WO2024155943A2

WO2024155943A2 - Immunogenic compositions for african swine fever virus

Info

Publication number: WO2024155943A2
Application number: PCT/US2024/012252
Authority: WO
Inventors: Waithaka MWANGI; Tae Kim
Original assignee: Kansas State University Research Foundation
Priority date: 2023-01-19
Filing date: 2024-01-19
Publication date: 2024-07-25
Also published as: WO2024155943A3

Abstract

The present disclosure provides immunogenic compositions or vaccines against African Swine Fever Virus (ASFV), methods of making and using such immunogenic compositions or vaccines, and methods of administering such immunogenic compositions or vaccines. In some forms, a plurality of ASFV antigens are combined together into a live-vectored multivalent immunogenic composition. In some forms, the live-vectored multivalent immunogenic composition is a multicistronic expression cassette containing at least one ASFV antigen.

Description

IMMUNOGENIC COMPOSITIONS FOR AFRICAN SWINE FEVER VIRUS

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63/480,658, filed January 19, 2023, which is incorporated by reference herein in its entirety.

GOVERNMENT LICENSE RIGHTS

[0002] This invention was made with government support under USDA-NIFA 2019-67015- 29835 awarded by United States Department of Agriculture. The government has certain rights in the invention.

SEQUENCE LISTING

[0003] This application contains a sequence listing, the contents of the electronic sequence listing (KSURF-40482WO-27214-00065.xml; Size: 371,000 bytes; Date of Creation: January 19, 2024) are hereby incorporated by reference in its entirety.

BACKGROUND

[0004] African Swine Fever Virus (“ASFV”) is a highly contagious hemorrhagic disease of pigs that resembles classical swine fever in its presentation of clinical signs and lesions. ASFV is enzootic in many African countries as well as in Sardinia. Beginning in 2007, ASFV spread into domestic and wild pig populations in western and southern Russia. This spread has continued into Europe and threatens pig populations throughout the continent.

[0005] ASFV is a large, enveloped, double-stranded DNA virus that replicates primarily in cells of the mononuclear phagocytic system. It is a member of the Asfarviridae family. Genotypes of ASFV are differentiated by sequence analysis of the genomes of viruses obtained from different geographic areas. The virus is highly resistant to a wide pH range and to a freeze/thaw cycle and can remain infectious for many months at room temperature or when stored at 4 degrees C. Virus in body fluids and serum is inactivated in 30 min at 60 degrees C, but virus in unprocessed pig meat can be inactivated by heating to 70 degrees for 30 minutes. Although ASFV can be adapted to grow in cells from different species, it does not replicate readily in any species other than swine. [0006] Peracute, acute, subacute, and chronic forms of ASFV occur and mortality rates vary from 0 to 100%. Acute disease is characterized by a short incubation period of 3 - 7 days, followed by high fever (up to 42 degrees C) and death in 5 - 10 days. Clinical signs include loss of appetite; depression; recumbancy; hyperemia of the skin of the ears, abdomen, and legs; respiratory distress; vomiting; bleeding from the nose or rectum; diarrhea; and abortion. Lesions are evidenced in the lymph nodes, kidneys, heart, as well as other organs. Some isolates produce an enlarged and friable spleen; straw-colored or blood-stained fluid in pleural, pericardial, and peritoneal cavities; or edema and congestion of the lungs. Chronic disease is characterized by emaciation, swollen joints, and respiratory problems. Because ASFV cannot be distinguished from classical swine fever by either clinical or postmortem examination, infection must be confirmed by PCR, ELISA, or indirect immunofluorescence.

[0007] There is no treatment or prophylactic for ASFV. Successful eradication involves the slaughter and disposal of all animals on the premises.

[0008] What is needed is a composition and accompanying methods of making and administering the composition that reduces the severity of and/or incidence of clinical and postmortem signs of ASFV infection.

SUMMARY OF THE DISCLOSURE

[0009] 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.

[0010] In one aspect, the present disclosure generally provides an efficacious African Swine Fever Virus (ASFV) immunogenic composition or vaccine.

[0011] In another aspect, the present disclosure generally provides methods for making and/or producing an efficacious African Swine Fever Virus immunogenic composition or vaccine.

[0012] 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.

[0013] In another aspect, the present disclosure generally provides methods for reducing the incidence and/or severity of clinical and postmortem signs of ASFV infection. [0014] In another aspect, the present disclosure generally provides a compatible DIVA Lateral Flow Device (LFD) for rapid diagnosis.

[0015] In some forms of all aspects, the immunogenic composition comprises at least one ASFV antigen. Preferably, the antigen is conserved among ASFV isolates. One preferred source for conserved antigens is from the Georgia 2007/1 ASFV isolate. In preferred forms, a plurality of antigens are combined together into a live-vectored multivalent immunogenic composition. In some forms, the live-vectored multivalent immunogenic composition is a multi ci stronic expression cassette. In some forms, the multi ci stronic expression cassette includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more individual antigens. In some forms, the multi ci stronic cassette includes an insert of at least 1 kBp, 2 kBp, 3 kBp, 4 kBp, 5 kBp, 6 kBp, 7 kBp, or 8 kBp. Some preferred antigens are selected from the group consisting of sequences having at least 80%, more preferably 85%, still more preferably 90%, even more preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity or sequence homology with a sequence selected from SEQ ID NOS. 1 - 101. Nucleic acid sequences encoding any one or number of SEQ ID NOS. 1 - 101 can be combined together to form a multi ci stronic expression cassette. In other words, the individual sequences can be combined together in any order and with any number of individual sequences being combined. Some preferred multicistronic expression cassettes express a sequence selected from any one of SEQ ID NOS. 158-201. As can be appreciated, each of the antigens forming each expression cassette can vary in their sequence identity to the individual sequences as described above, therefore, the overall sequence identity or homology required for an expression cassette is at least 80%, more preferably 85%, still more preferably 90%, even more preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% with a sequence encoding a sequence selected from SEQ ID NOS. 1 - 101. In some forms, an El coding sequence is added to the expression cassette. In some forms, at least one El coding sequence is added to an expression cassette containing a sequence encoding at least one of SEQ ID NOS. 1-101. Similarly, the overall sequence of the preferred multicistronic expression cassettes can vary to the same degree, both at the individual sequences forming the cassette as well as the overall sequence of combined sequences forming the cassette. Further, the individual sequences forming a multicistronic expression cassette can appear in any order. In some forms, the individual sequences are fused or included in-frame to create a chimeric antigenic sequence. In some forms, a self-cleaving peptide linker is included between the different antigens forming the multivalent composition. A preferred linker is provided herein as SEQ ID NO. 204. In some forms, a tail is added to the end. One preferred tail is provided herein as SEQ ID NO. 203. A preferred El sequence is provided herein as SEQ ID NO. 202.

[0016] In some forms of a live-vectored multivalent immunogenic composition of the disclosure, the multi ci str onic expression cassette utilizes a replication competent vector, such as an adenovirus. In some forms, the adenovirus is recombinant. In some forms, the adenovirus is human adenovirus. In some forms, the adenovirus is human type 5 (RcAd5). In still other forms, the multicistronic cassette utilizes an attenuated bovine parainfluenza virus type 3 genotype c (BPIV3c) as the vector. In addition to inducing strong cytotoxic T lymphocyte (CTL) immune responses, compositions of the disclosure generate IFN-y and antibody responses. In still other forms, a baculovirus or lentivirus vector is utilized.

[0017] In another aspect, the present disclosure generally provides nucleic acid sequences for expressing any of SEQ ID NOS. 1-131, SEQ ID NOS. 158-201, or sequences having at least 80%, more preferably 85%, still more preferably 90%, even more preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity or homology with any of SEQ ID NOS. 1-131 or 158-201. Such nucleic acid sequences can be placed in a vector for expression wherein the resultant construct is administered to a subject as described in this disclosure or the expressed antigens are recovered and used in accordance with methods of this disclosure or administered in accordance with methods of this disclosure.

[0018] In another aspect of the present disclosure, a fragment of a nucleic acid sequence or expressed antigen is provided. In preferred forms, the expressed antigen fragments comprise a portion of any of SEQ ID NOS. 1-131, 158-201, or the nucleic acid sequences encoding such fragments. In preferred forms, a nucleic acid fragment encoding any one or more of SEQ ID NOS. 1-131 or 158-201 includes at least 15, 21, 30, 45, 60, 75, 99, 120, 150, or 180 consecutive nucleotides encoding the specific sequence from SEQ ID NOS. 1-131 or 158-201 or sequences having at least 80%, more preferably 85%, still more preferably 90%, even more preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity or homology with any of SEQ ID NOS. 1-131 or 158-201. Similarly, a fragment of an antigen expressed by a nucleic acid sequence encoding any one of SEQ ID NOS. 1-131 or 158-201 or at least 80%, more preferably 85%, still more preferably 90%, even more preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity or homology with any of SEQ ID NOS. 1-131 or 158-201 will preferably include at least 5, 7, 10, 15, 20, 25, 33, 40, 50, or 60 consecutive amino acids from the specific expressed sequence.

[0019] Knowledge gleaned from previous and ongoing ASFV vaccine studies suggests that an efficacious subunit vaccine will preferably include a system capable of cytosolic antigen expression and amplification for effective priming and expansion of cytotoxic T lymphocytes (CTLs) which are required to eliminate infected cells. The ASFV Georgia 2007/1 isolate was selected because it is highly virulent and epidemiologically relevant. A chimeric antigen, designated KP1712, was also generated for development of a compatible DIVA lateral flow-based highly sensitive diagnostic tool.

[0020] 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 an 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.

[0021] In some forms of the present disclosure, at least one ASFV nucleic acid sequence encoding at least one of SEQ ID NOS. 1-101 is inserted into an appropriate vector for administration to a subject. After administration to the subject, the recombinant construct expresses at least one sequence in vivo. In preferred forms, more than one ASFV nucleic acid sequence is included in a multi ci str onic cassette and the entire cassette is inserted into the vector for administration and in vivo expression. In some forms, the cassette includes 1, 2, 3, 4, 5, 6, 7, 8, 9, or more DNA sequences encoding 1, 2, 3, 4, 5, 6, 7, 8, 9, or more of SEQ ID NOS. 1-101. In some forms, the cassette is inserted into a conventional vector. In some forms, the cassette is an adenovirus, baculovirus, or lentivirus vector, such as an adenovirus. In some forms, the adenovirus is recombinant. In some forms, the adenovirus is human adenovirus. In some forms, the adenovirus is human type 5 (RcAd5). In some forms, the vector is replication competent. In some forms, the vector is an attenuated bovine parainfluenza virus type 3 genotype c (BPIV3c). [0022] In some forms, the reduction in incidence and/or severity of infection by ASFV and/or the incidence and/or severity of clinical and postmortem signs of ASFV infection in a subject is determined by a comparison of an animal or group of animals that received at least one administration of a composition described herein to an animal or group of animals that did not receive at least one administration of a composition described herein. Preferably the reduction in incidence and/or severity in subjects receiving at least one administration of a composition described herein is at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or even 100% compared to the animal or group of animals that did not receive at least one administration. The reduction in incidence and/or severity can also be determined by the number of animals exhibiting clinical or postmortem signs of ASFV infection and/or by the number of such signs in individual animals or a group of animals and/or by a comparison of the severity of such signs in individual animals or a group of animals. Such methods of comparison are also effective for chronic disease caused by ASFV.

[0023] The preferred methods of the present disclosure will begin with synthesis of chimeric genes encoding multiple ASFV antigens codon-optimized for protein expression in swine cells. Some preferred sequences are selected from the group consisting of nucleotide sequences that encode SEQ ID NOS. 1-101, a fragment thereof, or sequences having at least 80%, more preferably 85%, still more preferably 90%, even more preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity or sequence homology to SEQ ID NOS. 1-101 or a fragment thereof. One or more nucleotide sequences encoding one or more than one of SEQ ID NOS. 1- 101 or a fragment thereof can be synthesized and cloned into a plasmid vector. The resultant construct(s) is used to generate recombinant viral expression construct(s). Protein expression by the constructs was evaluated by immunocytometric analysis using anti-HA and anti-FLAG monoclonal antibodies (mAbs), and ASFV-specific convalescent serum was used to validate authenticity of the expressed antigens. The multi ci str onic expression cassettes were formed using the constructs and modified to add, in-frame, a HA-tag at the N-termini and a FLAG-tag at the C- termini. In some forms, the expression cassette further includes a Kozaks sequence. In some forms, the tags added to the expression cassette are within the expression cassette and not at the N- or C- termini. The amino acid sequences of the multi ci stronic cassettes were used to design synthetic genes codon-optimized for protein expression in swine cells. Recombinant DNA and expression constructs were generated using the synthetic genes. [0024] In some forms, the methods of the present disclosure will begin with the isolation of

ASFV DNA. Any ASFV sequence can be used for purposes of the present disclosure. Some preferred sequences are selected from the group consisting of nucleotide sequences that encode SEQ ID NOS. 1-101 or a fragment thereof or sequences having at least 80%, more preferably 85%, still more preferably 90%, even more preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity or sequence homology to SEQ ID NOS. 1-101 or a fragment thereof. One or more nucleotide sequences encoding one or more than one of SEQ ID NOS. 1- 101 or a fragment thereof can be isolated and cloned into the vector. In some forms, the ASFV DNA is preferably amplified using PCR methods. The resulting DNA is then cloned into the transfer vector.

[0025] In one aspect of the present disclosure, a method for generating a recombinant viral construct containing ASFV DNA is provided. This method generally comprises the steps of: 1) cloning at least one recombinant ASFV DNA sequence into a transfer vector; and 2) shuttling the portion of the transfer construct containing the recombinant ASFV sequence into a viral vector, to generate the recombinant viral construct. As noted above, some preferred ASFV nucleotide or DNA sequences encode protein sequences having at least 80%, more preferably 85%, still more preferably 90%, even more preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity or sequence homology to SEQ ID NOS. 1-101 or a fragment thereof.

[0026] According to a further aspect, the ASFV DNA can be amplified prior to step 1) in vitro. In vitro methods for amplifying the ASFV DNA and cloning in vitro amplified ASFV DNA into a transfer vector and suitable transfer vectors are described above or known to a person skilled in the art. Thus according to a further aspect, the present disclosure relates to a method for generating a recombinant viral construct containing ASFV DNA and expressing at least one desired ASFV protein comprising the steps of: 1) Synthesis of codon-optimized gene or amplifying ASFV DNA in vitro, 2) cloning the ASFV DNA into a transfer vector; and 3) shuttling a portion thereof containing the recombinant ASFV DNA into a viral vector to generate the recombinant viral construct. In some forms, the ASFV DNA sequences encode protein sequences having at least 80%, more preferably 85%, still more preferably 90%, even more preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity or sequence homology to at least one sequence encoded by any one of SEQ ID NOS. 1-101 or a fragment thereof. In some forms, the recombinant construct is administered for in vivo protein expression and in other forms, the protein(s) is expressed, recovered, and administered to the subject.

[0027] In another aspect of the present disclosure, a method for preparing a composition, preferably an immunogenic composition, such as a vaccine, for invoking an immune response against ASFV is provided. Generally, this method includes the steps of generating a viral expression construct, wherein the construct comprises 1) recombinant DNA from at least one DNA sequence of ASFV that encodes at least one desired antigen, such as those described herein 2) infecting cells in growth media with the recombinant virus, 3) causing the virus to express the recombinant protein from ASFV, 4) recovering the expressed recombinant protein, 5) and, in some forms, preparing the composition by combining the recovered protein with a suitable adjuvant and/or other pharmaceutically acceptable carrier. It is understood that adjuvants are not required to practice the methods nor to combine with the antigens of the disclosure in compositions. In some preferred forms, the composition also includes at least a portion of the viral construct expressing said ASFV protein, and/or a portion of the cell culture supemate.

[0028] In another aspect of the present disclosure, a method for preparing an immunogenic composition, such as a vaccine, for invoking an immune response against ASFV comprises the steps of 1) expressing and recovering ASFV protein, and 2) admixing the recovered protein with a suitable pharmaceutical-acceptable or veterinary-acceptable carrier. 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 one preferred form, the composition provided herewith, contains ASFV protein recovered from in vitro cultured cells, wherein said cells were infected with a recombinant viral construct containing ASFV DNA and expressing ASFV protein, and wherein the cell culture was treated to inactivate the viral construct, and an equivalent concentration of a neutralization agent was added, and wherein both an adjuvant and physiological saline are also added. As with the other aspects, the ASFV protein preferably has at least 80%, more preferably 85%, still more preferably 90%, even more preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity or sequence homology to SEQ ID NOS. 1-101 or a fragment thereof. When included, the amount of physiological saline is preferably about 50 to about 90% (v/v), more preferably about 60 to 80% (v/v), still more preferably about 70% (v/v). Optionally, this method can also include the addition of a protectant. A protectant as used herein, refers to an anti-microbiological active agent, such as for example Gentamycin, Merthiolate, and the like. In particular adding a protectant is most preferred for the preparation of a multi-dose composition. Those anti-microbiological active agents are added in concentrations effective to prevent the composition of interest from any microbiological contamination or for inhibition of any microbiological growth within the composition of interest.

[0029] The methods of the present disclosure can also comprise the addition of any stabilizing agent, such as for example saccharides, trehalose, mannitol, saccharose and the like, to increase and/or maintain product shelf-life and/or to enhance stability.

[0030] In another aspect of the present disclosure, products resulting from the methods as described above are provided. In particular, the present disclosure relates to a composition of matter comprising a construct having therein at least one ASFV nucleic acid sequence. In some forms, the ASFV nucleic acid sequence(s) encodes a protein having at least 80%, more preferably 85%, still more preferably 90%, even more preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity or sequence homology to SEQ ID NOS. 1-101 or a fragment thereof. In some forms, the vector is a live vector. In some forms, the vector is a replication competent vector, such as a recombinant adenovirus. In still other forms, the multicistronic cassette utilizes an attenuated bovine parainfluenza virus type 3 genotype c (BPIV3c) as the vector. In some forms, the vector including the ASFV nucleic acid sequence(s) is administered to a subject as described herein. In other forms, the vector expresses the nucleic acid sequences in vitro and the resulting recombinant ASFV polypeptides or proteins are recovered and administered to a subject as described in this disclosure. Of course, it is understood that the ASFV polypeptide used in an immunogenic composition in accordance with the present disclosure can be derived in any fashion including isolation and purification, standard protein synthesis, and recombinant methodology.

[0031] In a further aspect of the present disclosure, an immunogenic composition effective for lessening the incidence and/or severity of clinical and/or postmortem symptoms or signs associated with ASFV infection comprising at least one ASFV nucleic acid is provided. Preferably, the ASFV nucleic acid encodes 1) any polypeptide that is at least 80%, more preferably 85%, still more preferably 90%, even more preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homologous to the polypeptide of any one of SEQ ID NOS: 1-101, or any combination thereof; 2) any immunogenic portion of the polypeptides of 1); and, 3) a polypeptide equivalent to (due to the degeneracy of the genetic code) one encoded by a DNA comprising the sequence of SEQ ID NOS: 1-101. As noted above, it is particularly preferred to have multiple sequences encoding any one of SEQ ID NOS: 1-101 included in the immunogenic composition and even more preferred to have them combined together in a multi ci str onic expression cassette. Additionally, it is preferred to include an El sequence with the ASFV nucleic acid sequence(s).

[0032] According to a further aspect, at least one ASFV protein is provided in the immunological composition at an antigen inclusion level effective for inducing the desired immune response, namely reducing the incidence of or lessening the severity of clinical signs resulting from ASFV infection. Preferably, the ASFV protein inclusion level is at least 25 pg antigen/ml of the final immunogenic composition (pg/ml), more preferably from about 25 to about 400 pg/ml.

[0033] The at least one nucleotide sequence, polypeptide, or polypeptide encoded by the multicistronic expression cassette is incorporated into a composition that can be administered to an animal susceptible to ASFV infection. In preferred forms, the composition may also include additional components known to those of skill in the art (see also Remington’s Pharmaceutical Sciences. (1990). 18th ed. Mack Publ., Easton).

[0034] Those of skill in the art will understand that the composition herein may incorporate known injectable, physiologically acceptable, sterile solutions. For preparing a ready-to-use solution for parenteral injection or infusion, aqueous isotonic solutions, such as e g. saline or corresponding plasma protein solutions are readily available. In addition, the immunogenic and vaccine compositions of the present disclosure can include diluents, isotonic agents, stabilizers, or adjuvants. Diluents can include water, saline, dextrose, ethanol, glycerol, and the like. Isotonic agents can include sodium chloride, dextrose, mannitol, sorbitol, and lactose, among others. Stabilizers include albumin and alkali salts of ethylenediaminetetraacetic acid, among others. Suitable adjuvants are those described above. Oral forms of the composition are also envisioned and in some forms, preferred.

[0035] 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. 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.

[0036] It will be found that the immunogenic compositions comprising live-vectored multivalent immunogenic compositions as provided herewith are very effective in reducing the severity of or incidence of clinical or postmortem signs associated with ASFV infections up to and including the prevention of such signs.

[0037] Another aspect of the present disclosure relates to a kit. Generally the kit includes a container comprising at least one dose of the live-vectored multivalent immunogenic composition of ASFV nucleotide sequences as provided in this disclosure.

[0038] In another aspect of the present disclosure, a kit is provided. Generally, the kit includes a container comprising at least one does of at least one ASFV recombinant protein as described herein. In some forms, one dose comprises at least 2 pg ASFV protein. Said container can comprise from 1 to 250 doses of the immunogenic composition. In some preferred forms, the container contains 1, 10, 25, 50, 100, 150, 200, or 250 doses of the immunogenic composition of ASFV protein. Preferably, each of the containers comprising more than one dose of the immunogenic composition of ASFV protein further comprises an anti-microbiological active agent. Those agents are for example, antibiotics including Gentamicin and the like. Thus, one aspect of the present disclosure relates to a container that comprises from 1 to 250 doses of the immunogenic composition of ASFV protein, wherein one dose comprises at least 2 pg ASFV protein, and Gentamicin, preferably from about 1 pg/ml to about 60 pg/ml of antibiotics, and more preferably less than about 30 pg/ml. In preferred forms, the kit also includes an instruction manual, including the information for the administration of at least one dose of the immunogenic composition of ASFV protein into animals, preferably pigs and piglets to lessen the incidence and/or severity of clinical symptoms associated with ASFV infection. Moreover, according to a further aspect, said instruction manual comprises the information of a second or further administration(s) of at least one dose of the immunogenic composition of ASFV, wherein the second administration or any further administration is at least 7, 8, 9, 10, 11, 12, 13, 14 days, or more beyond the initial or any former administration. Such timeframes are also preferred for any administration of the immunogenic compositions of this disclosure. In some preferred forms, said instruction manual also includes the information, to administer an immune stimulant. Preferably, said immune stimulant shall be given at least twice. Preferably, at least 14, more preferably at least 21, and even more preferably at least 28 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 of ASFV protein. 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. The kit may also comprise a second container, including at least one dose of the immune stimulant.

[0039] A further aspect of the present disclosure relates to the kits as described above, comprising the immunogenic composition of ASFV as provided herewith and the instruction manual, wherein the instruction manual further includes the information to administer the ASFV immunogenic composition together, or around the same time as, with an immunogenic composition that comprises an additional antigen effective for reducing the severity of or incidence of clinical signs related to another porcine pathogen. Preferably, the manual contains the information of when the ASFV-containing composition and the immunogenic composition that comprises an additional antigen are administered.

[0040] A further aspect, relates to the use of any of the compositions provided herewith as a medicament, preferably as a veterinary medicament, even more preferably as a vaccine. Moreover, the present disclosure also relates to the use of any of the compositions described herein, for the preparation of a medicament for lessening the incidence amd/or severity of clinical and/or postmortem symptoms or signs associated with ASFV infection. Preferably, the medicament is for the prevention of ASFV infection in swine, even more preferably in piglets.

[0041] A further aspect relates to a method for (1) the prevention of an infection, or reinfection with ASFV or (2) the reduction in incidence or severity of or elimination of clinical and/or postmortem signs or symptoms caused by ASFV in a subject, comprising administering any of the immunogenic compositions provided herewith to a subject in need thereof. In some forms, the subject is a mammal, such as a pig. It is understood that the reduction is in comparison to a subject that has not received an administration of a composition of the present disclosure. Preferably, one dose or two doses of the immunogenic composition is/are administered. A further aspect relates to the method of treatment as described above, wherein a second application or administration of the immunogenic composition is administered. Preferably, the second administration is done with the same immunogenic composition, preferably having the same amount of ASFV antigen (protein or live-vectored nucleic acid). Preferably, the second administration is done at least 7, 8, 9, 10, 11, 12, 13, 14, or more days beyond the initial administration, even more preferably at least 3-4 weeks beyond the initial administration. In preferred forms, the method is effective after just a single dose of the immunogenic composition and does not require a second or subsequent administration in order to confer the protective benefits upon the subject.

[0042] According to a further aspect, the present disclosure provides a multivalent combination vaccine which includes an immunological agent effective for reducing the incidence of or lessening the incidence and/or severity of ASFV infection, and at least one immunological active component against another disease-causing organism in swine.

[0043] In particular the immunological agent effective for reducing the incidence of or lessening the severity of ASFV infection is an ASFV antigen or protein as described herein. Preferably, said ASFV antigen encodes a protein having, or is a protein that has at least 80%, more preferably 85%, still more preferably 90%, even more preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology with any one of SEQ ID NOS: 1-101 and any combination thereof as provided herewith.

[0044] Preferably the other disease-causing organism in swine 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; Law sonia intracellularis; Leptospira spp. preferably Leptospira australis; Leptospira canicola; Leptospira grippotyphosa; Leptospira icterohaemorrhagicae; and Leptospira interrogans; Leptospira pomona; Leptospira tarassovi; Mycobacterium spp. preferably M. avium; M. intracellulare; and M.bovis; Mycoplasma hyopneumoniae (M hyd); Pasteurella multocida; Porcine circovirus; Porcine cytomegalovirus; Porcine Parvovirus; Porcine Reproductive and Respiratory Syndrome (PRRS) Virus; Pseudorabies virus; Rotavirus; Salmonella spp .; preferably S. thyhimurium; 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.

[0045] Nucleotide, polynucleotide or nucleic acid sequence will be understood according to the present disclosure as meaning both a double- stranded or single-stranded DNA in the monomeric and dimeric (so-called in tandem) forms and the transcription products of said DNAs.

[0046] It must be understood that the present disclosure does not relate to the genomic nucleotide sequences taken in their natural environment, that is to say in the natural state. It concerns sequences which it has been possible to isolate, purify or partially purify, starting from separation methods such as, for example, ion-exchange chromatography, by exclusion based on molecular size, or by affinity, or alternatively fractionation techniques based on solubility in different solvents, or starting from methods of genetic engineering such as amplification, cloning and subcloning, it being possible for the sequences of the disclosure to be carried by vectors.

[0047] Among said nucleotide sequences according to the disclosure, those coding for polypeptides, such as, for example, the sequences SEQ ID NOS. 1-101 or any fragment thereof and any combination thereof are contemplated. The nucleotide sequence fragments according to the disclosure can be obtained, for example, by specific amplification, such as PCR, or after digestion with appropriate restriction enzymes of nucleotide sequences according to the disclosure, these methods in particular being described in the work of Sambrook et al., 1989. Said representative fragments can likewise be obtained by chemical synthesis when their size is not very large and according to methods well known to persons skilled in the art.

[0048] Modified nucleotide sequence will be understood as meaning any nucleotide sequence obtained by mutagenesis according to techniques well known to the person skilled in the art, and containing modifications with respect to the normal sequences according to the disclosure, for example mutations in the regulatory and/or promoter sequences of polypeptide expression, especially leading to a modification of the rate of expression of said polypeptide or to a modulation of the replicative cycle.

[0049] In the present description, the terms polypeptide, peptide and protein are interchangeable.

[0050] It must be understood that the disclosure does not relate to the polypeptides in natural form, that is to say that they are not taken in their natural environment but that they can be isolated or obtained by purification from natural sources, or else obtained by genetic recombination, or alternatively by chemical synthesis and that they can thus contain unnatural amino acids.

[0051] Polypeptide fragment according to the disclosure is understood as designating a polypeptide containing at least 5 consecutive amino acids, preferably at least 10 consecutive amino acids or at least 15 consecutive amino acids.

[0052] The nucleotide sequences coding for a polypeptide according to the disclosure are likewise part of the disclosure.

[0053] The disclosure likewise relates to nucleotide sequences utilizable as a primer or probe, characterized in that said sequences are selected from the nucleotide sequences according to the disclosure.

[0054] Another subject of the present disclosure is a vector for the cloning and/or expression of a sequence, characterized in that it contains a nucleotide sequence according to the disclosure.

[0055] The vectors according to the disclosure, characterized in that they contain the elements allowing the expression and/or the secretion of said nucleotide sequences in a determined host cell, are likewise part of the disclosure. The vector must then contain a promoter, signals of initiation and termination of translation, as well as appropriate regions of regulation of transcription. It must be able to be maintained stably in the host cell and can optionally have particular signals specifying the secretion of the translated protein. These different elements are chosen as a function of the host cell used. To this end, the nucleotide sequences according to the disclosure can be inserted into autonomous replication vectors within the chosen host, or integrated vectors of the chosen host. Such vectors will be prepared according to the methods currently used by the person skilled in the art, and it will be possible to introduce the clones resulting therefrom into an appropriate host by standard methods, such as, for example, oral administration, injection, lipofection, transdermal administration, electroporation and thermal shock. The vectors according to the disclosure are, for example, vectors of plasmid or viral origin. Preferred vectors for the expression of polypeptides of the disclosure include adenovirus. In some forms, the adenovirus is recombinant. In some forms, the adenovirus is human adenovirus. In some forms, the adenovirus is human type 5 (RcAd5), or an attenuated bovine parainfluenza virus type 3 genotype 3C (BPIV3C).

[0056] Advantageously, the antigenic determinant is such that it is capable of inducing a humoral and/or cellular response. It will be possible for such a determinant to comprise a polypeptide according to the disclosure in glycosylated form used with a view to obtaining immunogenic compositions capable of inducing the synthesis of antibodies directed against multiple epitopes. Said polypeptides or their glycosylated fragments are likewise part of the disclosure. These hybrid molecules can be formed, in part, of a polypeptide carrier molecule or of fragments thereof according to the disclosure, associated with a possibly immunogenic part, in particular an epitope of the diphtheria toxin, the tetanus toxin, a surface antigen of the hepatitis B virus (patent FR 7921811), the VP 1 antigen ofthe poliomyelitis virus or any other viral or bacterial toxin or antigen. The procedures for synthesis of hybrid molecules encompass the methods used in genetic engineering for constructing hybrid nucleotide sequences coding for the polypeptide sequences sought. It will be possible, for example, to refer advantageously to the technique for obtainment of genes coding for fusion proteins described by Minton in 1984. Said hybrid nucleotide sequences coding for a hybrid polypeptide as well as the hybrid polypeptides according to the disclosure characterized in that they are recombinant polypeptides obtained by the expression of said hybrid nucleotide sequences are likewise part of the disclosure.

[0057] The disclosure likewise comprises the vectors characterized in that they contain one of said hybrid nucleotide sequences. The host cells transformed by said vectors, the transgenic animals comprising one of said transformed cells as well as the procedures for preparation of recombinant polypeptides using said vectors, said transformed cells and/or said transgenic animals are, of course, likewise part of the disclosure.

[0058] The disclosure likewise relates to a pharmaceutical composition comprising a compound selected from the following compounds: a) a nucleotide sequence according to the disclosure; b) a polypeptide according to the disclosure; c) a vector, a viral particle or a cell transformed according to the disclosure; d) an antibody according to the disclosure; and e) a compound capable of being selected by a selection method according to the disclosure; possibly in combination with a pharmaceutically acceptable carrier and, if need be, with one or more adjuvants of the appropriate immunity.

[0059] The disclosure also relates to an immunogenic and/or vaccine composition, characterized in that it comprises a compound selected from the following compounds: a) a nucleotide sequence according to the disclosure; b) a polypeptide according to the disclosure; c) a vector or a viral particle according to the disclosure; and d) a cell according to the disclosure.

[0060] In one embodiment, the vaccine composition according to the disclosure is characterized in that it comprises a mixture of at least two of said compounds a), b), c) and d) above and in that one of the two said compounds is related to the ASFV.

[0061] In another embodiment of the disclosure, the vaccine composition is characterized in that it comprises at least one compound a), b), c), or d) above which is related to ASFV.

[0062] A compound related to ASFV is understood herein as respectively designating a compound obtained from the genomic sequence of the ASFV. Preferably the compound will be selected from the group consisting of at least one nucleic acid sequence encoding for a sequence having at least 80%, more preferably 85%, still more preferably 90%, even more preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology with any one or more of SEQ ID NOS : 1 - 101 or a fragment thereof, and any combination thereof, a polypeptide sequence having at least 80%, more preferably 85%, still more preferably 90%, even more preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology with any one or more of SEQ ID NOS: 1-101 or a fragment thereof, and any combination thereof, and any combination of at least one nucleic acid sequence and at least one polypeptide sequence, as described herein.

[0063] The disclosure is additionally aimed at an immunogenic and/or vaccine composition, characterized in that it comprises at least one of the following compounds: 1) a nucleotide sequence encoding any one of SEQ ID NOS. 1-101 or one of their fragments or homologues; 2) a polypeptide selected from the group consisting of SEQ ID NOS. 1-101; 3) a vector or a viral particle comprising a nucleotide sequence of 1); 4) a transformed cell capable of expressing a polypeptide of 2); or 5) a mixture of at least two of said compounds. [0064] The disclosure also comprises an immunogenic and/or vaccine composition according to the disclosure, characterized in that it comprises said mixture of at least two of said compounds as a combination product for simultaneous, separate or protracted use for the prevention or the treatment of infection by ASFV including a reduction in the incidence and/or severity of clinical and/or postmortem signs or symptoms of ASFV infection.

[0065] The disclosure is likewise directed at a pharmaceutical composition according to the disclosure, for the prevention or the treatment of an infection by ASFV including a reduction in the incidence and/or severity of clinical and/or postmortem signs or symptoms of ASFV infection.

[0066] It is understood that “prevention” as used in the present disclosure, includes the complete prevention of infection by ASFV, but also encompasses a reduction in the severity of or incidence of clinical and/or postmortem signs associated with or caused by ASFV infection. Such prevention is also referred to herein as a protective effect.

[0067] The disclosure likewise concerns the use of a composition according to the disclosure, for the preparation of a medicament intended for the prevention or the treatment of infection by ASFV.

[0068] The live-vectored multivalent immunogenic compositions as described herein and the polypeptides of the disclosure entering into the immunogenic or vaccine compositions according to the disclosure can be selected by techniques known to the person skilled in the art such as, for example, depending on the capacity of said polypeptides to stimulate the T cells, which is translated, for example, by their proliferation or the secretion of interleukins, and which leads to the production of antibodies directed against said polypeptides.

[0069] In pigs, as in mice, in which a weight dose of the vaccine composition comparable to the dose used in man is administered, the antibody reaction is tested by taking of the serum followed by a study of the formation of a complex between the antibodies present in the serum and the antigen of the vaccine composition, according to the usual techniques.

[0070] The pharmaceutical compositions according to the disclosure will contain an effective quantity of the compounds of the disclosure, that is to say in sufficient quantity of said compound(s) allowing the desired effect to be obtained, such as, for example, the modulation of the cellular replication of ASFV. The person skilled in the art will know how to determine this quantity, as a function, for example, of the age and of the weight of the individual to be treated, of the state of advancement of the pathology, of the possible secondary effects and by means of a test of evaluation of the effects obtained on a population range, these tests being known in these fields of application.

[0071] According to the disclosure, said vaccine combinations will preferably be combined with a pharmaceutically or veterinary acceptable carrier and, if need be, with one or more adjuvants of the appropriate immunity.

[0072] According to another embodiment of the vaccine composition according to the disclosure, the nucleotide sequence, preferably a DNA, is complexed with DEAE-dextran (Pagano et al., 1967) or with nuclear proteins (Kaneda et al., 1989), with lipids (Feigner et al., 1987) or encapsulated in liposomes (Fraley et al., 1980) or else introduced in the form of a gel facilitating its transfection into the cells (Midoux et al., 1993, Pastore et al., 1994). The polynucleotide or the vector according to the disclosure can also be in suspension in a buffer solution or be combined with liposomes.

[0073] These compounds can be administered by the systemic route, in particular by the intravenous route, by the intramuscular, intradermal or subcutaneous route, or by the oral route. In a more preferred manner, the vaccine composition comprising polypeptides according to the disclosure will be administered by the intramuscular route, through the food or by nebulization only once, or several times, staggered over time.

[0074] Their administration modes, dosages and optimum pharmaceutical forms can be determined according to the criteria generally taken into account in the establishment of a treatment adapted to an animal such as, for example, the age or the weight, the seriousness of its general condition, the tolerance to the treatment and the secondary effects noted. Preferably, the vaccine of the present disclosure is administered in an amount that is protective or provides a protective effect against ASFV infection. In some forms, the composition of the disclosure is administered once or multiple times.

[0075] An immunologically effective amount of the vaccines or immunogenic compositions of the present disclosure is administered to a pig in need of protection against clinical signs of ASFV infection. The immunologically effective amount or the immunogenic amount that inoculates the pig can be easily determined or readily titrated by routine testing. An effective amount is one in which a sufficient immunological response to the vaccine is attained to protect the pig exposed to the virus which causes ASFV signs. Preferably, the pig is protected to an extent in which one to all of the adverse physiological symptoms or effects of the viral disease are significantly reduced, ameliorated or totally prevented.

[0076] The vaccine can be administered in a single dose or in repeated doses with single doses being preferred. Single dose vaccines provide protection after a single dose without the need for any booster or subsequent dosages. Protection can include the complete prevention of clinical signs of infection, or a lessening of the severity, duration, or likelihood of the manifestation of one or more clinical signs of infection.

[0077] Desirably, the vaccine is administered to a pig not yet exposed to the ASFV virus.

[0078] When administered as a liquid, the present vaccine may be prepared in the form of an aqueous solution, syrup, an elixir, a tincture and the like. Such formulations are known in the art and are typically prepared by dissolution of the antigen and other typical additives in the appropriate carrier or solvent systems. Suitable carriers or solvents include, but are not limited to, water, saline, ethanol, ethylene glycol, glycerol, etc. Typical additives are, for example, certified dyes, flavors, sweeteners and antimicrobial preservatives such as thimerosal (sodium ethylmercurithiosalicylate). Such solutions may be stabilized, for example, by addition of partially hydrolyzed gelatin, sorbitol or cell culture medium, and may be buffered by conventional methods using reagents known in the art, such as sodium hydrogen phosphate, sodium dihydrogen phosphate, potassium hydrogen phosphate, potassium dihydrogen phosphate, a mixture thereof, and the like.

[0079] Liquid formulations also may include suspensions and emulsions that contain suspending or emulsifying agents in combination with other standard co-formulants. These types of liquid formulations may be prepared by conventional methods. Suspensions, for example, may be prepared using a colloid mill. Emulsions, for example, may be prepared using a homogenizer.

[0080] Parenteral formulations, designed for injection into body fluid systems, require proper isotonicity and pH buffering to the corresponding levels of porcine body fluids. Isotonicity can be appropriately adjusted with sodium chloride and other salts as needed. Suitable solvents, such as ethanol or propylene glycol, can be used to increase the solubility of the ingredients in the formulation and the stability of the liquid preparation. Further additives that can be employed in the present vaccine include, but are not limited to, dextrose, conventional antioxidants and conventional chelating agents such as ethylenediamine tetraacetic acid (EDTA). Parenteral dosage forms must also be sterilized prior to use.

[0081] Another aspect of the present disclosure is the preparation of the combination vaccine(s) or immunogenic compositions. Such combinations can be between the different vaccine components described herein. For example, a vaccine of the present disclosure can include both protein portions and DNA portions of ASFV, as described herein, which are administered concurrently or separately. Additionally, the combinations can be between the ASFV vaccine components described herein and antigens of other disease-causing organisms, such as those described above.

[0082] According to the present disclosure, an effective amount of a combination vaccine administered to pigs provides effective immunity or a protective effect against microbiological infections caused by ASFV and at least one further pathogen. Preferred combinations of antigens for the treatment and prophylaxis of microbiological diseases in pigs are listed above.

[0083] According to a further embodiment, the combination vaccine is administered to pigs in one or two doses at an interval of about 2 to 4 weeks. For example, the first administration is performed when the animal is about 2 to 3 weeks to about 8 weeks of age. The second administration is performed about 1 to about 4 weeks after the first administration of the first vaccination. According to a further embodiment, revaccination is performed in an interval of 3 to 12 months after administration of the second dose. Administration of subsequent vaccine doses is preferably done on a 6 month to an annual basis. In another preferred embodiment, animals vaccinated before the age of about 2 to 3 weeks should be revaccinated. Administration of subsequent vaccine doses is preferably done on an annual basis. In the event that one of the components of the combination vaccine is effective after just a single dose, such component needs to only be administered a single time with the other component(s) administered according to their preferred regimen.

[0084] The following examples demonstrate certain aspects of the present disclosure. However, it is to be understood that these examples are for illustration only and do not purport to be wholly definitive as to conditions and scope of this disclosure. It should be appreciated that when typical reaction conditions (e g., temperature, reaction times, etc.) have been given, the conditions both above and below the specified ranges can also be used, though generally less conveniently. The examples are conducted at room temperature (about 23°C to about 28°C) and at atmospheric pressure. All parts and percentages referred to herein are on a weight basis and all temperatures are expressed in degrees centigrade unless otherwise specified. Further unless noted otherwise, all components of the disclosure are understood to be disclosed to cover “comprising”, “consisting essentially of’, and “consisting of’ claim language as those terms are commonly used in patent claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0085] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0086] 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.

[0087] FIGURE 1 A is a schematic representation of the organization of the multi ci str onic expression cassettes for the ASFV antigens fused in-frame to El depicting up to eight codon- optimized synthetic genes separated by the 2A autocleavable motif in a single cassette.

[0088] FIGURE IB is a set of photographs showing protein expression by recombinant virus constructs confirmed by immunocytometric analyses of infected HEK 293A cells using ASFV convalescent serum or GFP expression. Representative data is shown for four recombinant virus constructs, Ad-GFP, and a negative control.

[0089] FIGURE 1C is a set of photographs showing the evaluation of virus replication by immunofluorescence assay (IF A) using primary swine alveolar epithelial cells infected for forty hours at 0.1MOI with either Replication-Defective adenovirus-5 expressing p72-pl5-B602L or p62-p32-p54-EP153R-pl0 (RD-Ad5-3 and RD-Ad5-4, respectively), or infected with either RC- Ad5 expressing p72-pl5-B602L or p62-p32-p54-EP153R-pl0 (RC-Ad5-3 and RC-Ad5-4, respectively], and then probed with the ASFV convalescent serum. RD-Ad5-Luc and RC-Ad5- GFP served as negative controls. The presence of fluorescing species (light gray) in RD-Ad5-3, and-4 sera, and the concomitant absence of fluorescing species in the negative controls (dark gray), confirms evidence of viral replication.

[0090] FIGURE 2 is a diagrammatic representation of an exemplary experimental design and timeline in which age-matched commercial piglets are divided into four treatment groups: 1) RC-Ad5-ASFV plus adjuvant; 2) RC-Ad5-ASFV (without structural protein genes) plus adjuvant; 3) RC-Ad5-ASFV without adjuvant; and 4) RC-Ad5-GFP plus adjuvant (Negative control). In depictions of each group, piglets that were inoculated are depicted with a lighter shade of gray, while piglets depicted in a darker shade of gray denote non-immunized ‘contact spreaders.’ Following a one-week acclimation period, Day 0 sampling was done, and the treatment piglets received IM inoculation of the priming dose followed by boosts on days 21 and 35 post-priming. One week after the second boost, contact-challenge piglets received an IM injection of ASFV (Georgia 2007/1) which spread the virus to the co-mingled vaccinees. The pigs were monitored every day for clinical symptoms, with biweekly samples of blood, serum, and nasal swabs collected until termination due to sickness. Pigs that survived were euthanized on day 39 postchallenge when the study was terminated.

[0091] FIGURE 3 is a set of graphs tracking changes in bodyweight and temperature for each treatment group over the course of an exemplary immunization phase. Weekly measurements of body weight (A) and temperatures (B) during the immunization phase revealed no abnormalities. All pigs, including vaccinated, negative control, and non-immunized contact spreaders, gained body weight and maintained normal body temperatures throughout the immunization phase. A key is provided to show which line graph label corresponds to each pig ID number.

[0092] FIGURE 4 is a set of graphs illustrating seroconversion of immunized pigs to p62 antigen as determined by ELISA. Purified recombinant p62 (100 ng) expressed in Expi293F was coated on an ELISA plate. Sera from blood collected on days 0, 7, 14, 21, 28 and 35 post-priming [diluted 1 : 100] were tested for binding as judged by TMB substrate reactivity following incubation with goat anti-porcine IgG-HRP [1 : 10,000] secondary antibody. Asterisks (*) at day 21 denotes the first booster and (**) at day 35 denotes the second booster administered. Group 2 was negative for p62 since this antigen was not included in the antigen formulation.

[0093] FIGURE 5 is a set of photographs showing the evaluation, via IFA, of whether antibodies elicited by recombinant viral construct cocktail formulations recognized virus-infected macrophages. Antibodies elicited by the RC-Ad-ASFV cocktail formulations were tested for recognition of ASFV-infected primary swine macrophages by indirect Immuno-Fluorescence Antibody assay (IFA) using sera collected one week after the second boost. Representative data is shown for sera from two pigs from each treatment group (Groups 1-3) and from the RC-Ad5-GFP control (Group 4). Normal swine serum served as the negative control, whereas ASFV convalescent serum was the positive control. Sera from all the vaccinees, but not from the negative controls, recognized the ASFV-infected swine macrophages (Table 4). Darker shades of gray seen in the “Normal serum,” “Pig 42 (G4)” and “Pig 47 (G4)” images are characteristic of negative controls, where no ASFV-infected primary swine macrophages were found. Lighter shades of gray correspond to detection of ASFV-infected primary swine macrophages..

[0094] FIGURE 6A is graph illustrating pig survival post-challenge of vaccinated (Gl, G2, G3); sham treatment control (G4); and IM challenged contact spreader pigs (in red). At necropsy, internal organs and tissues from the survivors, but not the other pigs, had no lesions caused by ASFV. High viremia was detected in the samples collected from all the pigs from groups 1, 2, and 4. RD-Ad primed antibody responses against ASFV antigens p32, p54, pp62, and p72.

[0095] FIGURE 6B is a heat map displaying the daily clinical scores of each pig postchallenge in an exemplary immunization experiment. Contact challenge pigs are labeled with “CC,” and the day on which pigs were terminated is marked with a “T ” Pigs in group 3 terminated on day 39 only showed mild clinical symptoms.

[0096] FIGURE 6C is a heat map displaying viral load detected in blood collected from pigs in each test group in an exemplary immunization experiment. Viral load is presented as the logarithm (base 10) of the mean copy number per milliliter (CN/mL). Contact challenge pigs are labeled with “CC,” and the day on which pigs were terminated is marked with a “T ” Five of the six pigs in group 3 remained negative until day 39, when the study was terminated.

[0097] FIGURE 6D shows a series of bar graphs of data collected of viremia in tissues and fecal materials following necropsy of pigs from each test group in an exemplary immunization experiment. Viral load was enumerated via quantitative reverse transcription polymerase chain reaction (qRT-PCR) and is presented as the logarithm (base 10) of the mean copy number per milliliter (CN/mL). Each graph displays the viral load for individual tissues collected from each pig, with the specific tissues in question specified as labels below each graph.

[0098] FIGURE 6E shows IF A photographs of primary swine macrophages incubated with spleen homogenates and then probed with ASFV convalescent serum. Uninfected (No virus) and infected (Plus virus) macrophages served as negative and positive controls, respectively. Pigs with ID numbers 25, 27, 35, 37, 44 and 48 are from Group 3 of the exemplary immunization experiment described in FIG. 2, while pig 32 is from Group 2 and pig 39 is from Group 1. There was no rescuable ASF virus in spleen tissues from the 5/6 survivors (pig numbers 25, 27, 35, 44, and 48), except in the sample collected from pig 37 that succumbed, as judged by IFA of primary swine macrophages incubated with spleen homogenates and then probed with ASFV convalescent serum. Uninfected (No virus) and infected (Plus virus) macrophages served as negative and positive controls, respectively. The negative control IFA image shows no infected macrophages, while the positive control shows infected macrophages. The IFA images of samples corresponding to pigs 25, 27, 35, 44, and 48 do not show fluorescence corresponding to rescuable virus. The IFA images of samples corresponding to pigs 37, 32, and 39 do show fluorescence corresponding to rescuable virus. Samples from pig number 39 (group 1) and number 32 (group 2) that succumbed had rescuable virus. The outcome was validated by F) qRT-PCR (p72 DNA) enumeration of viral loads in the infected macrophages. Similar results were obtained when pericardium fluids were screened (G and H). In the IFA images of pericardium fluids in Figure 6G, the images of samples corresponding to pigs 25, 27, 35, 44, and 48 do not show fluorescence corresponding to rescuable virus, while the IFA images of samples corresponding to pigs 37, 32, and 39 do show fluorescence corresponding to rescuable virus.

[0099] FIGURE 6F is a bar graph showing average viral load in CN/mL in the infected macrophages as determined via qRT-PCR. “NV” and “PV” represent “no virus” (negative control) and “plus virus” (positive control) respectively. The same pigs whose data are depicted here are depicted in FIG. 6E.

[0100] FIGURE 6G shows IFA photographs of primary swine macrophages incubated with pericardium homogenates and then probed with ASFV convalescent serum. Uninfected (No virus) and infected (Plus virus) macrophages served as negative and positive controls, respectively. Pigs with ID numbers 25, 27, 35, 37, 44 and 48 are from Group 3 of the exemplary immunization experiment described in FIG. 2, while pig 32 is from Group 2 and pig 39 is from Group 1.

[0101] FIGURE 6H is a bar graph showing average viral load in CN/mL in the infected macrophages as determined via qRT-PCR. “NV” and “PV” represent “no virus” (negative control) and “plus virus” (positive control) respectively. The same pigs whose data are depicted here are depicted in FIG. 6G.

[0102] FIGURE 7A is a series of line graphs that display body weight data of pigs from all four test groups in the exemplary immunization experiment outlined in FIG. 2. Data was collected at a twice-weekly interval following the ASFV challenge until the end of the study. Remarkably, surviving pigs in Group 3 consistently demonstrated an increase in body weight, whereas pigs in the other groups exhibited a decrease in body weight. The body temperature post-challenge indicated that survivor pigs in Group 3 consistently maintained a normal range body temperature, while pigs in the other groups exhibited an increase in their body temperature before reaching the termination point. A key is provided to show which line graph label corresponds to each pig ID number.

[0103] FIGURE 7B is a series of line graphs that display temperature data of piges from all four test groups in the exemplary immunization experiment outline in FIG. 2. Data was collected at a twice-weekly interval following the ASFV challenge until the end of the study. Numbers in each graph’s legend correspond to the ID of the pig in question.

[0104] FIGURE 8 A shows IF A photographs of sera from protected and non-protected pigs, indicating that even at 1 : 1 dilution, all sera failed to neutralize ASFV or block infection of the primary swine macrophages. “No virus” and “plus virus” images are presented as negative and positive controls, respectively. The IFA images show fluorescence signals corresponding to infected primary swine macrophages in the ‘plus virus’ image and in the images corresponding to pigs 20, 36, 29, 25, 27, 35, 37, 44, 48, and 47, leaving the ‘negative control’ as the only image that did not show fluorescence data corresponding to primary swine macrophage infection. Data for all the pigs from group 3 are shown as well as representatives from the other groups; ASFV p72 DNA was detected by RT-qPCR analyses of samples from virus neutralization assays conducted using heat inactivated sera (B) or not inactivated sera (C) from the vaccinees and controls. Data from analyses of the sera from all the pigs is summarized in Table 5.

[0105] FIGURE 8B is a bar graph of the logarithmic mean viral load in CN/mL for the NV (negative control), PV (positive control), and four test groups in heat-inactivated serum as measured by RT-qPCR.

[0106] FIGURE 8C is a bar graph of the logarithmic mean viral load in CN/mL for the NV (negative control), PV (positive control), and four test groups in serum without heat inactivation as measured by RT-qPCR.

[0107] FIGURE 9A is a bar graph showing the measured percent positive of ASFV-specific Granzyme B+ CD8a+ T cells detected in peripheral blood mononuclear cells (PBMCs) from survivors on days 26 and 33 from the exemplary immunization experiment outlined in FIG. 2.

[0108] FIGURE 9B is a bar graph showing the measured percent positive of ASFV-specific Granzyme B+ CD8α+ T cells detected in splenocytes at the point of study termination.

[0109]

DETAILED DESCRIPTION

[0110] 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.

[0111] ‘A”, “an”, and “the” include the singular and plural forms thereof unless the context clearly indicates otherwise.

[0112] “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.

[0113] The terms “pig” and “piglet” are used interchangeably herein.

[0114] 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 γδ 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.

[0115] 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.

[0116] “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 di oleate; 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).

[0117] 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.

[0118] 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 polyhydroxylated 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. [0119] 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.

[0120] 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 8 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.

[0121] “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 NTH 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.

[0122] “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.

[0123] 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.

[0124] 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.

[0125] ‘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.

[0126] 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.

[0127] 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.

[0128] 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.

[0129] 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.

[0130] 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. EXAMPLES

[0131] The following examples illustrate embodiments of the disclosure. Nothing in these examples should be limiting to the disclosure as these are representative in nature.

EXAMPLE 1

[0132] Briefly, the ASFV Georgia 2007/1 proteins were used to generate forty-four cassettes (-1500 amino acid polypeptides or less) for optimal expression in adenovirus 5. Due to its large size, the pp220 polyprotein was split and used to generate two expression cassettes. The other two large polypeptides, NP1450L and G1340L, were used to generate one cassette for each. All the other polypeptides were combined to generate multi ci stronic expression cassettes. The polypeptides were used to generate codon-optimized synthetic genes that were modified to add HA- and FLAG-tags at the 5’ and 3’ ends, respectively, for tracking protein expression. The genes were cloned into pDONR221 shuttle vector (ThermoFisher Scientific) that was modified to add, in-frame, the gene encoding the adenovirus El (Ela and Elb) separated by a 2A autocleavable motif to allow generation of individual proteins [Figure 1A], The El protein is required for adenovirus replication in primary cells or in cells that lack El complementation. The recombinant pDONR.221 constructs were used to shuttle the expression cassettes into the adenovirus 5 plasmid backbone vector pAD/CMV/V5-DEST by LR recombination [ThermoFisher Scientific], Positive clones of the recombinant plasmid constructs were used to transfect HEK293A mammalian cells to assemble recombinant RcAd5 virus constructs and protein expression was validated by immunocytometric analyses using ASFV convalescent serum [Figure IB], Recombinant RcAd5 expressing GFP [RcAd5-GFP] was generated to serve as a negative control for the antigens [Figure IB], The recombinant viruses were amplified in HEK293A cells, purified by CsCl density gradient ultracentrifugation, dialyzed in PBS, tittered, and then frozen at -80°C.

[0133] Table 1 shows a series of multi ci stronic expression cassettes organized by construct designation and described by their ASFV antigens.

[0134] Table 1 : Multicistronic Expression Cassettes

[0135] Immunization of pigs: Safety, immunogenicity, and efficacy of the RcAd5- vectored ASFV prototype vaccine was evaluated in healthy female piglets [35-40 lbs.]. As shown in FIG. 2, age-matched commercial piglets were divided into four treatment groups: 1) RC-Ad5-ASFV plus adjuvant; 2) RC-Ad5-ASFV (without structural protein genes) plus adjuvant; 3) RC-Ad5-ASFV without adjuvant; and 4) RC-Ad5-GFP plus adjuvant (Negative control). Treatment groups are summarized in Table 2. Following a one-week acclimation period, Day 0 sampling was done, and the treatment piglets received intramuscular inoculation of the priming dose followed by boosts on days 21 and 35 post-priming. One week after the second boost, contact-challenge piglets received an IM injection of ASFV (Georgia 2007/1) which spread the virus to the co-mingled vaccinees. The pigs were monitored every day for clinical symptoms, with biweekly samples of blood, serum, and nasal swabs collected until termination due to sickness. Pigs that survived were euthanized on day 39 post-challenge when the study was terminated. The intramuscular injection of a prime dose occurred with 10⁹ IFU/each virus construct and the following booster doses occurred withlO¹⁰ IFU/each virus construct at 21- and 35-days post-priming. Each dose of the RcAd5-ASFV cocktail was split into two pools based on protein expression levels [High and low expressers] and injected separately around the neck region. Piglets were also monitored for changes in body temperature and body weight. These measurements were taken weekly. Body weight and temperature changes for each pig ID in each of the four test Groups are shown in FIG. 3 Body weights for piglets in all of Groups 1-4 increased steadily over a six-week period, with no outliers detected. Piglet temperatures in all Groups vacillated within a narrow normal range, indicating no abnormalities. The results were the same for piglets that were vaccinated, negative controls, and non-immunized contact spreaders.

[0136] Immunogen safety and tolerability: Following priming and post-boosting, the piglets were monitored once daily and the following parameters were recorded to document local and systemic adverse effects, if any, caused by the immunogen: inoculation site swelling, nasal discharge (shedding of the recombinant RcAd5 in nasal swabs was also monitored), and rectal temperature.

[0137] Table 2: Immunization Protocol

[0138] Post-immunization Readouts: Following priming, the immunized piglets seroconverted as judged by indirect ELISA using recombinant ASFV antigens. The induced antibody responses were significantly amplified by the booster dose [Fig. 4], Evaluation of ASFV antigen-specific IFN-y+/granzyme B+ T cell responses by flow cytometric analyses following Intracellular staining is pending.

[0139] Expression of ASFV antigens by recombinant Replication-Competent Adenovirus-5 (RC-Ad5) was confirmed by immunocytometric analyses of infected HEK-293A cells probed with ASFV convalescent serum or evaluated for GFP expression. Representative data is shown for four RC-Ad5 virus constructs encoding ASFV antigens (RC-Ad5-l, RC-Ad5- 3, and RC-Ad5-5), RC-Ad5-GFP, and a negative control. Results are shown in FIG. IB.

[0140] Virus replication was evaluated by IFA using primary swine alveolar epithelial cells infected for 40 h at 0.1MOI with either Replication-Defective adenovirus-5 expressing p72- p15-B602L or p62-p32-p54-EP153R-p! 0 (RD-Ad5-3 and RD-Ad5-4, respectively), or infected with either RC-Ad5 expressing p72-pl5-B602L or p62-p32-p54-EP153R-pl0 (RC-Ad5-3 and RC-Ad5-4, respectively], and then probed with the ASFV convalescent serum. RD-Ad5-Luc and RC-Ad5-GFP served as negative controls. Results are shown in FIG. 1C.

[0141] Recombinant RC-Ad5-ASFV Virus Shedding

[0142] Nasal samples collected one week after the second boost were negative for RC- Ad5 following virus rescue using primary swine alveolar epithelial cells or HEK-293 A cells evaluated by immunocytometric analyses. Cells exposed to nasal samples collected from the RC- Ad5-ASFV-immunized pigs were probed with anti-Ad5 polyclonal IgGs or ASFV convalescent serum, whereas RC-Ad5-GFP-infected cells were scored for GFP fluorescence. The positive controls were RC-Ad5-ASFV-infected cells, whereas uninfected cells served as negative controls. Results are summarized in Table 3.

[0143] Table 3: Recombinant RC-Ad5-ASFV Virus Was Not Shed

[0144] ASFV Challenge Two weeks after the last boost, challenge was initiated by injecting (IM) the two naive piglets in each group [Table 2: contact spreaders] with 50 TCID50 ASFV (Georgia 2007/1) and allowed to co-mingle with the treatment piglets for seven days when they were euthanized as they exhibited classical ASFV symptoms. This contact challenge model, which mimics natural virus spread and transmission during ASFV outbreak, has worked well in our studies. Following challenge, clinical scores, viremia, and time to death were determinants of vaccine efficacy. At termination, pigs were necropsied, and evaluated and scored by a pathologist blinded to the treatments. Tissue samples (Tonsil, lung, liver, spleen, kidney, heart, intestine, lymph nodes [submandibular, gastrohepatic, mesenteric, renal, and cranial mediastinal] were obtained to analyze and score pathological lesions. Viremia in blood and nasal/rectal swabs was determined by quantitative PCR. Following challenge, one out of the three ASFV subunit vaccine candidate formulations that were tested conferred protection to 5/6 (83%) of the vaccinated pigs [for 39 days post-challenge]. All controls and the pigs immunized with the other two formulations did not make it past 7 days post-challenge. The protected pigs performed extremely well and were putting on ~16 lb. /week and they weighed >200 lb. by the time the study was terminated [Fig. 6A],

[0145] All the vaccinees mounted antibody responses as judged by IgG ELISA using pp62 antigen. Group 2 was negative for pp62 since the cocktail used excluded this antigen. However, sera from all the test pigs, but not the negative controls, recognized ASFV-infected swine macrophages (Table 4). The primed B cell responses were strongly recalled upon boost on day 21. Notably, the IgG responses mounted by the vaccinees in groups 1 and 3, post-prime and post-boost, were similar. This outcome suggests that the Quil-A adjuvant used in group 1 did not impact IgG responses. The antibody responses mounted were mostly IgGs and low amounts of IgMs and IgAs.

[0146] Antibodies induced by the RC-Ad-ASFV recognized ASF Virus: Antibodies elicited by the RC-Ad-ASFV cocktail formulations were tested for recognition of ASFV-infected primary swine macrophages by indirect Immuno-Fluorescence Antibody assay (IF A) using sera collected one week after the second boost as shown in FIG. 5. Representative data is shown for sera from two pigs from each treatment group (Groups 1-3) and from the RC-Ad5-GFP control (Group 4). Normal swine serum served as the negative control, whereas ASFV convalescent serum was the positive control. Sera from all the vaccinees, but not from the negative controls, recognized the ASFV-infected swine macrophages (Table 4).

[0147] Table 4: Elicited Antibodies Recognized ASFV-infected Primary Swine Macrophages

[0148] Pig Survival Post-Challenge: Pig survival post-challenge of vaccinated (Gl, G2, G3); sham treatment controls (G4); and IM challenged contact spreader pigs (in red) that were injected with ASFV (Georgia 2007/1) are shown in FIG 6A. The contact spreaders were allowed to co-mingle with the treatment pigs for 6-7 days when they were euthanized as they exhibited ASF symptoms. Notably, 5/6 pigs in G3 treatment group survived (p<0.0001), but all the pigs from the other treatment groups succumbed.

[0149] Clinical scores post-challenge: The heat map in FIG. 6B displays the daily clinical scores of each pig post-challenge. The contact challenge (CC) pigs started to show ASF symptoms five days post-challenge. Thereafter, labored breathing, coughing/sneezing, and lethargy/recumbency became more apparent. Consequently, all the CC pigs were terminated on days 6-7 post-challenge. All the immunized pigs in groups 1 and 2 and the negative control pigs in group 4 developed clinical disease and were terminated by day 18 after the initiation of the challenge. One pig (number 37) in group 3 was terminated at day 11 after exhibiting severe ASF symptoms, but the other 5 pigs in this group only showed mild clinical symptoms and survived until the end of the study on day 39. The day pigs were terminated is denoted by a 'T'.

[0150] Post-challenge viremia in blood: As shown in FIG. 6C, ASF virus was detectable in whole blood collected from the contact challenge (CC) pigs by day 5 based on qRT-PCR (p72 DNA) and they were all terminated by day 7. The virus was detectable in some test pigs on day 8, but most of them had detectable virus on days 11-12 and some were terminated due to severe ASF. The two remaining pigs (numbers 50 and 47) had detectable virus DNA on day 15. All the vaccinees and the negative controls, except 5/6 pigs in group 3, had detectable virus DNA on the day they were terminated (marked with a ‘T’). ASF virus DNA in the blood collected from the 5/6 survivors in group 3 was last detected on day 26 following the initiation of challenge by contact with IM infected pigs and remained negative until day 39 when the study was terminated.

[0151] Viremia in terminal tissues: Referring to FIG. 6D, following necropsy, viremia in tissues and fecal materials was enumerated by RT-qPCR. Viral load is presented as the logarithm (base 10) of the mean copy number per milliliter (CN/mL). Each graph displays the viral load for individual tissues collected from each pig. Notably, no virus DNA was detected in the 5/6 pigs in group 3 that survived, but the lone pig in this group that succumbed to ASF (number 37) had high viral loads in all the samples tested.

[0152] Survivors had no viable virus: Referring to FIG. 6E, there was no rescuable ASF virus in spleen tissues from the 5/6 survivors (pig numbers 25, 27, 35, 44, and 48), except in the sample collected from pig 37 that succumbed, as judged by IFA of primary swine macrophages incubated with spleen homogenates and then probed with ASFV convalescent serum. Uninfected (No virus) and infected (Plus virus) macrophages served as negative and positive controls, respectively. Samples from pig number 39 (group 1) and number 32 (group 2) that succumbed had rescuable virus. The outcome was validated by qRT-PCR (p72 DNA) enumeration of viral loads in the infected macrophages, the data for which is shown in FIG. 6F. Similar results were obtained when pericardium fluids were screened (FIGS. 6G and 6H).

[0153] FIG. 7A shows the body weight data of pigs from all four groups, collected at a twice-weekly interval following the ASFV challenge until the end of the study. Remarkably, surviving pigs in Group 3 consistently demonstrated an increase in body weight, whereas pigs in the other groups exhibited a decrease in body weight. As seen in FIG. 7B, the body temperature post-challenge indicated that survivor pigs in Group 3 consistently maintained a normal range body temperature, while pigs in the other groups exhibited an increase in their body temperature before reaching the termination point.

[0154] IFA analyses like those shown in FIG 8A showed that sera from the protected and the non-protected pigs failed to neutralize ASFV or block infection of primary swine macrophages even at 1 : 1 dilution. Data for all the pigs from group 3 are shown as well as representatives from the other groups; ASFV p72 DNA was detected by RT-qPCR analyses of samples from virus neutralization assays conducted using heat inactivated sera (shown in FIG. 8B) or not inactivated sera (shown in FIG. 8C) from the vaccinees and controls. Data from analyses of the sera from all the pigs is summarized in Table 5.

[0155] Table 5: Elicited Antibodies Did Not Neutralize ASFV

[0156] Analysis of SLA Class I and Class II Haplotypes and Survival: SLA class I and II haplotypes of six pigs from group 1 (non-protected) and six pigs from group 3 (5/6 protected) were typed and compared to determine whether there was a correlation between SLA and protection. These two groups received the same RC-Ad5-ASFV virus constructs except that the cocktail used in group 1 was formulated with Quil A adjuvant. The animals used were three- way (LRYS/D) crossbred finishing pigs. LRYS/D: pigs were 25% Landrace (LR), 25% Yorkshire (YS), and 50% Duroc (D); SLA: Swine Leukocyte Antigen; Hp: Haplotype; ksu: Kansas State University; Blank: Alleles that cannot be identified with the study primer sets. There was no correlation found between the survivors and non-survivors as both groups share same SLA-I and SLA-II haplotypes. These results are summarized in Table 6.

[0157] Table 6: No Correlation Between Survival and SLA Class I and Class II Haplotypes

[0159] Analysis of Frequencies of SLA Class I and Class II Haplotypes: The 12

LRYS/D-crossbred finishing pigs in groups 1 and 3 comprised 11 SLA class I (SLA-I) haplotypes. Only two haplotypes (Hp-04.0 and Hp-35.0) explained 54.17% of the SLA-I diversity These two most abundant SLA-I haplotypes - Hp-04.0 (SLA-l*04:01-SLA-3*04:01- SLA-2*04:01) and Hp-35.0 (SLA-l*12:01,13:01-SLA-3*05:02-SLA-2*10:01), occurred at frequencies of 41.67 and 12.50%, respectively. SLA-6 sequencing revealed three genotypic variants, of which SLA-6*ksu01 occurred at a frequency of 66.67%, followed by SLA-6*ksuO2 (16.67%) and SLA-6*ksuO3 (16.67%). For SLA class II (SLA-II), also 11 haplotypes were found, of which the three haplotypes Hp-0.05, Hp- 0.02, and Hp-0.15b explained 50.00% of the SLA-II-diversity. The two most abundant SLA-II haplotypes, Hp-0.05 and (DRBl*05:01- DQBl*02:01-DQA*02:02:02) and Hp-0.02 (DRBl*02:01-DQBl*02:01-DQA*02:01), occurred at frequencies of 20.83 and 16.67%, respectively. Results are summarized in Table 7.

[0160] Table 7: Frequencies of SLA Class I and Class II Haplotypes

[0161] Immune response species were measured in survivor pigs on days 26 and 33 postchallenge. As seen in FIGS. 9A and 9B, live lymphocytes were measured and reported as percent-positive ASFV-specific Granzyme B⁺ CD8a⁺ T cells. FIG. 9A shows measured results for pig IDs 25, 27, 35, 44, and 48, at 26 and 33 days post-challenge respectively as measured in peripheral blood mononuclear cells. FIG. 9B shows measured results for pig IDs 25, 27, 35, 44 and 48 at 39 days post-challenge as measured in splenocytes.

[0162] The following multi ci stronic cassettes were generated:

1. P220.1-2A-E1 Includes fragment of SEQ ID NO. 1 , (2) 2A autocleavable motifs, E1 polypeptide and histidine tag

39. M448R-2A-E423R-2A-MGF505-1R-2A-E1

Claims

What is claimed is:

1. An immunogenic composition comprising a chimeric gene comprising at least one nucleic acid sequence encoding for a sequence having at least 80% sequence homology with any one or more of SEQ ID NOS: 1-101, and any combination thereof.

2. The immunogenic composition of claim 1, wherein the chimeric gene includes at least two of said nucleic acid sequences.

3. The immunogenic composition of claim 2, wherein said chimeric gene includes a self-cleaving peptide linker between at least two of each of the at least two nucleic acid sequences.

4. The immunogenic composition of claim 1, wherein said immunogenic composition is in the form of a multi ci stronic cassette.

5. The immunogenic composition of claim 1, wherein said chimeric gene is inserted into a vector.

6. The immunogenic composition of claim 5, wherein said vector is selected from the group consisting of an adenovirus, baculovirus, or lentivirus vector.

7. The immunogenic composition of claim 6, wherein said vector is a human adenovirus or an attenuated bovine parainfluenza virus type 3 genotype c (BPIV3c).

8. The immunogenic composition of claim 1, wherein said chimeric gene is selected from the group consisting of SEQ ID NOS. 102 - 131, 158-201, or 205- > .

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

10. The immunogenic composition of claim 9, wherein said another disease-causing organism is selected from the group consisting of Actinobacillus pleuropneumonia, Adenovirus; Alphavirus such as Eastern equine encephalomyelitis viruses; Bordetella bronchi septica, Brachyspira spp., preferably B. hyodyentheriae; B. piosicoli, Brucella suis, preferably biovars 1, 2, and 3; Classical swine fever virus; Clostridium spp., preferably Cl. difficile, C 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. preferably M. avium; M. intr acellular e; and M.bovis; 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. thyhimurium; and 5. 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.

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

12. An immunogenic composition comprising at least one recombinant sequence selected from the group consisting of sequences having at least 80% sequence identity to one of SEQ ID NOS. 1-101.

13. The immunogenic composition of claim 12, further comprising an antigen from another disease-causing organism.

14. The immunogenic composition of claim 13, 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. preferably M. avium; M. intr acellular e; and M.bovis; Mycoplasma hyopneumoniae (M hyo); Pasteurella multocida; Porcine circovirus; Porcine cytomegalovirus; Porcine Parvovirus; Porcine Reproductive and Respiratory Syndrome (PRRS) Virus; Pseudorabies virus; Rotavirus; Salmonella .s/yz; preferably S. thyhimuriunr, 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.

15. The immunogenic composition of claim 12, further comprising at least one pharmaceutical-acceptable carrier.

16. 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.

17. The method of claim 17, 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.

18. The method of claim 17, wherein the clinical or postmortem sign or symptom of ASFV is selected from the group consisting of loss of appetite, depression, recumbency, hyperemia of the skin of the ears, abdomen, and/or legs, respiratory distress, vomiting, bleeding from the nose or rectum, diarrhea, abortion, lesions in at least one internal organ, an enlarged and/or friable spleen, straw-colored or blood-stained fluid in pleural, pericardial, and peritoneal cavities, edema and congestion of the lungs, and any combination thereof.

19. 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 12 to an animal in need thereof.

20. 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 12.