL3, two pig experiments (one-month vaccination-challenge and two-month vaccination-challenge) were conducted. In one-month vaccination-challenge experiment, five pigs were vaccinated (i.m., once) with 104TCID50 VNUA-ASFV-LAVL3, three contact pigs (without vaccination) were added to test the shedding of the virus, and three control pigs were kept in different room. After one-month, all pigs were challenged with 103HAD50 of wild-type VNUA-ASFV-05L1 strain.

[0118]

[0116] Similar to the one-month vaccination-challenge experiment, in two-month vaccination-challenge experiment, five pigs were vaccinated (i.m., once) with 104TCID50 VNUA- ASFV-LAVL3, three contact pigs (without vaccination) were added to test the shedding of the virus, and three control pigs were kept in a different room. After two-month, all pigs were challenged with IxlO3HAD50 of the wild-type VNUA-ASFV-05L1 strain. Blood and serum samples were collected at 0, 30, and 60 days post-vaccination (DPV), and at various DPC (0, 3, 5, 7, 9, 11 , 14, 17, 21 , 25, and 28). The presence of clinical signs (anorexia, depression, fever, purple skin discoloration, staggering gait, diarrhea, and cough), body temperature, and survival rate were monitored daily throughout the experiment.

[0119]

[0117] DNA Extraction, Quantitative ASFV Real-Time PCR and Genome Sequencing: qRT-PCR was used to detect ASFV DNA in oral fluid, rectal swabs, blood, and tissue samples collected from the experimental pigs. DNA extraction was performed using an automated King Fisher™ Duo Prime DNA / RNA extraction system (Thermo Fisher Scientific, Waltham, MA, USA) with MagMAX CORE nucleic acid purification kit (Life Sciences, NY, USA) following the manufacturer’s instructions. Subsequently, ASFV DNA was quantified using the Platinum SuperMix-UDG kit (Invitrogen, Waltham, MA, USA) on CFX Optus 96 Realtime PCR system (BioRad, Hercules, CA, USA). Specific primers and a probe targeting the ASFV p72 gene, developed by Haines et.al., were employed. Samples with Ct values <40 were considered positive for ASFV.

[0120]

[0118] The ASFV genome next-generation sequencing and de novo genome assembly were performed as described previously. LAV whole genome analysis and comparison with the parental virus VNUA-ASFV-05L1 (GenBank accession number MW465755.1) were performed using Genome Workbench from NCBI and CLC Sequence Viewer 8.0.0 (Qiagen, Hilden, Germany).

[0121]

[0119] ASFV Specific Antibody Detection: T A commercial ASF blocking ELISA kit (INGEZIM PPA COMP AC H.PPA.k3, Ingenasa, Madrid, Spain) was used to detect anti-ASFV antibodies in serum samples. The assay was performed following the manufacturer’s instructions. Each sample’s competition percentage (S / N%) was calculated according to the manufacturer’s instructions, with interpretations as follows: > 50% positive, 40-50% doubtful, and <40% negative.

[0122]

[0120] ELISPOT and ELISA to Evaluate IFN-Y and IL-10 Cellcular Responses in Pigs: Interferon gamma (IFN-y) production by peripheral blood mononuclear cells (PBMC) of pigs was assessed using an enzyme-linked immunospot assay (ELISPOT). Briefly, PBMCs were isolated from each pig using density-gradient centrifugation with 1.077 g / mL Ficoll-Paque PREMIUM density gradient media (Cytiva, Marlborough, MA, USA) in SepMateTM tubes (STEMCELL Technologies Inc., Cambridge, MA, USA). PBMC suspensions (2* 105 cells / well) were added to Multi ScreenHTS IP Filter Plates (Millipore Sigma, Lenexa, KS, USA) pre-coated with anti-pig IFN-y antibodies (BD Biosciences, San Jose, CA, USA). PBMCs were incubated at 37°C for 18 hrs with the following stimuli (100 pL / well): phorbol myristate acetate (PMA, 25 ng / mL) / ionomycin (2.5 pg / mL) (Millipore Sigma, Lenexa, KS, USA) combination as positive control, VNUA-ASFV- 05L1 (105 HAD50 / mL) as re- stimulation agent, and complete culture media as unstimulated control. After washing, biotinylated anti-pig IFN-y (BD Biosciences, San Jose, CA, USA), HRP Streptavidin (BD Biosciences, San Jose, CA, USA), and fresh NovaRED Peroxidase Substrate (Vector Labs, Newark, CA, USA) were added as per the manufacturer’s instructions. The reaction was stopped by rinsing the plate with deionized water, ant the number of IFN-y spots was quantified using a CTL Spot Reader (CTL, New York, NY, USA).

[0123]

[0121] The concentration of porcine IL-10 in serum samples of pigs were measured by porcine IL-10 Quantikine ELISA kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's instructions.

[0124]

[0122] Statistical Analysis: Statistical analysis was performed using GraphPad Prism 6.0 (GraphPad Software, CA, USA). The data from assays for virus titration in cell cultures and blood samples in experimental pigs at different time points and efficacy studies were expressed as the mean log titer ± SD (Standard deviation) for each group and analyzed with Student’s t-test. The data for antibody and cellular responses were expressed as mean readings ± SD for each group. The significance of differences between the experimental groups was analyzed with analysis of variance (ANOVA) followed by Turkey’s post-test. For all statistical analyses, p values less than 0.05 were considered as statistically significant.

[0125]

[0123] VNUA-ASFV-LAVL3 loses hemadsorption activity during adaptation in 3D4 / 21 cells: VNUA-ASFV-LAVL3 was generated by serially passaging the virulent VNUA- ASFV^ strain (which had already undergone 70 passages in PAM cells) in 3D4 / 21 cells for a total of 50 times. We oberved moderate viral growth in 3D4 / 21 cells, reaching titers of approximately 104to 105TCIDso / mL by passage 12. The moderate-to-high titer (around 105TCIDso / mL) was maintained throughout subsequent passages to passage 50, with the highest titer (106TCIDso / mL) observed at 72 hpi. Compared to the parental VNUA-ASFV-L2 strain, VNUA- ASFV-LAVL3 exhibited comparable growth kinetics in both PAMs and 3D4 / 21 cells, with less than a 0.5 loglO difference in viral titers at each time point (Figure 1A, B). Notably, during adaptation and passaging in 3D4 / 21 cells, VNUA-ASFV-LAVL3 lost the hemadsorption activity characteristic of its parental virus, VNUA-ASFV-L2 (Figure 1C, D).

[0124] Genetic Characterization of VNUA-ASFV-LAVL3: Whole-genome sequencing revealed that the genome of VNUA-ASFV-LAVL3 comprises 179,678 nucleotides with a GC content of 36,34%. Comparative analysis with the wild-type VNUA-ASFV-05L1 (GenBank accession number MW465755.1) identified a notable deletion encompassing a region spaning from 8, 555 bp to 17, 972 bp in the genome of VNUA-ASFV-05L1. This deletion resulted in the loss of 10 known genes (285L, MGF 110-8L, MGF 100-1R, MGF 110-9L, MGF 110-1 IL, MGF 110-14L, MGF 110-12L, MGF 110-13L, MGF360-4L, and MGF360-6L), partial sequence loss of the X69R gene, and the deletion of 11 uncharacterized sequences (Figure 2).

[0126]

[0125] Additionally, insertions, and mutations leading to amino acid substitutions or protein disruptions were discovered in the genome of VNUA-ASFV-LAVL3 (Table 1). Of particular interest were three alteration within the EP402R (CD2v) gene: a single nucleotide substitution (72, 222 thymine to cytosine) and two single nucleotide deletions (72, 844 thymine and 72, 965 cytosine). These changes potentially disrupt the CD2v protein, which plays a crucial role in the hemadsorption of swine red blood cells (RBCs).

[0127] Table 1. Mutations of VNUA-ASFV-LAVL3 using the reverse-complement sequence of wildtype VNUA-ASFV-05L1 (SEQ ID NO. 2) as a Reference Sequence

[0128]

[0126] VNUA-ASFV-LAVL3 exhibits high safety in pigs: Pigs inoculated with 103,

[0129] 104 and 105 TCID50 of VNUA-ASFV-LAVL3 exhibited a temporary low fever (<40.6 °C) between 9-11 DPI (peak at 10 DPI) (Figure 3 A), 8-11 DPI (peak at 10 DPI) (Figure 3C), and 5-9 DPI (peak at 7 DPI) (Figure 3E), respectively. Apart from this, vaccinated pigs maintained normal body temperature throughout the 28-day observation period and showed no obvious ASFV-specific clinical signs. In contrast, control pigs inoculated with the parental VNUA- ASFV-L2 virus developed fever as early as 3 DPI (peak at 5 DPI with 41-41.8oC) (Figure 3 A, C,

[0130] E) followed by severe ASF clinical signs (anorexia, coughing, depression, purple skin discoloration, respiratory symptoms, diarrhea) and died within 8-9 DPI (Figure 3A, C, E).

[0131]

[0127] VNUA-ASFV-LAVL3 vaccinated pigs showed very low viremia, with Ct values ranging from 34.62-37.51 (low dose) between 5-17 DPI, and 31.52-35.17 (high dose) between 3- 21 DPI. This viremia rapidly declined, becoming almost undetectable by 17 DPI (Figure 3B, D,

[0132] F). On the other hand, control pigs inoculated with the parental VNUA-ASFV-L2 virus reached peak viremia (Ct value ~ 15) at 7 DPI, remaining high until death by 8-9 DPI (Figure 3B, D, F). Additionally, ASFV was undetectable in oral fluid or rectal swabs from vaccinated pigs throughout the 28-day observation period (Data not shown)..

[0133]

[0128] Vaccination with VNUA-ASFV-LAVL3 confers full protection in pigs: To evaluate the potential of VNUA-ASFV-LAVL3 as a live-attenuated virus vaccine, pigs vaccinated with different doses of the VNUA-ASFV-LAVL3 were challenged intramuscularly with IxlO3HAD50 of the highly virulent wild-type VNUA-ASFV-05L1 strain at 28 DPI. All vaccinated groups maintained normal rectal temperatures after the challenge, unlike control pigs whose temperatures rapidly increased to 41.8°C at 5 DPC (Figure 4A). The challenge virus was almost undetectable in blood samples of all vaccinated groups. Only one pig in the 103TCID50 dose group showed low viral titer (Ct value 37.84) at 7 DPC (Figure 4B). All vaccinated pigs remained healthy and fully protected against the virulent challenge, achieving 100% survival throughout the 28-day DPC observation. In contrast, the challenge virus was detected in control pigs as early as 3-5 DPC, rapidly increasing by 5-7 DPC (Figure 4B). They developed typical ASF clinical signs at 5-9 DPC and succumbed to death by 8-9 DPC.

[0134]

[0129] VNUA-ASFV-LAVL3 vaccination prevents replication of wild-type ASFV and protects against pathological lesions in pigs: To further validate the protective efficacy conferred by VNUA-ASFV-LAVL3 in pigs, we investigated the presence of virulent challenge strain and associated pathological lesions in various organs of vaccinated pigs at 28 DPC and control pigs at 8-9 DPC. At 28 DPC, organs (brain, heart, lung, liver, stomach, spleen, kidney, bladder, tonsil, lymph node, and bone marrow) collected from control pigs displayed high ASFV titers as determined by real-time PCR analysis (Table 2). In contrast, pigs vaccinated with VNUA-ASFV- LAVL3 at various doses (103- 105TCIDso) showed undetectable levels of ASFV in all examined organs at 28 DPC (Table 2). This data suggests that VNUA-ASFV-LAVL3 vaccination effectively prevents both viral replication and the development of pathological lesions in pigs challenged with the virulent ASFV strain.

[0135] Table 2: ASFV detection in organs of VNUA-ASFV-LAVL3 vaccinated pigs and control pigs

[0136] Quantitative ASFV Real-Time PCR

[0137] [130J Note the following definitions for acronyms used in Table 2: (LLN) inguinal lymph node; (MLN) mesenteric lymph node; (SLN) submaxillary lymph node; (BM) bone- marrow; (SI) small intestine; (LI) large intestine; negative results for ASFV DNA with Realtime PCR; positive results for ASFV DNA with Real-time PCR.

[0138]

[0131] Post-mortem examination of non-immunized control pigs challenged with the wildtype VNUA-ASFV-05L1 revealed severe gross lesions and pathological signs of acute ASF, as previously described. These pigs displayed extensive macroscopic lesions, including necrosis and hemorrhage in various organs: mandibular lymph nodes, lungs, mesentery, spleen, renal cortex, pericardium, and myocardium. Additionally, non-immunized pigs exhibited dark and enlarged spleens, swollen livers and gallbladders, and interstitial pulmonary edema (Figure 5A). Conversely, pigs vaccinated with VNUA-ASFV-LAVL3 showed no clinical signs, pathological lesions, or abnormalities in any examined organs at necropsy (Figure 5B).

[0139]

[0132] Pigs VNUA-ASFV-LAVL3 induce robust ASFV-specific humoral and cellular immune responses: Pigs vaccinated with the dose of 103, 104, and 105TCID50 of the VNUA- ASFV-LAVL3 strain developed anti-ASFV antibodies as early as 7 days post vaccination (DPV). At 28 DPV (0 DPC), the blocking percentage (%) of ASFV-specific antibodies reached approximately 83.6-103.7%, whereas no ASFV specific antibodies were detected in the control animals prior to the challenge (Figure 6A, B, C). Following the challenge with wild-type VNUA- ASFV-05L1, the antibody levels peaked at 7 DPC (35 DPI), and remained consistent up to 28 DPC (56 DPI) at the end of the experiment (Figure 6A, B, C). ELISPOT assay results showed that positive controls exhibited spot counts exceeding 200. PBMCs of VNUA-ASFV-LAVL3- vaccinated pigs produced IFN-y after stimulation with the wild-type VNUA-ASFV-05L1. The numbers of spot-forming cells from VNUA-ASFV-LAVL3-vaccinated pigs were significantly (p <0.0001) higher than the cells from non-vaccinated control pigs (Figure 6D).

[0140]

[0133] Pigs can be protected against wild-type ASFV challenge for up to 2 months after a single vaccination with VNUA-ASFV-LAVL3: We evaluated the ability to confer longterm protection (1-month and 2-months) of the VNUA-ASFV-LAVL3 using a single dose (104TCID50) in pigs. As expected, all vaccinated pigs developed consistently ASFV specific antibody response with sustained immune response up to 2 months post-vaccination. All contact pigs were negative for ASFV specific antibody responses and ASFV till the moment of challenge, indicating no ASFV infection (Data not shown). After challenge, 2 out of 5 vaccinated pigs in 2-months vaccination group showed low level of ASFV (Realtime PCR Ct value ranging from 37.51 to 36.18) at 7 and 9 DPC, and showed negative at later time-points post-challenge. All vaccinated pigs survived, remained healthy, and no clinical signs of ASFV infection after challenging with wild-type VNUA-ASFV-05L1 (Table 3). The control pigs and contact pigs developed fever at 3 DPC. Subsequenctly clinical signs (anorexia, coughing, depression, purple skin discoloration, severe respiratory symptoms, and diarrhea) of acute ASF were observed. All control pigs and contact pigs became viremic at 5-7 DPC and were euthanized at 8-11 DPC (Table 3). Table 3; Long-term protection conferred by a single vaccination of VNUA-ASFV-LAVL3

[0141]

[0134] Analysis of Preceding Examples: Live attenuated vaccines, generated by serial passaging of a virus in cultured cells, have proven highly effective in preventing numerous viral diseases, including smallpox, polio, measles, mumps, and yellow fever. These LAVs elicit robust cellular and humoral immune responses, often providing lifelong protection with minimal dosing (one or two doses). Carefully control passage numbers during cell culture is crucial to balance viral replication and immune induction for developing cell passaged LAVs. Excessive passaging can weaken the vaccine by eliminating essential protective genes. Through careful cell culture and controlled passage times, we have successfully generated a HAD-positive ASF LAV candidate. Here, we report a non-HAD ASF LAV candidate, VNUA-ASFV-LAVL3, developed using the same approach. The cell-adapted VNUA-ASFV-LAVL3 can efficiently infect and replicate in 3D4 / 21 cells with 0.5% DMSO in the culture medium (Fig. IB). Earlier studies highlighted the potential of non-HAD ASFV strains for LAV development. The naturally attenuated non-HAD ASFV / L60 and Lvl7 / WB / Riel strains displayed significantly lower virulence than their parental virulent HAD strains. Consistent with these findings, the non-HAD VNUA-ASFV-LAVL3 exhibited highly attenuated properties, and showing promise for inducing protective immunity against wild-type genotype II ASFV in pigs (Fig. 3, 4, 5).

[0135] The EP402R gene, encoding the CD2v protein, is responsible for the ASFV distinctive HAD phenomenon in ASFV. We identified a single nucleotide substitution (72, 222 thymine to cytosine) and two single nucleotide deletions (72, 844 thymine and 72, 965 cytosine) in the EP402R gene of VNUA-ASFV-LAVL3 (Table 1), leading to frameshift variant in the CD2v protein, shedding light on the genetic basis for the loss of haemadsorption with VNUA- ASFV-LAVL3 strain. Additionally, the MGF region in ASFV plays a crucial role in viral virulence, and deletions within this region are associated with attenuated phenotypes. Fullgenome sequencing revealed deletions of 10 genes (285L, MGF 110-8L, MGF 100-1R, MGF 110-9L, MGF 110-1 IL, MGF 110-14L, MGF 110-12L, MGF 110-13L, MGF360-4L, and MGF360-6L) and a partial deletion in the X69R gene within the MGF region of VNUA-ASFV- LAVL3 (Table 1), potentially contributing to its attenuated phenotype in pigs.

[0142]

[0136] A coordinated host response involving both cellular and humoral immunity is essential for combating ASFV infection. Cellular immunity, particularly T cell-mediated responses, plays a pivotal role in controlling viral replication by targeting infected cells. ELISPOT assays demonstrated that VNUA-ASFV-LAVL3 induced robust cellular immune responses (ASFV-specific IFN-y) in pigs. While the precise role of antibodies in ASFV protection remains unclear, their presence is considered crucial for defense. Pigs vaccinated with VNUA-ASFV-LAVL3 at doses of 103, 104, and 105TCID50 developed anti-ASFV antibodies as early as 7 DPV, consistent with previous findings of antibody detection within 7-14 days postinfection in pigs infected with low or moderately virulent ASFV strains. However, the exact mechanisms of how cellular and humoral immune responses work together in combating ASFV requires further investigation.

[0143]

[0137] A potential concern with LAVs is the risk of shedding and potential transmission or reversion to virulence. In our study, no transmission of VNUA-ASFV-LAVL3 from vaccinated pigs to unvaccinated contact pigs was observed, even when challenged 1- or 2-months postvaccination. All contact pigs remained negative for ASFV-specific antibodies until challenge. Furthermore, VNUA-ASFV-LAVL3 DNA was undetectable in organs of vaccinated pigs (Table 2). Notably, a single dose (104TCIDso) of VNUA-ASFV-LAVL3 effectively protected pigs against wild-type ASFV challenge for up to 2 months (Table 3).

[0144]

[0138] FIGS. 18A and 18B show cytokine responses induced in control and vaccinated pigs up to 42 days post-challenge. Vaccinated pigs were vaccinated with 103, 104, or 1CP doses of VNUA-ASFV-LAVL3 passaged 128 times. Significant levels of IL-10 (Thl) were observed in the serum of control pigs post-challenge. A slightly increased level of IFN-y was observed in pigs vaccinated with VNUA-ASFV-LAVL3, but no significant difference in IFN- y was observed in non-vaccinated pigs post-challenge.

[0145]

[0139] Table 4 below provides a summary of the results of testing five groups of pigs on various vaccine dosage regimes against a control group tested solely with Roswell Park Memorial Institute 1640 (RPMI) medium alone. Pigs arrived at the facitily at 6-weeks old and were vaccinated at 7 weeks.

[0146] Table 4. Outcomes for Challenge Groups

[0140] Table 5 provided below shows the safety, transmission, and protection of a single does of VNUA-ASFV-LAVL3 at 1 and 2 months post-vaccination.

[0147] Table 5. Safety, Transmission, Protection 1 and 2 months after vaccination

[0148]

[0141] Table 6 provided below shows the safety, transmission, and protection of a single does of VNUA-ASFV-LAVL3 at 1 and 2 months post -vaccination.

[0149] Table 6; Safety, Transmission, Protection 1 and 2 months post-vaccination

[0150]

[0142] Table 7 provided below shows the safety, transmission, and protection of a single does of VNUA-ASFV-LAVL3 at 1 and 2 months post-vaccination.

[0151] Table 7; Safety, Transmission, Protection 1 and 2 months post-vaccination

[0152]

[0143] Table 8 provided below shows the safety, transmission, and protection of a single does of VNUA-ASFV-LAVL3 at 1 and 2 months post -vaccination. Table 8; Safety, Transmission, Protection 1 and 2 months post-vaccination

[0153]

[0144] Table 9 provided below shows safety and efficacy data for a single dose of

[0154] VNUA-ASFV-LAVL3 in sows at a collaborative research farm.

[0155] Table 9: Safety, Efficacy of vaccination in sows at collaborative research farm

[0156]

[0145] Table 10 provided below shows safety and efficacy data for a single dose of

[0157] VNUA-ASFV-LAVL3 in sows at a collaborative research farm.

[0158] Table 10: Safety, Efficacy of vaccination in sows at collaborative research farm

[0159]

[0146] Table 11 provided below shows data related to protection provided by a single dose of VNUA-ASFV-LAVL3 against the virulent hybrid / recombinant ASFV Genotype 1 and 2 (VNUA-ASFV-G12).

[0160] Table 11 : Safety, Efficacy of vaccination in sows at collaborative research farm

[0161]

[0147] Table 12 provided below shows data related to protection provided by a single dose of VNUA-ASFV-LAVL3 against the virulent recombinant ASFV Genotype 1 and 2 (VNUA-ASFV-G12).

[0162] Table 12: Safety, Efficacy of vaccination in sows at collaborative research farm

Claims

What is claimed is:

1. An immunogenic composition comprising an attenuated modified live, killed, or inactivated African Swine Fever Virus (ASFV) having at least one missing, disrupted, or modified gene or sequence in comparison to a wild-type ASFV, wherein administration of said attenuated modified live, killed, or inactivated ASFV reduces the incidence of and / or severity of at least one sign or symptom caused by ASFV in an animal or group of animals in need thereof.

2. The immunogenic composition of claim 1, wherein the at least one missing, disrupted, or modified gene or sequence is selected from the group consisting of 285L, X69R, A104R, F1055L, CD2v, M1249L, C45L, B962L, B385R, B407L, G1340L, G1211R, CP2475L, CP530R, NP1450L, D250R, D1133L, S183L, S273R, P1192R, H233R, H240R, E248R, I177L, MGF 110-8L, MGF 100-1R, MGF 100-9L, MGF 110-1 IL, MGF 110-14L, MGF 110-12L, MGF 110-13L, MGF 360-4L, MGF360-6L, MGF 300-2R, MGF 505-2R, MGF 360-18R, and an uncharacterized region of SEQ. ID NO. 1 selected from the group consisting of base pair (bp) positions 6,343 to 6,344, 87, 9,004 to 9,131, 9516 to 9,732, 10,108 to 10,265, 11, 139 to 11,428, 11,789 to 11,877, 12,244 to 12,432, 12,793 to 12,878, 13,703 to 13,882, 15,047 to 15,862, 16,991 to 17,868, and any combination thereof.

3. The immunogenic composition of claim 1, wherein the missing, disrupted, or modified gene or sequence is selected from the group consisting of an MGF gene, an L gene, and an uncharacterized gene or sequence.

4. The immunogenic composition of claim 1, wherein the missing, disrupted, or modified gene or sequence is selected from the group consisting of an MGF gene; an L gene; an uncharacterized gene or sequence; at least one MGF gene and one L gene; at least one MGF gene and at least one uncharacterized gene or sequence; at least one uncharacterized gene or sequence and at least one L gene; at least one MGF gene, one L gene, and one uncharacterized gene or sequence; at least one MGF 360 gene and / or at least one MGF 110 gene; MGF 360-6L, MGF 360-4L, and at least one L gene; at least one MGF 360 gene, at least one L gene, and at least one uncharacterized gene or sequence; MGF 360-6L, X69R, 285L, and at least one uncharacterized gene or sequence; MGF110-13L, MGF 360-4L, and at least one uncharacterized gene or sequence; MGF 360-4L, MGF 100-1R, and MGF 110-7L; 285L, A104R, and X69R; at least one MGF gene and the A104R gene; at least one MGF gene and the X69R gene; at least one MGF gene and the 285L gene; at least one MGF gene, the X69R gene, and the 285L gene; at least one MGF gene, the X69R gene, and the F1055L gene; at least one MGF gene, the II 17L gene, and the 285L gene; at least one MGF gene, the X69R gene, the F1055L gene, and the 285L gene; at least one MGF gene, one L gene, and the Fl 055L gene; at least one MGF gene, one L gene, and the X69R gene; at least one MGF gene, one L gene, and the 285L gene; at least one MGF gene, one L gene, the X69R gene, and the 285L gene; at least one MGF gene, one L gene, the X69R gene, and the F1055L gene; at least one MGF gene, one L gene, the F1055L gene, and the 285L gene; at least one MGF gene, one L gene, the X69R gene, the F1055L gene, and the 285L gene; at least one MGF gene, one uncharacterized gene, and the F1055L gene; at least one MGF gene, one uncharacterized gene, and the X69R gene; at least one MGF gene, one uncharacterized gene, and the 285L gene; at least one MGF gene, one uncharacterized gene, the X69R gene, and the 285L gene; at least one MGF gene, one uncharacterized gene, the X69R gene, and the F1055L gene; at least one MGF gene, one uncharacterized gene, the F1055L gene, and the 285L gene; and at least one MGF gene, one uncharacterized gene, the X69R gene, the F1055L gene, and the 285L gene.

5. The immunogenic composition of claim 1 further comprising an antigen from another disease-causing organism.

6. The immunogenic composition of claim 5, wherein said another disease-causing organism is selected from the group consisting of Actinobacillus pleuropneumonia, Adenovirus; Alphavirus such as Eastern equine encephalomyelitis viruses; Bordetella bronchiseptica, Brachyspira spp., preferably B. hyodyentheriae; B. piosicoli, Brucella suis. preferably biovars 1, 2, and 3; Classical swine fever virus; Clostridium spp., preferably CL difficile, Cl. perfringens types A, B, and C, Cl. novyi, Cl.septicum, CL tetani; Coronavirus, preferably Porcine Respiratory Corona virus; Eperythrozoonosis suis; Erysipelothrix rhsiopathiae; Escherichia coli; Haemophilus parasuis, preferably subtypes 1, 7 and 14: Hemagglutinating encephalomyelitis virus; Japanese Encephalitis Virus; Lawsonia intracellularis; Leptospira spp.: preferably Leptospira australis; Leptospira canicola; Leptospira grippotyphosa; Leptospira icterohaemorrhagicae; and Leptospira interrogans; Leptospira pomona; Leptospira tarassovi; Mycobacterium spp. preferablyM. avium; M. intracellulare ; and M.hovis Mycoplasma hyopneumoniae (M hyo) Pasteurella multocida, Porcine circovirus; Porcine cytomegalovirus; Porcine Parvovirus; Porcine Reproductive and Respiratory Syndrome (PRRS) Virus; Pseudorabies virus; Rotavirus; Salmonella spp:, preferably S. thyhimurhinr, and S. choleraesuis; Staph, hyicus; Staphylococcus spp. preferably Streptococcus spp., preferably Strep. suis, Swine herpes virus; Swine Influenza Virus; Swine pox virus; Swine pox virus; Vesicular stomatitis virus; Virus of vesicular exanthema of swine; Leptospira Hardjo,' and / or Mycoplasma hyosynoviae.

7. The immunogenic composition of claim 1, further comprising at least one pharmaceutical- acceptable carrier.

8. The immunogenic composition of claim 1 comprising at least one nucleotide sequence having at least 90% sequence identity to SEQ ID NO. 1.

9. The immunogenic composition of claim 8, further comprising at least one pharmaceutical- acceptable carrier.

10. The immunogenic composition of claim 1, wherein the at least one sign or symptom of ASFV is selected from the group consisting of loss of high fever, decreased appetite and weakness, red blotchy skin or skin lesions, coughing and difficulty breathing, vomiting, reddening or darkening of the skin and particularly ears and snout, gummed up eyes, abortion and still births and weak litters, weakness and unwillingness to stand, and any combination thereof.

11. A method of decreasing the incidence of or severity of at least one clinical or postmortem sign or symptom of African Swine Fever Virus (ASFV) comprising the step of administering at least one dose of the immunogenic composition of claim 1 to an animal in need thereof.

12. The method of claim 11, wherein the incidence or severity is decreased at least 10% in comparison to an animal that has not received the immunogenic composition of claim 1.

13. The method of claim 11, wherein the at least one sign or symptom of ASFV is selected from the group consisting of loss of high fever, decreased appetite and weakness, red blotchy skin or skin lesions, coughing and difficulty breathing, vomiting, reddening ordarkening of the skin and particularly ears and snout, gummed up eyes, abortion and still births and weak litters, weakness and unwillingness to stand, and any combination thereof.

14. The method of claim 11, wherein the immunogenic composition of claim 1 is administered via a method selected from the group consisting of intravenous, intravascular, intramuscular, intranasal, intraarterial, intraperitoneal, oral, subcutaneous, transdermal, or intrathecal routes.

15. The method of claim 11, comprising the step of administering at least two doses of the immunogenic composition of claim 1.

16. The method of claim 15, wherein the at least two doses are separated by a period of at least three weeks.

17. A method of decreasing the incidence of or severity of at least one clinical or postmortem sign or symptom of African Swine Fever Virus (ASFV) comprising the step of administering at least one dose of the immunogenic composition of claim 8 to an animal in need thereof.

18. The method of claim 19, wherein the incidence or severity is decreased at least 10% in comparison to an animal that has not received the immunogenic composition of claim 8.

19. The method of claim 17, wherein the at least one sign or symptom of ASFV is selected from the group consisting of loss of high fever, decreased appetite and weakness, red blotchy skin or skin lesions, coughing and difficulty breathing, vomiting, reddening or darkening of the skin and particularly ears and snout, gummed up eyes, abortion and still births and weak litters, weakness and unwillingness to stand, and any combination thereof.

20. The method of claim 17, wherein a blocking percentage of ASFV-specific antibodies reaches no less than 80% by 28 days after the step of administering at least one dose of the immunogenic composition of claim 8.

21. The method of claim 20, wherein the blocking percentage of ASFV-specific antibodies is no less than 80% 56 days after the step of administering at least one dose of the immunogenic composition of claim 8.

22. The method of claim 20, wherein the blocking percentage of ASFV-specific antibodies is no less than 100% 56 days after the step of administering at least one dose of the immunogenic composition of claim 8.

23. The method of claim 17 wherein an ELISPOT assay conducted 28 days after the step of administering at least one dose of the immunogenic composition of claim 8 exhibits a spot count exceeding 200.

24. The immunogenic composition of claim 1 comprising at least one nucleotide sequence having at least 98% sequence identity to SEQ ID NO. 1.

25. The immunogenic composition of claim 1 wherein the attenuated modified live, killed, or inactivated ASFV exhibits no hemadsorption activity in cells derived from pigs.

26. The immunogenic composition of claim 25 wherein the cells derived from pigs are 3D4 / 21 cells.

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